extfieldsofstudy: Chemistry Medicine

text:
The X-ray Structure of the PurR-Guanine-purF Operator Complex Reveals the Contributions of Complementary Electrostatic Surfaces and a Water-mediated Hydrogen Bond to Corepressor Specificity and Binding Affinity*

The purine repressor, PurR, is the master regulatory protein of de novo purine nucleotide biosynthesis in Escherichia coli. This dimeric transcription factor is activated to bind to cognate DNA operator sites by initially binding either of its physiologically relevant, high affinity corepressors, hypoxanthine (K d = 9.3 μm) or guanine (K d = 1.5 μm). Here, we report the 2.5-Å crystal structure of the PurR-guanine-purF operator ternary complex and complete the atomic description of 6-oxopurine-induced repression by PurR. As anticipated, the structure of the PurR-guanine-purFoperator complex is isomorphous to the PurR-hypoxanthine-purF operator complex, and their protein-DNA and protein-corepressor interactions are nearly identical. The former finding confirms the use of an identical allosteric DNA-binding mechanism whereby corepressor binding 40 Å from the DNA-binding domain juxtaposes the hinge regions of each monomer, thus favoring the formation and insertion of the critical minor groove-binding hinge helices. Strikingly, the higher binding affinity of guanine for PurR and the ability of PurR to discriminate against 2-oxopurines do not result from direct protein-ligand interactions, but rather from a water-mediated contact with the exocyclic N-2 of guanine, which dictates the presence of a donor group on the corepressor, and the better electrostatic complementarity of the guanine base and the corepressor-binding pocket.

The purine repressor, PurR, is a 341-amino acid residue, dimeric DNA-binding protein from Escherichia coli. In E. coli, PurR functions as the master regulatory protein for de novo purine nucleotide biosynthesis and, to a lesser extent, de novo pyrimidine nucleotide biosynthesis and salvage, by repressing transcription of over 18 genes (1). In addition, PurR regulates the transcription of several genes encoding proteins necessary for polyamine and nucleotide biosynthesis (2,3). Finally, PurR is autoregulated (4,5).
PurR belongs to the highly homologous lactose repressor (LacI) family of transcription regulators (6,7). Nearly all LacI members function by binding to similar pseudo-palindromic operator sites typically 16 -18 base pairs long. Structurally, LacI members can be divided into two functional domains: an N-terminal DNA-binding domain (ϳ60 residues) and a C-terminal oligomerization effector-binding domain (Ͼ250 residues). Effector binding serves as a signal of the metabolic state of the cell, and whereas most LacI proteins bind with high affinity to their operator sites only in the absence of effector binding, the specific interaction of PurR with its DNA operator sites is dependent on corepressor binding. PurR is unique in having two physiologically relevant corepressors, hypoxanthine, to which it binds with a K d of 9.3 M, and guanine, to which it binds with higher affinity (K d ϭ 1.5 M) (8).
To describe the interactions between PurR and its corepressors and operator sites, we determined the structure of a PurR-hypoxanthine-purF operator complex (9). The structure revealed that PurR has a bipartite structure: a C-terminal corepressor-binding domain (CBD), 1 which spans residues 61-341 and is responsible for dimerization and corepressor binding and specificity, and an N-terminal DNA-binding domain, which spans residues 1-60. The latter domain is composed of a classical helix-turn-helix motif that is closed off by a loop and another helix. By engaging in a series of direct and water-mediated contacts, this three-helix bundle provides the major groove binding specificity of PurR. It is followed by another loop and a fourth helix, the hinge helix. The hinge helix, with a 2-fold related mate, inserts into the DNA minor groove and makes additional specific contacts. Furthermore, hinge helix insertion kinks the purF operator by permitting the interdigitation of the side chains of Leu 54 (and its 2-fold mate, Leu 54Ј ) between the central CpG base pair step of the operator (9).
The CBD is oblate in shape and consists of two topologically similar ␣/␤-subdomains (the N-and C-terminal subdomains) that are connected by three crossover connections. The CBD is structurally similar to the bacterial periplasmic binding proteins (10 -12). As observed in the periplasmic binding proteins, PurR binds hypoxanthine in the cleft between the CBD subdomains through a series of polar, nonpolar, and aromatic interactions (9). Direct and water-mediated contacts are provided by the side chains of Tyr 73 , Phe 74 , Arg 190 , Thr 192 , Phe 221 , and Asp 275 . The ability of PurR to utilize hypoxanthine or guanine, but not adenine and xanthine, indicates the critical importance of the chemical nature of the exocyclic 2-and 6-groups in * This work was supported by United States Public Health Service Grants GM49244 (to R. G. B.) and GM24658 (to H. Z.) and a National Defense Science and Engineering Graduate Fellowship in Biosciences (to M. A. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The binding specificity. The key PurR residue that specifies an oxygen at the purine exocyclic 6-position is Arg 190 . The guanidinium group of Arg 190 donates two hydrogen bonds to the O-6 acceptor of hypoxanthine (9). Thus, the side chain donors of Arg 190 dictate the requirement for hydrogen bond acceptors at this purine position. However, the mechanism of discrimination at the exocyclic 2-group was unclear, as was the stereochemical basis by which PurR binds guanine more tightly than hypoxanthine (8).
To begin the structural elucidation of the allosteric mechanism of PurR, which transduces the signal of corepressor binding by the CBD to effect DNA binding by the distal DNAbinding domain, the structure of the corepressor-free CBD was determined (13). From this and the hypoxanthine-bound PurR structure, the role of that purine corepressor binding was discerned. Upon binding hypoxanthine, there is a dramatic rearrangement in the tertiary and quaternary structures of the CBD N-terminal subdomain that results in the juxtaposition of the two hinge regions of PurR, which are over 40 Å away (13). Corepressor binding is essential for this juxtaposition as it shifts the equilibrium from the open form of PurR, in which the N-and C-terminal subdomains of the CBD are rotated far apart, to the closed conformation. Although likely, it was not known if guanine binding causes the identical structural changes that result in this allosteric shift.
To address the issues of the corepressor specificity and the higher affinity of PurR for guanine and its possible consequences on the intramolecular signaling pathway and allostery of this protein, we have determined the structure of a PurRguanine-purF operator complex at 2.5-Å resolution. As expected, the PurR-purF operator contacts and DNA bending are nearly identical to those seen in the PurR-hypoxanthine-purF operator complex (9). Strikingly, the structure reveals that the higher affinity of guanine for PurR (over hypoxanthine) and the discrimination against xanthine as a corepressor arise from 1) a corepressor-water-protein hydrogen bonding network, which involves the exocyclic N-2 hydrogen bond donor of guanine and the hydrogen bond acceptors, O-⑀ of Glu 222 and the backbone carbonyl oxygen of Phe 221 , and 2) the intrinsic negative electrostatic environment of the corepressor-binding pocket, which favors the more positive guanine as the corepressor.

EXPERIMENTAL PROCEDURES
Crystallization and Data Collection-PurR was overexpressed in E. coli and purified by DE53 cellulose and heparin-agarose chromatography as described (14). Crystals of the PurR-guanine-purF operator complex were grown by the hanging-drop vapor diffusion method, which was used previously to crystallize the PurR-hypoxanthine-purF operator complex (15). Briefly, protein at a concentration of 0.25-0.50 mM was saturated with guanine and mixed 1:1 (v/v) with a 0.50 mM solution of the 16-base pair purF operator, which has an additional 5Ј-nucleoside overhang on both strands, the identity of which is irrelevant to crystallization. The protein/corepressor/DNA solution was mixed 1:1 with the reservoir solution, which was 25% polyethylene glycol 4000, 0.40 M ammonium sulfate, 25 mM hexamminecobalt chloride, and 0.10 M ammonium phosphate, pH 7.5. Crystals initially grow as thin two-dimensional plates and, within a period of 2 weeks to several months, melt and are replaced by large rhombohedral crystals (typically 0.6 ϫ 0.2 ϫ 0.3 mm). The PurR-guanine-purF operator crystals are isomorphous to the PurR-hypoxanthine-purF operator crystals and take the space group C222 1 , with unit cell dimensions of a ϭ 176.0, b ϭ 95.08, and c ϭ 81.51 Å. There is one PurR monomer, one guanine molecule, and a purF operator half-site in the asymmetric unit.
X-ray intensity data were collected at room temperature with an Area Detector Systems Corp. multiwire area detector (16) and a Rigaku RU200-H rotating anode operating at 40 kV and 150 mA. The data from two crystals were processed and merged with the software provided by Area Detector Systems Corp. The R merge of the data to 2.5 Å is 6.29%, and the data are 87% complete from 10.0 to 2.5 Å and 78% from 2.7 to 2.5 Å. The redundancy of the data is 3-fold. The I/(I) is 7.4 for the entire data set and 1.4 for the data in the resolution shell between 2.7 and 2.5 Å.
Refinement-The previously determined structure of the PurR-hypoxanthine-purF operator complex minus the hypoxanthine and solvent molecules was used as the starting model for refinement (9). Rigid body refinement was carried out on the model followed by XYZ and B factor refinement as implemented in TNT (17). The current model, refined from 10.0 to 2.5 Å, has an R factor of 18.0% and displays excellent stereochemistry with only two residues, Ser 124 and Asp 275 in the unfavorable region of the Ramachandran plot (18). As observed previously, the unfavorable / angles of Asp 275 are necessary for its interaction with the N-9 atom of the corepressor (9). Although not directly in contact with the corepressor, Ser 124 is located directly above the corepressor-binding pocket and takes this conformation to avoid steric clash with the side chain of Arg 190 . 2 The root mean square deviations of bond lengths and bond angles are 0.02 Å and 2.23°, respectively. An F o Ϫ F c omit map, in which the guanine base, residues 221-222, and Wat 1 were omitted from the model and followed by 20 cycles of positional and thermal parameter refinement, is shown in Fig.  1   The map was calculated using structure factors from a structure in which guanine, Wat 1 , Phe 221 , and Glu 222 were removed, and an additional 20 cycles of refinement were carried using TNT (17). The contour level of the map is 3.5 . The hydrogen bonds formed between the water molecule (Wat 1 ) and the carbonyl oxygen of Phe 221 and the carboxyl side chain oxygen of Glu 222 are shown. Such a donor-acceptor arrangement leaves only hydrogen bond acceptors and results in a water-specific interaction with the exocyclic N-2 of guanine and discrimination against O-2 of xanthine.

RESULTS AND DISCUSSION
The current model of the PurR-guanine-purF operator complex includes residues 3-340 of the PurR monomer, one guanine molecule, a purF operator half-site, and 20 solvent molecules (Fig. 2). The atomic structures of PurR and the purF operator are essentially identical to those seen in the PurRhypoxanthine-purF operator complex and display a root mean square deviation of 0.25 Å for all atoms except solvent (9). Therefore, guanine and hypoxanthine induce the same DNA binding conformation of PurR. The N-terminal DNA-binding domain (residues 3-60) consists of a helix-turn-helix-loop-helix motif, which is responsible for DNA major groove binding specificity, and a hinge helix, which binds, with its 2-fold related mate, in the minor groove of the DNA. The insertion of two ␣-helices into the narrow minor groove is made possible by the partial interdigitation of the hinge helix residue Leu 54 (and Leu 54Ј ) within the central CpG base pair step. DNA bending, which was calculated with CURVES (19) and was exclusive of the first and last base pairs of the 16-base pair operator because of their poor electron density, revealed a 55°kink in the PurR-guanine-purF operator complex resulting from this side chain partial insertion. This bend is essentially identical to the 54°bend observed in the structure of the PurR-hypoxanthine-purF operator complex (9,20). Moreover, no significant changes in any base pair or base pair step parameter were observed.
The CBD consists of two subdomains: the CBD N-terminal subdomain, which consists of a core of six ␤-strands (␤A-␤E and ␤J) surrounded by four ␣-helices (I-III and IX), and the CBD C-terminal subdomain, which consists of a core of five ␤-strands (␤F-␤I and ␤K) surrounded by five ␣-helices (IV-VIII) (9). Three crossover peptides connect the two subdomains and serve as a floor to the corepressor, which is bound in the cleft between the two CBD subdomains. As seen in the periplasmic binding proteins, these peptidic linkages act as a hinge to allow movements of subdomains upon ligand association and dissociation (9 -12). All contacts observed between PurR and the corepressor hypoxanthine in the PurR-hypoxanthine-purF operator complex are also present in the PurR-guanine-purF operator complex. Stacking interactions occur between guanine and the side chains of Tyr 73 and Phe 221 , which sandwich the corepressor, and Phe 74 . Hydrogen bonds are provided by Thr 192 , Asp 275 , and Arg 190 . O-␥ of Thr 192 hydrogen bonds with N-7 of the guanine ring (2.73 Å, O-␥-N-7) and also makes van der Waals contact with the corepressor C-8 via its side chain methyl group. Asp 275 makes a hydrogen bond with N-9 of the purine (2.92 Å, O-␦2-N-9). The importance of Arg 190 in corepressor binding specificity at the exocyclic 6-position was revealed in the PurR-hypoxanthine-purF operator complex (9). In the PurR-guanine-purF operator complex, Arg 190 also specifies an oxygen at the exocyclic 6-position by providing two hydrogen bonds to this exocyclic amine from its side chain N-and NH-1 atoms (3.03 Å, N--O-6; and 2.72 Å, NH-1-O-6). Arg 190 contributes further to corepressor binding affinity by hydrogen bonding to a water molecule that is simultaneously hydrogen-bonded to N-1 of the guanine ring (3.15 Å, H 2 O-N-1; and 2.72 Å, H 2 O-NH 2 ). The essentially identical binding observed between PurR and hypoxanthine and guanine in the PurR-hypoxanthine-purF operator and PurR-guanine-purF operator complexes and the identical corepressor-mediated conformations of both complexes reveal how PurR utilizes two related molecules as corepressors. However, these interactions do not explain the nearly 7-fold higher affinity of guanine for PurR or the mechanism by which PurR discriminates at the purine exocyclic 2-position, i.e. against xanthine.
On the basis of the location of the side chain of Glu 222 in the PurR-hypoxanthine-purF operator complex (9), we anticipated that upon guanine binding by PurR, this side chain would swing into the corepressor-binding pocket, eject a tightly bound water molecule (Wat-1), and make a direct hydrogen bond with the exocyclic N-2 atom. However, in the PurR-guanine-purF operator complex, the position of the side chain of Glu 222 has changed little, and the water molecule is still present, although slightly shifted in position (Fig. 1). Rather, now the water molecule is positioned perfectly to hydrogen bond to the exocy-  (Fig. 1). This leaves only the two acceptor lone pairs of this water molecule, one of which interacts with the guanine N-2 atom and presumably the other with a hydrogen bond donor from the bulk solvent. Such a hydrogen bonding network would disfavor the binding of xanthine, which has an exocyclic acceptor atom (O-2) at this position. Consistent with this hypothesis is the in vitro binding of xanthine to PurR, which is ϳ200-fold weaker than guanine binding (data not shown). Remarkably, this water molecule is found not only in the PurR-guanine-purF operator and the PurR-hypoxanthine-purF operator complexes, but also in the corepressor-free form of PurR, where the identical water-protein hydrogen bonds are made (13). This demonstrates that this tightly bound water

FIG. 2. Structure of the PurR-guanine-purF operator complex.
Labeled are the helix-turn-helix-loop-helix (HTHLH) domain, which is responsible for major groove specificity (colored red); the hinge helix motif, which mediates minor groove specificity (colored blue); the Nterminal subdomain of the corepressor-binding domain, residues 61-159 and 294 -310 (colored yellow), which is critical in intramolecular signaling; and the C-terminal subdomain of the corepressor-binding domain (colored green), residues 160 -298 and 311-341, which imparts dimer stability. The guanine corepressor is shown as balls and sticks. The figure was generated with MOLSCRIPT (39). molecule acts as an extension of the protein.
The importance of water-mediated contacts in ligand binding specificity is now well documented. Notable examples include members of the periplasmic binding protein family such as the arabinose-binding protein (21,22), lysine/arginine/ornithinebinding protein (23), and oligopeptide-binding protein (OppA) (24); enzymes, e.g. ␣-chymotrypsin (25); and DNA-binding proteins as exemplified in the TrpR-tryptophan-trp operator complex (26,27). In the arabinose-binding protein, two hydrogenbonded water molecules, located in the binding pocket, aid in L-arabinose binding while creating an unfavorable interaction with the C-5 methyl group of D-fucose, thus explaining the low affinity of the latter. The near equal affinity of the arabinosebinding protein for D-galactose is explained by the replacement of one of these water molecules with the D-galactose -CH 2 OH group (21,22). A similar situation is observed in the lysine/ arginine/ornithine-binding protein, where water molecules assist in the binding of lysine, ornithine, and arginine. However, one of the water molecules must be displaced to permit binding of the larger arginine ligand (23). In contrast, OppA provides an example of the use of solvent in decreasing ligand binding specificity through the accommodation of multiple substrates with different peptide side chains in voluminous hydrated cavities from which water molecules are readily gained or lost (24). In the structure of ␣-chymotrypsin complexed with a turkey ovomucoid inhibitor, it was demonstrated that a water molecule, also present in the apo structure, mediated an ionic in-teraction that was critical for inhibitor binding specificity (25). Thus, as in the case of PurR, this water molecule can be considered as a constitutive element of the protein. Finally, the TrpR-tryptophan trp operator complex reveals quite dramatically the use of water molecules to nearly completely specify protein-DNA binding specificity (26,27). Thus, through its small molecular size, which allows it to pack around ligands of different size and shape, and its ability to provide hydrogen bond donors as well as acceptors, water provides a key element in the formation of a wide variety of protein-ligand complexes.
In addition to the direct water-mediated discrimination against xanthine, an examination of the electrostatic potential of the PurR corepressor-binding pocket and its proximity reveals a second possible source of unfavorable xanthine binding as well as the chemical basis of the more favorable binding of guanine over hypoxanthine. PurR displays a negative electrostatic potential surface near the exocyclic 2-position of the guanine ring (Fig. 3). Glu 220 , Glu 222 , and Glu 70 and Glu 70Ј from the other subunit contribute to the negative potential of the binding pocket of PurR. This surface is complemented by N-2 of guanine, which imparts a significant positive potential to this base. Hypoxanthine, which lacks any exocyclic group at the ring 2-position, is nearly neutral, but is still weakly positive. In contrast to the physiological corepressors, xanthine possesses a negative electrostatic potential at its exocyclic O-2 position, which likely contributes further to its very low affinity (Fig. 4). A similar electrostatic complementarity is observed between Shown is a cut-away surface representation revealing the electronegative potential of the PurR corepressor-binding pocket, which encompasses the electropositive exocyclic N-2 of the guanine. This figure was generated with GRASP (40). the exocyclic N-2 atom of the guanine base of GDP and ARF-1 (28), where the N-2 atom is enclosed in a positive pocket in the protein. This suggests that such electrostatic complementarity may play a significant role in purine-protein binding and recognition. In addition to the patch of negative electrostatic potential near the exocyclic 2-atom of guanine, there is another locus of negative potential within the corepressor-binding pocket, which is contributed by Glu 248 and Asp 275 and complements the positive potential of the N-9 atom of the purine rings of guanine and hypoxanthine (Fig. 4). Beyond its contribution to binding, the charged environment of PurR could serve to steer its guanine and hypoxanthine corepressors into their bound positions within the binding pocket. Indeed, electrostatic focusing by narrow clefts appears to be a general phenomenon, having been perhaps best documented for copper,zinc-superoxide dismutase (29) and trypsin (30). Thus, a tight water-mediated hydrogen bond and the negative electrostatic potential of the corepressor-binding pocket, which complements guanine better than hypoxanthine, provide a thermodynamic rationale for the increased affinity of PurR for guanine over hypoxanthine while allowing PurR to maintain the same activated conformation for both corepressors. Combined, these interactions are consistent with the small favorable free energy change (⌬⌬G Ϸ Ϫ1 kcal/mol, as calculated by the relationship ⌬⌬G ϭ ϪRT ln(K dGua /K dHyp ) observed for PurR-guanine binding over PurR-hypoxanthine binding.
PurR is unique in being the only repressor with two physiologically relevant corepressors to have had their three-dimensional structures as well as its corepressor-free form determined at atomic resolution (9,13). Structures are known for the corepressor-free and -bound forms of only two other repressors: the tryptophan repressor (TrpR) (27, 31-33) and the methionine repressor (MetJ) (34 -36). The TrpR and MetJ repressors are similar in that they both bind their corepressors within their DNA-binding domains. In the case of TrpR, the L-tryptophan corepressor reorients the flexible DNA-binding helixturn-helix reading heads to allow them to fit into successive major grooves. Moreover, the L-tryptophan makes direct contacts with the DNA phosphate backbone. For MetJ, the binding of its corepressor, S-adenosylmethionine, which is located ϳ12 Å from the DNA backbone, does not appear to result in any conformational change or structural rearrangement of the MetJ protein (34,35). Rather, the electrostatic potential of MetJ becomes decidedly more positive and might effect, albeit indirectly, the high affinity DNA binding (36). Clearly, PurR is different and binds its corepressors within a corepressor-binding pocket, which is at least 40 Å from the DNA-binding domain. Corepressor binding activates DNA binding allosterically by repositioning the hinge regions of each PurR monomer such that they may interact, undergo a coil-to-helix transition (37, 38), and bind the DNA minor groove.
In summary, the structure of the PurR-guanine-purF operator complex has established that binding of either hypoxanthine or guanine induces the same activated conformation of PurR, which can then bind the pur regulon operators with high affinity. The structure also reveals the likely basis for the higher affinity of PurR for its guanine corepressor versus hypoxanthine and the stereochemical mechanism of xanthine discrimination: 1) water-mediated readout of the exocyclic 2-position of the purine and 2) better protein-corepressor electrostatic complementarity, notably about the exocyclic 2-position of the purine ring.


extfieldsofstudy: Biology Medicine

text:
Direct Ca2+-dependent heterophilic interaction between desmosomal cadherins, desmoglein and desmocollin, contributes to cell-cell adhesion.

Human fibrosarcoma cells, HT-1080, feature extensive adherens junctions, lack mature desmosomes, and express a single known desmosomal protein, Desmoglein 2 (Dsg2). Transfection of these cells with bovine Desmocollin 1a (Dsc1a) caused dramatic changes in the subcellular distribution of endogenous Dsg2. Both cadherins clustered in the areas of the adherens junctions, whereas only a minor portion of Dsg2 was seen in these areas in the parental cells. Deletion mapping showed that intact extracellular cadherin-like repeats of Dsc1a (Arg1-Thr170) are required for the translocation of Dsg2. Deletion of the intracellular C-domain that mediates the interaction of Dsc1a with plakoglobin, or the CSI region that is involved in the binding to desmoplakin, had no effect. Coimmunoprecipitation experiments of cell lysates stably expressing Dsc1a with anti-Dsc or -Dsg antibodies demonstrate that the desmosomal cadherins, Dsg2 and Dsc1a, are involved in a direct Ca2+-dependent interaction. This conclusion was further supported by the results of solid phase binding experiments. These showed that the Dsc1a fragment containing cadherin-like repeats 1 and 2 binds directly to the extracellular portion of Dsg in a Ca2+-dependent manner. The contribution of the Dsg/ Dsc interaction to cell–cell adhesion was tested by coculturing HT-1080 cells expressing Dsc1a with HT-1080 cells lacking Dsc but expressing myc-tagged plakoglobin (MPg). In the latter cells, MPg and the endogenous Dsg form stable complexes. The observed specific coimmunoprecipitation of MPg by anti-Dsc antibodies in coculture indicates that an intercellular interaction between Dsc1 and Dsg is involved in cell–cell adhesion.

Dsc1a with anti-Dsc or -Dsg antibodies demonstrate that the desmosomal cadherins, Dsg2 and Dsc1a, are involved in a direct Ca 2 ϩ -dependent interaction. This conclusion was further supported by the results of solid phase binding experiments. These showed that the Dsc1a fragment containing cadherin-like repeats 1 and 2 binds directly to the extracellular portion of Dsg in a Ca 2 ϩ -dependent manner. The contribution of the Dsg/ Dsc interaction to cell-cell adhesion was tested by coculturing HT-1080 cells expressing Dsc1a with HT-1080 cells lacking Dsc but expressing myc-tagged plakoglobin (MPg). In the latter cells, MPg and the endogenous Dsg form stable complexes. The observed specific coimmunoprecipitation of MPg by anti-Dsc antibodies in coculture indicates that an intercellular interaction between Dsc1 and Dsg is involved in cell-cell adhesion.
S tructurally related desmosomes and adherens junctions, collectively termed adhering junctions, are involved in anchoring the cytoskeleton to the plasma membrane, intercellular cell type-specific adhesion, and signaling (Geiger and Ayalon, 1992;Schmidt et al., 1994;Klymkowsky and Parr, 1995;Peifer, 1995;Cowin and Burke, 1996;Gumbiner, 1996). Classic and desmosomal cadherins are featured in both adherens and desmosome junctions. It is widely accepted that classic cadherins mediate homophilic calcium-dependent cell-cell adhesion (Nose et al., 1990;Grunwald, 1993;Shapiro et al., 1995;Brieher et al., 1996;Nagar et al., 1996). An exception to this rule, heterophilic binding of the chicken B-cadherin to LCAM, has been documented (Murphy-Erdosh et al., 1995). On the intracellular face of the plasma membrane, cadherins are integrated into plaques consisting of junctional-specific proteins. These proteins function in the formation of anchoring sites for microfilaments and intermediate filaments (in adherens junctions and desmosomes, respectively) and are critical for adhesion and signaling properties of cadherins (Nagafuchi and Takeichi, 1988;Ozawa et al., 1989;Green and Jones, 1990;Geiger and Ayalon, 1992;Schmidt et al., 1994).
In contrast with adherens junctions that may contain only one cadherin isoform, desmosomes always include cadherins from two subfamilies, desmogleins (Dsg1-3) 1 and desmocollins (Dsc1-3). Alternative splicing increases Dsc diversity producing long (Dsc a) and short (Dsc b) isoforms that differ in their intracellular domains (Garrod, 1993;Koch and Franke, 1994). Recent experiments with chimeric proteins consisting of the gap junction protein connexin32 and the intracellular regions of desmosome cadherins indicate that Dsg and Dsc have different functions. The CoDsc chimera containing the intracellular portion of Dsc1a nucleated the formation of the intracellular desmosomal plaques. The cytoplasmic domain of Dsg1, in a similar construct, displayed a dominant negative effect on desmosome formation (Troyanovsky et al., 1993(Troyanovsky et al., , 1994. Involvement of the desmosomal cadherins in cell-cell adhesion was underscored by cell culture observations that antibodies against the extracellular regions of Dsc interfered with the formation of the epithelial sheet (Cowin et al., 1984). Also, auto anti-Dsg antibodies caused a blistering skin disease (Stanley, 1995). Perturbation in epidermal cell-cell interactions was found in transgenic animals producing a dominant negative form of Dsg (Allen et al., 1996). Structural similarity between the extracellular repeats of Dsg and Dsc with those of the classic cadherins involved in homophilic adhesion provides additional support for the adhesive functions of the desmosomal cadherins. However, in contrast with classic cadherins, desmosomal cadherins, alone or in combination, failed to support cell-cell adhesion upon expression in nonadhesive fibroblast-like cells (Amagai et al., 1994;Chidgey et al., 1996;Kowalczyk et al., 1996). This suggests that the functional properties of classic and desmosomal cadherins are distinct despite their overall structural homology. Moreover, the molecular mechanism of coassociation of the different desmosomal cadherins in the actual desmosome is not well understood. While direct interactions between desmosomal cadherins were not documented, it seems likely that they are essential for desmosome assembly.
Several observations suggest that efficient desmosome formation, and hence interactions between desmosomal cadherins, may require the function of the classic cadherins (Wheelock and Jensen, 1992;Lewis et al., 1994;Amagai et al., 1995). To investigate this possibility, we expressed bovine Dsc1a in HT-1080 cells that feature extensive adherens junctions and produce endogenous Dsg2. We found that expression of Dsc1a in these cells induces the formation of stable complexes between Dsc and Dsg. In addition, we show a direct Ca 2 ϩ -dependent interaction between the extracellular regions of two desmosomal cadherins. These observations suggest that heterophilic interactions between desmosomal cadherins are important for targeting these proteins to desmosomes and for cell-cell adhesion.

Plasmid Construction
The plasmid BlDc1a encoding the entire sequence of the mature bovine Dsc1a isoform was obtained by subsequent insertions in the Bluescript vector of the XhoI-NotI and XhoI-KpnI inserts cut out from the plasmids BDC 7.5 and BDC 6.1, respectively (Koch et al., 1991). The sequence coding for the leader peptide lacking in BlDc1a was amplified from plasmid pDGK5(B), encoding bovine Dsg1 (Koch et al., 1990). It was inserted upstream to the Dsc1a sequence in BlDc1a between the unique HindIII and NarI sites, resulting in plasmid BlLDc1a. The fusion protein encoded by this plasmid contains the entire sequence of the Dsg-derived leader peptide and the entire sequence of the mature Dsc1a. In Dsc1a only the first amino acid residue Arg is replaced with Glu to create a Dsg-specific processing site. The same pDGK5(B) plasmid was used for construction of BlDc(1-170Dg) that contains the coding sequence for the leader peptide and for the Glu 1 -Arg 170 sequence of the mature Dsg1 preceding the corresponding sequence of Dsc1a. Deletion mutagenesis for constructing BlDc ⌬ (697-761) and BlDc ⌬ (597-609) was done using PCR-mediated site-directed mutagenesis. BlMPg, containing the entire sequence of human plakoglobin tagged on 5 Ј end by 6myc epitope, was constructed using a plasmid HPG Ca 2.1 (Franke et al., 1989) and a plasmid CS26MT provided by Dr. R. Kopan (Washington University, St. Louis, MO). The HindIII-XbaI inserts of all Bluescript subclones were further subcloned into the eukaryotic expression vector pBEHpac18 (Horst et al., 1991) containing the puromycin resistance gene and SV-40 early promoter element.
To express Dsg and Dsc extracellular fragments Dc12M (Arg 1 -Asp 212 ), Dg12F (Glu 1 -Asp 212 ), and Dg123F (Glu 1 -Val 335 ), in Escherichia coli , the QIA-expression system (Qiagen, Chatsworth, CA) was used. The corresponding sequences of the bovine Dsg1 and Dsc1a were amplified, ligated either with single myc or flag (Sigma Chemical Co., St. Louis, MO) sequences, and then inserted in a pQE18 vector. Correct amplification and cloning of all recombinant plasmids was checked by restriction endonu-clease mapping and nucleotide sequencing. Plasmid CMVSyPg encoding a chimeric protein consisting of the entire synaptophysin and plakoglobin has been described .

Solid Phase and Reconstitution Assays
The in vitro solid phase assay was described previously . In brief, Dsg fragments isolated as described previously  were diluted in loading buffer (20 mM Tris HCl, pH 7.8, 1 mM DTT with or without 2 mM EDTA) and immobilized on a 96-well dish and incubated with increasing amounts of Dc12M. Binding was detected by an ELISA assay with myc 9E10 mAb. The solid phase assay was always performed in the absence or presence of 2 mM EDTA added in each solution of the binding assay. This EDTA concentration did not change the affinity of the 9E10 antibody to the myc epitope, as shown by direct ELISA assay.
For the reconstitution assay the Dc12M fragment was mixed with the Dsg fragments, Dg12F, or Dg123F in 1.5 ml PBS in final concentration of 1 g/ml. For control, Dc12M was not added. Samples were incubated 15 min, and then subsequently treated with 75 l 9E10 anti-myc antibodies and with 15 g protein A-Sepharose (Pharmacia, Piscataway, NJ) suspended in PBS. The beads were then washed five times with PBS supplemented with 1% Triton X-100. The immunoprecipitates were analyzed by immunoblotting with different primary antibodies in conjunction with an enhanced chemiluminescence detection system (Boehringer Mannheim Biochemicals, Indianapolis, IN).

Expression of Desmocollin 1a in Human Fibrosarcoma HT-1080 Cells Induces Intracellular Redistribution and Lateral Clustering of Endogenous Desmoglein 2
Although interactions between desmosomal cadherins are likely to be involved in desmosome assembly and in cellcell adhesion of epithelial tissues, expression of Dsg and/or Dsc in the adhesion-deficient L-fibroblasts did not produce a detectable increase in intercellular adhesion (Amagai et al., 1994;Chidgey et al., 1996;Kowalczyk et al., 1996). Absence of the functional adherens junctions in L-fibroblasts could be one of several reasons contributing to the failure to demonstrate interactions between desmosomal cadherins. From this point of view, human fibrosarcoma HT-1080 cells present an advantage for studying Dsc-Dsg interactions. Supporting previous observations (Sacco et al., 1995), we have demonstrated (Figs. 1 and 2) that, in contrast with L-fibroblasts, HT-1080 cells exhibit prominent intercellular contacts containing adherens junctions proteins N-cadherin and ␤ -catenin. Surprisingly, we found that HT-1080 cells, despite their fibroblast-like phenotype, synthesize Dsg2 at essentially the same level as epithelial A-431 cells (Fig. 1). By immunofluorescence analysis (see list of the antibodies in Materials and Methods), HT-1080 cells were devoid of other desmosomal proteins such as plakoglobin, desmoplakin, and all known isoforms of Dscs (Fig. 1). Immunofluorescent staining with either monoclonal or polyclonal anti-Dsg antibodies revealed that a major portion of Dsg2 is randomly distributed on the surface of the HT-1080 cells (Fig. 2 a ). Compared with A-431 cells, only a small fraction of Dsg2 can be detected at cell-cell contact regions where it is colocalized with N-cadherin and ␤ -catenin ( Fig. 2 a Ј ). Dsg2 was sensitive to the treatment of HT-1080 cells with trypsin (not shown), confirming its cell surface localization.
Since desmosomes contain two distinct cadherins, Dsg and Dsc, we examined whether expression of the fulllength bovine Dsc1a can bring about lateral clustering of the endogenous Dsg2 in HT-1080 cells. Double immunofluorescence analysis using polyclonal anti-Dsg2 and monoclonal anti-Dsc1 2.10 antibodies revealed a redistribution of endogenous Dsg2 in HT-1080 cells stably transfected with Dsc1a (HTDc cells). In these cells both desmosomal cadherins Dsg2 and Dsc1a were colocalized in large clusters of cell-cell contact regions (Fig. 2, b and b Ј ) that were also positive for ␤ -catenin (Fig. 2, c and c Ј ). Notably, codistribution of both desmosomal cadherins was found exclusively at cell-cell contacts. For example, Dsc-positive polymorphic vesicles detected in the cytoplasm of the transfected cells were devoid of Dsg (Fig. 2).
To determine the contribution of the intracellular and extracellular domains of Dsc to the assembly of the clusters of desmosomal cadherins, we transfected HT-1080 cells with several mutants of Dsc1a (Fig. 3 A ). Two deletion mutants were constructed and stably expressed in HT-1080 cells. The first mutant, Dsc ⌬ (697-761), lacked the entire sequence of the C-domain mediating interaction of Dsc1a with plakoglobin. The second mutant, Dsc ⌬ (597-609), had an internal deletion of the CSI region involved in binding to desmoplakin (Troyanovsky et al., 1994 b ). Both deletions had no effect on the capacity of Dsc1a to form lateral clusters that incorporated endogenous Dsg (Fig. 2,  d and d Ј ). Expression of Dsc in HT-1080 cells did not induce synthesis of endogenous desmoplakin or plakoglobin as determined by either immunofluorescence microscopy or immunoblotting (not shown). Taken together, these observations suggested that known intracellular desmosomal proteins are not required for relocation of Dsg2 in HTDc cells. We then constructed a chimeric protein Dc(1-170Dg) in which the sequence (Arg 1 -Thr 170 ) corresponding to the first, and approximately half of the second, extracellular cadherin-like repeats of Dsc1a was replaced with the homologous sequence of the bovine Dsg1 (Fig. 3 A ). This chimeric protein was faithfully delivered to the cell surface upon expression in HT-1080 cells; however, it failed to trigger the clustering of endogenous Dsg (Fig. 2, e and e Ј ). Thus the extracellular region of Dsc1a, but not its intracellular domain, is required to effect lateral clustering of Dsg2 in HT-1080 cells.

Desmosomal Cadherins, Dsg and Dsc, Are Involved in Ca 2 ϩ -dependent Heterophilic Interactions
The ability of the Dsc1a to interact with Dsg2 in HTDc cells was tested in coimmunoprecipitation experiments ( Fig. 3 B ). The total NP-40 lysates of the control HT-1080 cells, and cells stably expressing Dsc1a or its mutants, were immunoprecipitated with anti-Dsc antibodies. The presence of Dsg in the immunoprecipitates was monitored by immunoblot analysis. These experiments revealed that Dsc1a was able to coprecipitate Dsg2 from the HTDc cell lysate (Fig. 3 B ). Substitution of the homologous sequence from bovine Dsg1 for the extracellular region of Dsc (the Dsc[1-170Dg] mutant) completely abolished Dsg/Dsc interactions in the coimmunoprecipitation assay, whereas deletions of the intracellular domain of Dsc, Dsc ⌬ (697-761) and Dsc ⌬ (597-609), had no effect. Therefore, the first two cadherin-like domains of the extracellular portion of Dsc1 are required for Dsg binding. N-cadherin and ␤catenin were not detected in immunoprecipitates obtained with anti-Dsc or -Dsg antibodies (not shown), indicating the absence of a strong interaction between desmosomal and classic cadherins.
To address the question of whether Dsg2 directly interacts with Dsc1a or these interactions are mediated by an additional protein(s), the control and HTDc cells were metabolically labeled before immunoprecipitation. A prominent 120-kD band reacting with anti-Dsc antibodies was present in the immunoprecipitates obtained from HTDc cells using mAb 2.10 (Fig. 3 C ). Only Dsg2 consistently coimmunoprecipitated with Dsc1a as a protein with a molecular mass of 160 kD that reacted with both anti-Dsg 3.10 and G129 antibodies (Fig. 3 C ). An association between Dsg and Dsc was also detected in similar immunoprecipitation experiments using anti-Dsg antibodies (Fig.  3). The absence of the other specific bands in addition to Dsg or Dsc in these immunoprecipitates suggests a direct interaction between these two proteins in the HTDc cells. A disproportionately small amount of coprecipitating protein in these experiments could be caused, in part, by low solubility of the Dsg/Dsc complexes compared with free forms of both cadherins. Indeed, a significant amount of both desmosomal cadherins was found in the pellets after NP-40 extraction (not shown).
It is well documented that interaction of classic cadherins is Ca 2 ϩ dependent (Ozawa et al., 1990;Geiger and Ayalon, 1992;Gumbiner, 1996;Brieher et al., 1996). To test whether Dsc/Dsg interactions detected in HTDc cells were sensitive to Ca 2 ϩ , the cells were lysed in the presence of an increasing concentration of EGTA. The data presented at Fig. 4 show that 2 mM EGTA reduced the amount of Dsc/Dsg complexes over twofold. EGTA, however, was unable to completely abolish interactions between the two desmosomal cadherins.
Since immunoprecipitation experiments revealed that the first two cadherin-like domains of Dsc are required for interactions with Dsg, we constructed and expressed the recombinant protein Dc12M in E. coli. This protein consists of the cadherin-like domains 1 and 2 of Dsc followed by the myc epitope and histidine hexamer (Fig. 5 A ). To determine whether the Dsg domain(s) is involved in the interaction with Dc12M protein, we constructed the Dsg fragments Dg12F and Dg123F containing its extracellular cadherin-like domains 1 and 2 or 1, 2, and 3, respectively. Carboxyl termini of the Dsg-derived polypeptides were tagged by the FLAG epitope and polyhystidine (Fig. 5 A). SDS-PAGE and immunoblot analysis of the recombinant proteins purified by Ni-agarose chromatography showed that the samples contained homogenous polypeptides of the expected molecular mass of 30 kD for Dg12F and Dc12M, and 45 kD for Dc123M (Fig. 5 A).
To analyze direct interactions between Dc12M and Dsg fragments we used a solid phase assay developed previ-ously for examination of plakoglobin-cadherin binding . Dsg fragments were immobilized on a 96-well dish and incubated with increasing amounts of Dc12M. Binding was detected by subsequent ELISA assay using anti-myc antibody. In this assay the Dc12M polypeptide interacts with Dg12F very weakly, and only in the presence of calcium ions (Fig. 5 B). Significant binding, however, was observed between Dc12M and Dg123F proteins containing extracellular domains 1-3. Addition of 5 mM EGTA also reduced but did not completely abolish these interactions (Fig. 5 B).
As an alternative approach, the molecular interactions between different Dsg and Dsc fragments were tested in a reconstitution assay. Fig. 5 C shows a representative experiment in which fragments Dc12M were mixed with fragments Dg12F or Dg123F and immunoprecipitated using anti-myc antibody. The results of these experiments are in good agreement with those obtained in the solid phase binding assay, demonstrating the strong interaction between Dc12M with Dg123F and Dg12F fragments.

Heterophilic Binding of Desmosomal Cadherins Contribute to Cell-Cell Adhesion
Endogenous expression of N-cadherin interferes with the measurement by conventional aggregation assays of the adhesive properties of HT-1080 cells that are due to  the expression of transfected desmosomal cadherins. To test the contribution of the Dsg/Dsc interaction to cell-cell adhesion, HT-1080 cells expressing Dsc⌬(697-761) were cocultured with HT-1080 cells lacking any form of Dsc but expressing myc-tagged plakoglobin (MPg). In the latter cells, plakoglobin and endogenous Dsg form stable complexes that can be detected by coimmunoprecipitation (Fig. 6 B). Thus, in this coculture system, specific coimmunoprecipitation of MPg by anti-Dsc antibodies in a Ca 2ϩdependent fashion will be indicative of intercellular interactions mediated by desmosomal cadherins (Fig. 6 A). The direct complex between Dsc and MPg could not be formed because of the absence of the plakoglobin binding site in Dsc⌬(697-761). A coculture of MPg-and Dsc(1-170Dg)expressing cells was used as an additional negative control. To verify that the subpopulations of the corresponding cells in both cocultures were present in an approximately equal ratio, aliquots of the cellular lysates were subjected to Western blot analysis with myc-and Dsc-specific antibodies before immunoprecipitation. Experiments shown in Fig. 6 C demonstrate that Dsc⌬(697-761), but not Dsc(1-170Dg), interacts with Dsg/MPg complexes present in the opposing cellular subpopulation of the coculture. In a separate experiment (Fig. 7) the HTDc cells were cocultured with HT-1080 cells stably producing the chimeric protein SyPg . This protein, as we reported previously, binds to classic and desmosomal cadherins. As a result, it is incorporated into cell-cell junctions of the transfected cells. Double immunofluorescence microscopy of these cocultures using polyclonal anti-synaptophysin and monoclonal anti-Dsc antibodies showed that Dsc1a was specifically incorporated into the junctions arising between cells from two subpopulations of Dsc1apositive and -negative cells.
In addition, to show that interactions between desmosomal cadherins were present at the intracellular interface, we investigated whether the amount of the Dsc/Dsg complex is proportionate to cell density in culture. Intercellular contacts in HT-1080 cells at %57ف confluence are more extensive than in those at low density. Thus, HT-1080 cells expressing Dc⌬(697-761) were plated at a density of 12 and 75% confluence and left overnight to allow intracellular contacts to stabilize. The lysates of low and high density cultures containing the same amount of total protein were immunoprecipitated with an anti-Dsc antibody, and immunoprecipitates were analyzed by immunoblotting (Fig. 6 D). A five-to sixfold reduction in the amount of Dsg in Dsc immunoprecipitates was obtained from cell cultures plated at low as compared with high densities. This shows that Dsg/Dsc interaction in coculture correlates well with the number of intercellular contacts. It is important to note that, while plated at low density, HT-1080 cells always contain some number of cell-cell contacts that cause coimmunoprecipitation of Dsg.

Discussion
A number of studies have shown that expression of Dsg and/or Dsc in adhesion-deficient L-fibroblasts fails to produce detectable levels of intercellular adhesion (Amagai et al., 1994;Chidgey et al., 1996;Kowalczyk et al., 1996). Although intercadherin interactions are very likely to be involved in desmosome assembly and in cell-cell adhesion of the epithelial tissues, no physical association between these two desmosomal cadherins was reported. The major reasons for failure to demonstrate interactions between desmosomal cadherins might have been (a) insolubility of the corresponding complexes in the epithelial cells, and (b) malfunction of Dsg and Dsc in L-fibroblasts in the ab- sence of functional adherens junctions and classic cadherins in particular. As an alternative model for study of the interaction between Dsc and Dsg, we chose fibrosarcoma HT-1080 cells. In contrast with L-fibroblasts, they have prominent intercellular contacts containing N-cadherin and ␤-catenin. HT-1080 cells produce a single desmosomal protein, Dsg2, at the same level as in epithelial A-431 cells. In these cells, however, only a minor portion of this cadherin is localized to cell-cell junctions.
Here we have shown that expression of the full-length Dsc1a in HT-1080 cells results in a redistribution of endogenous Dsg2. Both desmosomal proteins, Dsg2 and Dsc1a, were efficiently incorporated into the cell-cell contacts of HTDc cells, where they become associated with adherens junction proteins, such as N-cadherin and ␤-catenin. Coimmunoprecipitation experiments revealed that in these cells Dsc1a is able to interact directly with Dsg2, but not with N-cadherin. Notably, formation of Dsc/Dsg complexes was found in the absence of major desmosomal proteins, plakoglobin and desmoplakin, that were undetected in parental HT-1080 cells and in HTDc cells. In addition, deletions of plakoglobin or desmoplakin binding sites in Dsc1a had no effect on its ability to interact with Dsg2, demonstrating that known cytoplasmic interactions of Dsc1a are not required for Dsg/Dsc association. In contrast, replacement of the extracellular domain of Dsc1a with the corresponding sequence of Dsg completely abolished both Dsg redistribution and Dsg/Dsc complex formation in coimmunoprecipitation. Furthermore, association between Dsg and Dsc was calcium dependent, which is characteristic of interactions involving extracellular domains of cadherin. In vitro binding experiments provided further support for a direct interaction between the extra-cellular segments of two desmosomal cadherins. Using two different binding assays, we have found that Dc12M, a Dsc fragment containing extracellular repeats 1 and 2, binds directly to the extracellular region of Dsg. Chelation of Ca 2ϩ ions decreased but did not completely abolish this binding. Our data are not sufficient to conclude whether Dsg/Dsc heterodimers are formed through head-to-head or side-toside interactions (see hypothetical models of Dsg-Dsc complexes in Fig. 6). Recently, Brieher et al. (1996) showed that lateral dimers of the extracellular region of the C-cadherin in vitro are relatively stable after the removal of Ca 2ϩ ions. Similarly, incomplete inactivation of the Dsg-Dsc interaction even in the presence of a high concentration of EGTA suggests that the cadherin dimers have lateral alignment. However, in the solid phase assay, the Dc12M fragment binds more strongly to the Dsg fragment containing extracellular domain 3. This observation may be interpreted to mean that Dsg-Dsc forms antiparallel head-to-head complexes in which the extracellular domains 1 and/or 2 of Dsc bound to domain 3 of Dsg. Additional experiments are required to determine the exact structural features of Dsg/Dsc dimers.
Another important feature of Dsc/Dsg complexes is revealed by the fact that MPg coimmunoprecipitated with anti-Dsc antibodies from the coculture of Dsc⌬(697-761)and MPg-expressing HT-1080 cells. This unequivocally demonstrated that interacting Dsc and Dsg are derived from opposing cells. The number of Dsc/Dsg complexes directly correlates with the propagation of cell-cell contacts in HTDc coculture. Furthermore, clusters incorporating Dsc and Dsg are also assembled along the contacts with Dsc-negative cells. These observations are consistent with the idea that these complexes are formed only on the in- terface between two neighboring cells. Thus, heterophilic interactions between desmosomal cadherins are involved in intercellular adhesion of epithelial cells, and corresponding complexes can be functional elements in desmosome assembly. In support of this assumption we found that the bovine Dsc1a as well as its mutants Dsc⌬(697-761) and Dsc⌬(597-609) were efficiently incorporated into human desmosomes upon expression in epithelial A-431 cells (Chitaev, N.A., unpublished results). In contrast, the mutant Dsc(1-170Dg), unable to interact with Dsg in HT-1080 cells, was also unable to form desmosomes in A-431 cells. The question remains, however, how Dsg/Dsc complexes assemble in a mature desmosome. The function of the intracellular desmosomal plaque proteins, such as plakoglobin and desmoplakin, may be necessary for segregation of the Dsg/Dsc complexes from adherens junctions and for further desmosome assembly (Hinck et al., 1994;Allen et al., 1996;Bornslaeger et al., 1996;Chitaev et al., 1996;Demlehner et al., 1996;Ruiz et al., 1996;Troyanovsky et al., 1996). The subsequent expression of these desmosomal proteins in HTDc cells is likely to provide an excellent system for examining the molecular mechanisms involved in this process.
The low level of Dsg/Dsc interactions in sparse cultures of HTDc cells is consistent with the data of Kowalczyk et al. (1996) showing the absence of detectable intercadherin interactions when both Dsg and Dsc are coexpressed in mouse fibroblasts lacking adherens junctions. The requirement of adherens junctions for intercadherin interactions was suggested by observations that the malfunction of adherens junctions, induced either by E-cadherin antibodies or by dominant negative mutants of the classic cadherins, delays desmosome assembly in keratinocytes after raising the calcium concentration (Wheelock and Jensen, 1992;Lewis et al., 1994;Amagai et al., 1995). In HT-1080 cells, Dsc/Dsg clusters were found in areas where N-cadherin-␤-catenin complexes are abundant. Therefore, it is reasonable to hypothesize the existence of cross talk between ad-herens junctions and desmosomes, which allows Dsg/Dsc interactions only after the establishment of the cell typespecific contacts mediated by classic cadherins.


Meeting Report

I In n s si il li ic co o m me ee et ts s i in n v vi iv vo o Technological developments have had a profound impact on biology during the past decade, spectacularly augmenting our ability to survey and interrogate biological phenomena. In particular, they have increased capacity for data generation by several orders of magnitude and made computation a necessary partner of biology. The sixth meeting in the biennial series of bioinformatics conferences co-sponsored by Georgia Institute of Technology in Atlanta and the Oak Ridge National Laboratory addressed the challenges that this technology-driven avalanche of data pose to bioinformatics-increasing the complexity of long-standing problems and creating new ones. G Ge en no om me e a al li ig gn nm me en nt t a an nd d g ge en ne e p pr re ed di ic ct ti io on n Sequence alignment is unquestionably one of the 'founding problems' in bioinformatics. The availability of sequenced genomes of many species has highlighted the need for methods of making reliable multiple alignments of whole genomes. The alignment of entire genome sequences is much harder to achieve than the alignment of amino-acid sequences of individual proteins, because of the much longer sequences involved (ranging from megabases to tens of megabases), complex evolutionary relationships among the genomes (such as duplications, deletions and translocations) and heterogeneous mutation rates along the sequence. Different methods often produce discrepant alignments with the same set of genomic sequences, and Martin Tompa has attempted to navigate through this complexity. Instead of proposing yet another method for multiple sequence alignment, he presented an approach to evaluating the quality of a given multiple alignment. This is a seemingly more modest goal; he was, however, able to identify high-quality and reliable regions in the multiple alignment, which is very important because downstream comparative genome analysis is compromised by incorrect alignments. Tompa presented data showing that about 10% of the positions in multiple alignments of the human genome with other vertebrate genomes-a widely used technique in comparative genomic studies-are likely to be incorrect. Gene prediction in genomic sequences presents similar problems. Current methods for predicting the exonic structures of protein-coding genes from genomic sequences are generally based on computational models that capture our understanding of the way proteins are encoded in genomes. However, recent surveys of the transcriptional activity of the human genome, …

Introduction
The use of automation and robotic modules in a range of pharmaceutical and other fine chemical analysis laboratories was the subject of a meeting organized by the Automatic Methods Group and the Joint Pharmaceutical Analysis Group, held at the Royal Pharmaceutical Society's headquarters in London, on 7 December 1995. Current scope and opportunities for the future were considered, speakers from diverse fields discussed a range of successful applications. An equipment exhibition was also organized, enabling participants to see recent developments in the automation of dissolution testing and the analytical processing of metered-dose inhalers.

Abstracts of papers presented
State of the art in analytical robotics Kevin Warwick University of Reading In the opening presentation, Professor Kevin Warwick (Department of Cybernetics, University of Reading) updated the traditional concepts of robots (from those of either humanoid machines or standard production line arms) by describing intelligent, decision-making systems which feature a number of different information sensors to control movement and to select actions to be carried out. Modern robotic systems are now capable of learning by experience and are able to pass on this information to other robots. Various new developments were described, incorporating a number of new technologies, perhaps the most impressive of which concerned the process control of banknote production. In order to prevent forgery, the colour combinations used in the printing of banknotes are as complicated as possible and, although some tolerances are allowable, there are limits. Accordingly, a suitable robotic system for this application needs an appropriate feedback mechanism which allows a correct quality judgement to be made.
Similarly, cigarette manufacture relies on largely uninterrupted operation and it is extremely important to be able to predict or to schedule stoppages. This multivariable situation is difficult for humans to control, however, but predictability can be obtained by using an appropriate computer-based algorithm. Professor Warwick discussed the applicability of artificial neutral networks in attempting to mimic some of the operations carried out by the human brain. As such networks are good at modelling non-linear systems, they can be used directly to control robot manipulators or to assess the influence of a large number of process variables. The use of such technology, typically in a manufacturing or laboratory environment, will result in improved process control and operational performance.

Microbiological assays
Professor Ken Coleman SmithKline Beecham Some automated applications of microbiological assays were described by Ken Coleman (SmithKline Beecham, Brockham Park) in which the minimum inhibitory concentration of an antibiotic is established using a screening experiment involving, typically, as many as 40 organisms.
The first application featured the automation of the Hamilton AT + incorporating an X-Y arm capable of holding up to 12 syringes, and a liquid level detection system. This facilitated preparation of microlitre plates more rapidly than was achievable manually (typically, a reduction from 12 minutes to four minutes). The overall process of plate preparation (remembering that 40 organisms are routinely involved in the screening procedure) and plate reading was able to be completed in minutes, rather than several hours, with the automated system--which also incorporated in orca robot to stack plates for the Hamilton AT +, and a barcode reader for sample identification.
A second approach involved the use of a Zymate robotic system with image analysis facility, multi-point inoculator and an agar dispenser. This system also proved suitable for the kind of high-volume screening applications common to many microbiological laboratories, with the additional advantage that correct plate alignment could be assured routinely.

Progress and speculation
Ken Leiper Glaxo Laboratories 'History repeats itself--because we don't learn from it!' was the opening provided by Dr Ken Leiper (Glaxo Wellcome, Barnard Castle) in his provocative presentation which reviewed progress in, and considered the future of, the application and automation of analytical measurement.
The development of automated measurement has shown considerable progress over the last three or four decades, 0142-0453/97 $12.00 (C) 1997 Taylor & Francis Ltd from the dedicated auto-analysers of the 1950s to the vastly more sophisticated intelligent solutions of the present day where chemometric data handling techniques and robotic control are common. Moreover, analytical science itself is now generally recognized as having a major part to play in the way in which measurement techniques develop. The changing environment in which most businesses operate is forcing a reappraisal of the way in which automated measurements are being carried out. Corporate objectives and regulatory requirements are becoming increasingly more demanding, with products needing to be introduced more rapidly, with increased efficiency, more consistent quality and generally with reduced head count. Current measurement philosophy seems to fall some way short of the mark when these new business objectives are considered. Typically, laboratory-based methodology is developed in research and will need to be transferred into development, into manufacture--and perhaps even beyond, into a stability or contract group. Moreover, such methodology tends to be product-focused, with significant sample preparation, and gives little or no information about process performance. Demonstration of compliance, from the regulatory perspective, also seems to rely on highly bureaucratic procedures involving extensive checking and documentation.
Thus, the role of the analytical scientist is to provide an effective measurement service which helps to fulfil corporate objectives. This necessitates a measurement philosophy which establishes the relationship between 'need', 'measurement' and 'technology'.
Methodology is required which features little or no sample preparation, which may be easily transferredparticularly to the production area, if necessary--and which is self-calibrating and self-validating. Two examples were provided of such an approach; an automated antibiotic assay system which allowed measurement of several product quality parameters within a cycle time of only five minutes with overall software control to provide and maintain system integrity; and the use of rear infrared spectroscopy to provide chemical and physical information directly on the sample matrix (for example, during powder blending or tabletting studies). Such approaches, where emphasis is placed on process measurement rather than purely on the product, provide advantages in terms of reduced costs, improved process reliability, ease of method transfer and increased assurance of process and product compliance. Changes are required, however, in terms of recognition of business needs, improved liaison between suppliers and academia and the development of an improved skills base.
On-line near infra-red

Malcolm Crook
Process Analysis and Automation Dr Malcolm Crook (Process Analysis and Automation, Farnborough) initially discussed the benefits which are 52 achievable from the improved control provided by process analysis. These include the production of materials of consistent quality, increased throughput and efficiency, reduction in waste and time--as well as a demonstrable improvement in regulatory/compliance aspects.
In-line process analysis is particularly beneficial in that rapid (real time) results may be obtained, no sampling steps are required and operations generally minimize the exposure of staff to hazardous materials. The suitability of near infra-red spectroscopy for in-line analysis was emphasised in view of the speed and applicability of the technique, its relative insensitivity towards process debris, colour of sample, etc. and the fact that it utilizes sensible path lengths (unlike infra-red spectroscopy, for example).
Incorporation of fibre optic probes further enhances the capabilities of in-line process analysis using near infra-red spectroscopy. Both reflectance and transmission probes can be used, while fibre optic bundles up to or 2 km in length are also available. Such bundles can also be multiplexed, so that fibres may be made to point to different channels by simple perturbation of the electromagnetic field in which they are held. One consequence of the use of near infra-red spectroscopy is the need to apply some chemometric data analysis to the measurements obtained. Near infra-red bands are generally broad, poorly resolved and often difficult to assign. The application, therefore, of chemometric techniques such as 'partial least squares', for example, can prove extremely useful in correlating spectral information with chemical composition, physical aspects and general product quality characterization. Automation in water analysis (a laboratory for the 21st century) Robin Andrew North West Water North West Water's automated water analysis laboratory was described by Dr Robin Andrew (North West Water Ltd, Laboratory Services). The company provides water and waste-water services over an area of 14,000 km 2 and has about seven million customers. The Laboratory Services group provides an analytical service to the company on a range of materials (raw water, wastewaters, trade effluents, sludges, soils etc.) and with a range of analyses (organic and inorganic species, bacteria, parasites, etc.)--all within a very heavily regulated environment.
Typically, the Laboratory Services group handles over 250,000 samples per year and, with the benefits of automation, has reduced the number of its laboratories from 30 to and its staff from 260 to 110 since 1989.
The single major laboratory was designed with the objectives of improved quality of data, faster reporting of analytical results and lower operating costs. These aims have been achieved by establishing segregated areas, each dedicated to a particular type of analysis, through which samples are transported via a standard conveyor belt system. Different robotic units are used in each analytical area, typically 'industrial' type robots or 'gantry' style systems. Barcode readers are used for sample identification. Within the laboratory, some analytical instrumentation has deliberately not been fully integrated, in order to avoid becoming too committed to particular systems.
Overall control of the laboratory is provided by a LIMS, with manual intervention in the event that any of the automated checking and validation procedures should fail.
A number of lessons were learned during the design and implementation of the automated water analysis laboratory: clear objectives are essential, these laboratories need a lot of space, flexibility is vital, system integration requires careful planning and, crucially, staff involvement is the most important part of the exercise. process) has been modified to incorporate a Jouan centrifugal evaporator which can take up to 10 samples, significantly improving the productivity of the operation. Improvements in other parts of the analytical procedures (for example greater use of auto-injectors and the introduction of a chromatographic data handling system) have resulted in an overall increase in productivity of the laboratory. Automated approaches based on existing technology have been used, and have allowed staff time to be used more effectively. The laboratory undertakes a variety of analyses (drug residues, toxicants, environmental contaminants, pesticide residues, etc.), most of which is funded by MAFF and which is, theretbre, subject to significant financial constraint. Thus, any moves towards automation have had to be aimed at increasing throughput, but without incurring any large capital expenditure.
Among other activities, the Pesticides Group provides surveillance data on the major constituents of the diet of the UK population as part of a continuous monitoring programme. Thus, a variety of commodities are examined and a large range (well over 100) of pesticides are involved. This has resulted in the development and application of eleven significantly different methods of analysis (incorporating different techniques) to allow this range of pesticides to be monitored. In order to automate these activities, emphasis has been placed on increasing efficiency, with reduced costs, increased sample throughput and greater staff productivity. Examination of the various stages involved in the pesticide analysis procedure revealed that improvements were possible in the sample clean-up step, where fats or plant colourants are separated from the pesticide residues using gel permeation chromatography. Incorporation of a Gilson 232 sample handling system has improved this aspect significantly, allowing up to 60 samples to be run overnight. In addition, a sample concentration step (involving an expensive and time-consuming nitrogen evaporation A review of the application of an automated solution involving a Zymate robotic system was provided by Dr Gordon Plummer (Zeneca Pharmaceuticals). Analysis of samples arising from drug kinetic studies needs to be automated in view of reduced drug development times, increased regulatory expectations and greater economic constraints.
The sample preparation step of such analyses is the ratedetermining factor so, to address this problem, Zeneca introduced a Zymate system into the laboratory in 1984.
They now have eight such systems, capable of performing a wide variety of analytical tasks, although most have been dedicated to specific applications. In particular, the robotic systems are engaged in liquidliquid and solid-phase extraction's, allowing reductions in analysis time from ten minutes originally to less than three minutes now. As such, this has demonstrated the benefits of keeping up-to-date with developments and of up-grading the systems as new technology becomes available.
Extensive review of over several years has shown comparability of robot performance using the same modules, with good correlation between data generated automatically and manually. More extensive computer validation is required these days, however. In terms of cost benefit, the robotic system was found to pay for itself in less than one and a half years when used for complex analyses and in less than three years for simpler applications, when used continuously for three to four days per week. Dedicated resources are needed, however, if further applications are to be developed and good technical and engineering back-up is also required.
The self-service spectroscopy laboratory: a blueprint for the future?

Don Clark
Pfizer Central Research, Sandwich The principles of 'open access' analytical chemistry were given by Mr Don Clark (Pfizer Central Research, Sandwich) in a lively concluding presentation. Pfizer have used automation to help overcome the problem of increasing turnaround times in their Physical Sciences Laboratory, allowing the customers to collect and interpret their own data using spectroscopic instruments.
JMR was the first technique to be automated and has progressed from the use of a Nicolet QA 300 system to systems linked to Zymate's robot. This has improved capacity dramatically, freeing up the analysts' time to do more innovative tasks.
The development of an 'open access' mass spectrometer proved slightly more difficult in that commercial instrument manufacturers had not considered such a system at that time. Pfizer were able to collaborate extensively with VG, however, to design a suitable system which eventually fulfilled their needs--and provided the instrument manufacturer with a commercial product. Polarimetry is used extensively in Pfizer's laboratory and this technique has also now been automated successfully, based around a Perkin Elmer 341 system with custommade turntable from Peerless, to allow 'open access'.
Some 'smart' software developments have also been included for fault/error detection (for example, the detection of air bubbles in the system). The approach which has been adopted within the Physical Sciences Laboratory has been very well-received by the customers and allows more data, of improved quality, to be generated with less resource. Liaison and collaboration with instrument manufacturers is important in terms of helping to realize the true benefits of an automated approach.

Conclusion
Evidently there is no single way in which robotics or laboratory automation should be used to improve productivity or data quality--a variety of approaches are possible and significant levels of success may be achieved.
One important conclusion which may be reached, however, is that experiences gained within one field of application frequently translate into other areas. Thus, the analytical scientist, charged with the task of introducing the benefits of automation into the laboratory, would be well-advised to consider the successes gained by colleagues working in different sectors. This, and the importance of concentrating efforts on analytical measurements providing information about the process, rather than the product, were the key messages emanating from this well-attended and thought-provoking meeting.


extfieldsofstudy: Medicine Biology

s2fieldsofstudy: Biology Chemistry

text:
Activation of PKCα Downstream from Caspases during Apoptosis Induced by 7-Hydroxystaurosporine or the Topoisomerase Inhibitors, Camptothecin and Etoposide, in Human Myeloid Leukemia HL60 Cells*

We previously demonstrated that the anticancer agent and protein kinase C (PKC) inhibitor 7-hydroxystaurosporine (UCN-01) induces apoptosis independently of p53 and protein synthesis in HL60 cells. We now report the associated changes of PKC isoforms. PKCα, βI, βII, δ, and ζ activities were measured after immunoprecipitation of cytosols from UCN-01-treated HL60 cells. UCN-01 had no effect on PKCζ and inhibited kinase activity of PKCβI, βII, and δ. PKCα activity was initially inhibited at 1 h, and subsequently increased as cells underwent apoptosis 3 h after the beginning of UCN-01 treatment. Camptothecin (CPT) and etoposide (VP-16) also markedly enhanced PKCα activity during apoptosis in HL60 cells. However, CPT did not affect PKCβI, βII and ζ, and activated PKCδ. PKCα activation was not due to increased protein levels or proteolytic cleavage but was associated with PKCα autophosphorylation in vitro and increased phosphorylationin vivo. We also found that not only PKC δ but also PKC βI was proteolytically activated in HL60 cells during apoptosis. The PKCα activation and hyperphosphorylation were abrogated byN-benzyloxycarbonyl-Val-Ala-Asp(O-methyl)-fluoromethylketone (z-VAD-fmk) under conditions that abrogated apoptosis. z-VAD-fmk also prevented PKCδ and βI proteolytic activation. Together these findings suggest that caspases regulate PKC activity during apoptosis in HL60 cells. At least two modes of activation were observed: hyperphosphorylation for PKCα and proteolytic activation for PKC δ and βI.

In the present study, we investigated PKC isoform activities during UCN-01-induced apoptosis in human myeloid leukemia HL60 cells. HL60 cells were chosen for these studies because they undergo rapid apoptosis to various agents including UCN-01. This apoptosis is p53-independent as HL60 cells are p53 null (22) and does not require protein synthesis (14,23). It is preceded by transient activation of cyclin B1/Cdc2 kinase (14,24). The changes of PKC activity were also examined in HL60 cells undergoing apoptosis after treatment with the topoisomerase inhibitors, CPT and etoposide (VP-16). We found that UCN-01 inhibited PKC␤I, ␤II, and ␦ activity in drug-treated cells as well as in vitro. For PKC␣, UCN-01 first inhibited the kinase activity and then activated PKC␣. CPT-induced apoptosis was associated with marked increase in PKC␣ and ␦ activities. The mechanisms of PKC activation and their relationship to caspases (ICE-like proteases) are investigated. Cell Culture-Human promyelocytic leukemia HL60 cells were grown at 37°C in the presence of 5% CO 2 in RPMI 1640 medium supplemented with 10% fetal bovine serum (Life Technologies, Inc., Gaithersburg, MD), 2 mM glutamine, 100 units/ml penicillin, and 100 g/ml streptomycin.
PKC Assays-Immunocomplex beads were obtained by incubating different anti-PKC antibodies with cytosolic extracts in 20 l of reaction buffer containing 20 mM Tris-HCl, pH 7.4, 10 mM MgCl 2 , 10 M cold ATP, 0.4 mg/ml histone H1, 40 g/ml PS, 3.3 M dioleoylglycerol, 5 Ci of [␥-32 P]ATP in the absence or presence of 1.2 mM CaCl 2 depending on the PKC isoform activity measured. Incubations were carried out at 30°C for 10 min. Samples were loaded onto 12% SDS-PAGE gels (NOVEX, San Diego, CA) and electrophoresed at 120 V for 2 h. For quantification of kinase activity in immunoprecipitates, gels were dried, and the extent of histone H1 phosphorylation was measured using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).
Western Blot Analysis of PKCs-Cytosolic samples were electrophoresed at 120 V on 12% SDS-PAGE gels and electrophoretically transferred to Immobilon membranes (Millipore, Bedford, MA) for 2 h at 30 V. The membranes were blocked overnight in phosphate-buffered saline-Tween 20 (PBS-T) containing 5% non-fat dried milk. Probing with selected anti-PKC antibodies (1 g/ml in PBS-T) for 1 h was followed by incubation with horseradish peroxidase-labeled anti-rabbit IgG (1:1000 dilution). After washing in PBS-T, membranes were developed using the enhanced chemiluminescence (ECL) detection system (NEN Life Science Products).
PKC␣ Phosphorylation in Cells-HL60 cells were spun and resuspended at 10 6 cells/ml in phosphate-free RPMI 1640 medium supplemented with 10% dialyzed fetal bovine serum once. After 1 h, 50 Ci/ml [ 32 P]orthophosphate was added to cell cultures for 1 h. Then cells were chased in isotope-free medium for 1 h prior to UCN-01 treatment. Cytosol extracts were boiled in electrophoresis sample buffer and analyzed by 10% SDS-PAGE gels. Gels were dried and autoradiographed at Ϫ70°C.
Detection of DNA Fragmentation and Reconstituted Cell-free System-DNA fragmentation related to apoptosis was measured by filter elution assay as described previously (8,25).

UCN-01-and CPT-induced Apoptosis Are Associated with
Increased PKC Activity in HL60 Cells-Our previous studies indicated that UCN-01 and CPT are potent inducers of apoptosis in HL60 cells (14,27). After 3 h of drug treatment, 60 -90% of the cells exhibit apoptosis with typical morphological changes and internucleosomal fragmentation. To investigate whether PKC was involved in apoptosis induced by these anticancer drugs, we treated HL60 cells with UCN-01 or CPT and extracted cytosol and membrane fractions at various times. The synthetic peptide, MBP4 -14, was used as a specific and sensitive substrate to assay PKC activity (26). We found that phosphorylation of MBP4 -14 first decreased during the first hour of UCN-01 treatment compared with control and then increased progressively above control values 3 h after the beginning of treatment. CPT increased the phosphorylation of MBP4 -14 already after 1 h of treatment (Fig. 1). These measurements were performed in cytosolic fractions. In membrane fractions, there was no significant difference between basal and stimulated levels of MBP4 -14 phosphorylation and no change of MBP4 -14 phosphorylation after exposure to UCN-01 or CPT for 3 h (not shown). These data indicate that PKC activity is induced by UCN-01 and CPT treatment in HL60 cells undergoing apoptosis.
UCN-01-and CPT-induced Apoptosis Are Associated with Up-regulation of PKC␣ Activity-The above data raised the question as to what kinds of PKC isoforms were activated by UCN-01 or CPT. MBP4 -14 is selective for PKC␣, ␤I, ␤II, and ␦ isoforms but has been reported to be a poor substrate for other isoforms (26) . Protein G-Sepharose beads were first washed with nucleus buffer and after with kinase buffer. Histone H1 kinase reactions were carried out at 30°C for 10 min and were terminated by adding SDS-PAGE sample buffer. Proteins were separated by 12% SDS-PAGE. Gels were dried and exposed in PhosphorImager cassettes.
know that UCN-01 inhibits PKC␣, ␤, ␥, ␦, and ⑀ and does not inhibit PKC in vitro. Since PKC␥ was undetectable in HL60 cells and PKC⑀ was expressed only very weakly using Western blotting (data not shown) (1), we used anti-PKC␣, ␤I, ␤II, , and ␦ antibodies and immunoprecipitation to determine the effects of UCN-01 and CPT on PKC isoform activities. Fig. 2 shows that UCN-01 inhibited all the PKC isoforms tested at 1 h of treatment, which is consistent with the known anti-PKC activity of UCN-01 in vitro (10). However, at 3 h, PKC␣ activity was markedly stimulated (5-fold) by UCN-01. CPT increased PKC␣ and ␦ activities but had no effects on PKC␤I and ␤II activities. Neither UCN-01 nor CPT affected PKC activity (data not shown). These data indicate that apoptosis induced by UCN-01 and CPT treatment is associated with marked PKC␣ activation.

Suppression of UCN-01-and CPT-induced Apoptosis by the Caspase Inhibitor, z-VAD-fmk, Is Associated with Lack of PKC␣
Activation-Caspases (ICE-like proteases) are key mediators of apoptosis (28 -30), and we previously showed that the caspase inhibitor z-VAD-fmk prevents UCN-01-induced apoptosis in HL60 cells (14). Fig. 3A, shows that z-VAD-fmk also prevents apoptotic DNA fragmentation induced by CPT in HL60 cells. However, z-VAD-fmk does not inhibit the DNA fragmentation induced by UCN-01-and CPT-activated cytosol in cell-free system (8,14,25) (Fig. 3B). Under these conditions, we examined whether z-VAD-fmk affected the PKC changes induced by UCN-01 or CPT in HL60 cells undergoing apoptosis. We found that z-VAD-fmk blocked the activation of PKC␣ induced either by UCN-01 or CPT. However, z-VAD-fmk did not suppress the CPT-induced activation of PKC␦ (Fig. 4A) and had no effects on  4. Effects of UCN-01, CPT, VP-16, and z-VAD-fmk on PKC␣ activity in HL60 cells. HL60 cells were exposed to either 10 M UCN-01 or 1 M CPT in the absence or presence of 50 M z-VAD-fmk for 3 h (panels A and B). HL60 cells were treated with 50 M VP-16 for the indicated times (panel C). Cytosol fractions from untreated or treated cells were immunoprecipitated with the indicated anti-PKC␤ antibodies (panel A) or anti-PKC␣ and ␦ antibodies (panels B and C). PKC activity was measured using histone H1 as substrate.
FIG. 5. In vitro effects of UCN-01, CPT, and z-VAD-fmk on PKC␣ activity. A, cytosols from untreated HL60 cells were immunoprecipitated with anti-PKC␣ antibody. UCN-01 or CPT was added to the immunoprecipitates. Then histone H1 kinase activity was measured. B, cytosol was prepared from untreated cells or from cells treated with CPT or UCN-01 for 3 h. Cytosols were then incubated with 50 M z-VAD-fmk for 30 min at 30°C. Samples were immunoprecipitated with anti-PKC␣ antibody. Histone H1 kinase activity was measured. DMSO, dimethyl sulfoxide. the inhibitions of PKC␤I, ␤II, and ␦ activities induced by UCN-01 (Fig. 4). We next tested whether PKC␣ was activated in response to the topoisomerase II inhibitor, VP-16. We found that VP-16 also activated PKC␣ in HL60 cells undergoing apoptosis (8) (Fig. 4C). These data suggested that apoptosis induced by different pathways (topoisomerase inhibition for CPT or VP-16 or protein kinase inhibition for UCN-01) is associated with PKC␣ activation in HL60 cells. The observation that z-VAD-fmk blocked PKC␣ activation suggests that PKC␣ is downstream from caspases during apoptosis in HL60 cells.
Apoptotic PKC␣ Activation Is Not Due to a Direct Effect of UCN-01 or CPT, and Suppression of PKC␣ Activation Is Not Due to a Direct Effect of z-VAD-fmk on PKC␣ Activity-To further investigate the mechanisms by which UCN-01 or CPT regulate PKC␣ activity, we first tested whether UCN-01 or CPT directly affected PKC␣ activity using in vitro kinase assays. PKC␣ immunoprecipitates obtained from control cytosol were tested for kinase activity with UCN-01 or CPT. Fig. 5A shows that UCN-01 did not activate but rather inhibited PKC␣ activity directly. CPT had no direct effect on PKC␣ activity (Fig. 5A). z-VAD-fmk was also tested in this system and did not exhibit a direct effect on PKC␣ activation induced by UCN-01 or CPT (Fig. 5B). These results indicated that the PKC changes observed in cytosols from cells treated with UCN-01 or CPT in the absence or presence of z-VAD-fmk were indirect.
We next examined whether the activation of PKC␣ by UCN-01 or CPT and the effects of z-VAD-fmk might be due to alterations of the PKC␣ protein. Western blot analyses were carried out on cytosols from untreated, UCN-01-or CPTtreated HL60 cells using anti-PKC␣ antibody. PKC␣ protein levels remained unchanged after drug-treatments (Fig. 6A). Thus, PKC␣ activation by UCN-01 and CPT was not due to changes in PKC␣ protein levels. We also measured the other PKC isoforms. Protein levels of PKC␤I, ␤II, and ␦ did not change significantly (Fig. 6, B-D). Interestingly, UCN-01 and CPT induced PKC␤I and ␦ cleavages when HL60 cells underwent apoptosis, and z-VAD-fmk blocked these proteolytic effects. Proteolytic activation of PKC␦ by caspases is consistent with results obtained in U-937 cells (21). However, our data suggest that PKC␤I is also cleaved by caspases during apoptosis in HL60 cells.
Since neither proteolytic activation nor change in protein levels appear to account for PKC␣ activation, we next tested the effects of UCN-01 and CPT on the PKC␣ autophosphorylation. It is indeed known that autophosphorylation is one of the important modes of regulation of PKC activity (31). Autophosphorylation assays for PKC␣ were carried out essentially as described for histone H1 phosphorylation except that histone H1 was omitted from the reactions. Fig. 7A shows that treatment of HL60 cells for 3 h with either UCN-01 or CPT increased PKC␣ autophosphorylation (Fig. 7A) and that z-VADfmk blocked this increased autophosphorylation (Fig. 7B). Finally, we performed experiments to test in vivo phosphorylation of PKC␣ in HL60 cells treated with UCN-01. Fig. 8 shows that UCN-01 increased PKC␣ phosphorylation after 1 h of treatment and that z-VAD-fmk inhibited the UCN-01-induced PKC␣ phosphorylation. These results suggest that caspase activation during apoptosis in HL60 cells is upstream from PKC␣ autophosphorylation and activation.

DISCUSSION
The results of Fig. 1 show that although UCN-01 is a PKC inhibitor and CPT is a topoisomerase I inhibitor, both of them induced activation of PKC. Using immunoprecipitation assay for PKC activities, we found that UCN-01 inhibited PKC␤I, ␤II, and ␦ activities in whole cells as well as in vitro but that PKC␣ was activated in HL60 cells undergoing apoptosis. Apoptosis induced by CPT, a DNA topoisomerase I inhibitor was also associated with activation of PKC␣ as well as PKC␦ activation.
Caspases play a central role in the apoptosis pathways (28 -30). z-VAD-fmk, a cell-permeable caspase inhibitor with broad specificity, blocks apoptosis induced by various stimuli including treatment by UCN-01 or CPT. Our results demonstrate  8. UCN-01-induced apoptosis is associated with PKC␣ phosphorylation in vivo. HL60 cells were incubated in phosphatefree RPMI 1640 medium supplemented with 10% dialyzed fetal bovine serum for 1 h. Fifty Ci/ml [ 32 P]orthophosphate was then added for 1 h. Cells were washed once with phosphate-free medium for 1 h and then 10 M UCN-01 was added. Cytosols were prepared at the indicated times and were immunoprecipitated with anti-PKC␣ antibody. The beads were boiled in sample buffer and analyzed by SDS-PAGE gel electrophoresis. The gel was dried and exposed to Kodak Rx film at Ϫ70°C. that z-VAD-fmk selectively inhibited the activation of PKC␣ induced by UCN-01 and CPT. On the other hand, z-VAD-fmk did not affect the inhibition of PKC␤I, ␤II, and ␦ activities by UCN-01 and the activation of PKC␦ by CPT. At the same time, we found that z-VAD-fmk blocked the proteolytic cleavages of PKC␤I and PKC␦, suggesting that PKC␦ cleavage is not the only mechanism for PKC␦ activation. For PKC␣, we found no evidence that PKC␣ activation is associated with proteolytic cleavage.
Our data (Fig. 5) rule out that UCN-01, CPT, or z-VAD-fmk directly affected PKC␣. PKC␣ activation is not due to changes in PKC␣ protein levels since control and cells treated with UCN-01 or CPT in the absence and presence of z-VAD-fmk had approximately equal amounts of PKC␣ protein as detected by Western blot. Phosphorylation has emerged as an important mode of regulation for PKC (3,32). For example, mutation of Thr to Ala in PKC␣ (Thr-497) results in an inactive kinase (33), and PKC autophosphorylation has been shown to regulate PKC activity by trans-phosphorylation at the activation loop followed by autophosphorylation (31,32). Our results show that UCN-01 and CPT activated autophosphorylation of PKC␣ and that z-VAD-fmk reversed such effects. Using [ 32 P]orthophosphate metabolic labeling, we also found increased phosphorylation of PKC␣ in HL60 cells undergoing apoptosis and blockade of these effects by z-VAD-fmk. These findings suggest that alterations of PKC␣ phosphorylation might be important for modulating PKC␣ activity and might be regulated by caspases.
The mechanisms (pathways) by which UCN-01 and CPT induce phosphorylation and activation of PKC␣ are yet unknown. We previously showed that both CPT and UCN-01 induce transient and unscheduled cyclin B1/Cdc2 kinase activation before the onset of apoptosis in HL60 cells treated with CPT or UCN-01 (14,24). CPT and DNA damaging agents have also been shown to activate other protein kinases including DNA-dependent protein kinase (34), jun kinase (35,36), and c-Abl (36). Further investigation will be required to establish the connections between activation of various protein kinases and caspases during apoptosis and to establish the functional significance of PKC␣ activation in the apoptotic response of HL60 cells.