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Mutations in the Glutathione-gated KefC K+ Efflux System of Escherichia coli That Cause Constitutive Activation*

The kefC gene of Escherichia coli encodes a potassium efflux system that is gated by glutathione (GSH) and by GSH adducts. Independently isolatedkefC mutations that result in spontaneous activation of the efflux system have been analyzed. Three mutations affect residues located adjacent to the conserved Rossman fold in the carboxyl-terminal domain. Two mutations lie in a sequence predicted to form a cytoplasmically located loop in the membrane domain of KefC. All of the mutants retain normal regulation by the YabF protein and by GSH adducts. A mutation in the Rossman fold, R416S, alters the normal regulation of KefC by GSH. In contrast to the wild-type protein, which is inactive in the presence of GSH, the R416S mutant is only active in the presence of GSH or its analogue, ophthalmic acid. Other mutations in this region or elsewhere in the protein have their spontaneous activity augmented by depletion of the GSH pool. These data identify a specific role for the carboxyl-terminal domain of KefC in regulating KefC activity and are discussed in the light of recent data that suggest that GSH adducts can bind within a Rossman fold.

The KefB and KefC potassium efflux systems play a major role in protection of Escherichia coli cells against the toxicity of electrophiles. The addition of electrophiles to E. coli cells, or the stimulation of their synthesis in the cytoplasm, elicits the activation of these two efflux systems through the formation of glutathione (GSH) 1 adducts (1)(2)(3). KefB and KefC are maintained in an inactive state by the presence of reduced GSH or its non-sulfhydryl analogue, ophthalmic acid (4 -7). Activation, leading to potassium loss from the cell, occurs when the GSH is conjugated to electrophiles through the sulfhydryl group (1,8). Slower efflux is observed when GSH is removed from cells by mutations affecting the gshA gene (7). KefC is, therefore, a ligand-gated transport system with both negative (GSH) and positive (GSH adducts) effectors. This feature is common in many ion channels, and the organization of the protein is also consistent with a channel-like mechanism.
KefC belongs to a family of membrane proteins that includes Na ϩ /H ϩ antiports and regulatory proteins. 2 One of the significant differences within this group of proteins is the greater complexity of organization of the KefC protein. The aminoterminal domain is similar in hydrophobicity to many other transport proteins; however, there is located within it a potential voltage-gated sequence (RXXX) 5 followed by a series of hydrophobic residues. This is a structural feature similar to that found in many mammalian voltage-gated K ϩ channels (10,12). The protein lacks the distinctive P-loop that would place it in the voltage-gated K ϩ channel superfamily (10). Attached to the carboxyl terminus of the membrane protein via an acidic linker peptide is a "soluble" carboxyl-terminal domain, which contains a Rossman fold that has high sequence similarity to yeast glyceraldehyde-3-phosphate dehydrogenase (11). Finally, genetic evidence supports a homo-oligomeric organization for KefC (13) and the existence of a separate regulatory subunit, YabF for the E. coli KefC system with homologues identified for KefB and the KefX systems of E. coli and Haemophilus influenzae, respectively (10). These features reflect the complexity required to retain appropriate control over KefC activity.
Here we report the analysis of mutations that cause a spontaneous activation of KefC. Five alleles have been identified; two are found in the membrane-located domain of the protein and three are close to the Rossman fold domain. Further analysis of one of the mutations in the Rossman fold motif (R416S) shows that the mutation alters the normal regulation by GSH. These data are consistent with a model in which the carboxylterminal domain of KefC plays a major role in regulating the activity of the protein and may be the location of the GSHbinding site.

MATERIALS AND METHODS
Reagents-All of the chemical reagents were purchased from Sigma or BDH and were of analytical grade when possible. Ophthalmic acid was obtained from Bachem Ltd. (United Kingdom). The chemicals used for preparation of complex growth medium were supplied by Oxoid. Restriction enzymes, Taq DNA polymerase, and universal sequencing primers were supplied by Boehringer Mannheim. The Qiagen plasmid preparation kits were obtained from Qiagen. Other primers designed and used in this study were purchased from Genosys Biotechnologies, Inc. or supplied by Dr. P. Carter. The Wizard PCR Preps DNA purification system was obtained from Promega. The PRISM Ready Reaction DyeDeoxy Terminator cycle sequencing kit was obtained from Applied Biosystems Ltd. The Muta-Gene phagemid in vitro mutagenesis kit, version 2, was purchased from Bio-Rad.
Bacterial Strains, Plasmids, and Bacteriophage-All of the strains and plasmids used in this study are listed in Table I. Phage M13K07 used in this study was purchased from Bio-Rad. P1kc phage was used for transductions.
Media for Growth of Strains-The minimal medium Kx (where x indicates the approximate concentration [mM] of potassium) was used for the culture of cells for potassium transport experiments (14). Solid medium containing less than 1 mM potassium was prepared by repeated * 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  washing of the agar in 1 M NaCl to remove contaminating potassium ions in the agar and followed by rinsing with distilled water prior to sterilization. The complex medium LK was prepared as described previously (15). The medium used in the preparation of uracil-containing single-stranded DNA was prepared as described (16). Where required, media were supplemented with ampicillin (25 g ml Ϫ1 ), tetracycline (25 g ml Ϫ1 ), chloramphenicol (12.5 g ml Ϫ1 ), or kanamycin (25 g ml Ϫ1 ).
Growth Conditions-Cells were grown at 37°C in LK complex medium (15) for use in DNA manipulations. For potassium transport experiments, cells were grown in K 120 minimal medium at 37°C. The carbon source for growth was glucose (0.2% w/v). Strains were stored on high potassium medium to prevent the occurrence of suppressor mutations. Supplementation of strains with GSH or ophthalmic acid was achieved by addition of fresh 100 mM stock to a final concentration of 1 mM.
Measurement of Potassium Efflux from Bacterial Strains-Potassium efflux from bacterial strains was recorded as described previously (7,11).
DNA Manipulations-Restriction enzyme digestion and DNA ligation and transformation were carried out using standard protocols (16). Plasmid preparations were made using the Qiagen plasmid preparation kits.
PCR Analysis of kefC Mutants-PCR primers were designed using the published sequence of kefC (11). The primers used to amplify yabF were designed using a sequence obtained from the genome sequencing project (see Ref. 17; accession number P31577 GB.Eco110K). Primer pairs were designed to amplify yabF and kefC in 350 -400-base pair products that overlapped in sequence by approximately 100 base pairs to ensure that there would be no gaps in the sequence. PCR amplification used single colonies of the appropriate strain inoculated into the reaction mix to provide template DNA. Reactions were carried out in a total volume of 100 l (containing each primer at 0.25 M dNTP (200 M each) 2.5 units of Taq DNA polymerase and reaction buffer as supplied by Boehringer Mannheim) and overlaid with approximately 30 l of mineral oil. Once optimized so that only one product was observed by agarose gel electrophoresis, the PCR reaction was purified using the Wizard PCR Preps DNA purification system prior to sequencing. The PRISM Ready Reaction DyeDeoxy Terminator cycle sequencing kit was used to perform cycle sequencing reactions on PCR products or plasmids, using the protocol outlined by the supplier of the kit. Samples were run on an Applied Biosystems 373A DNA sequencer. The cloning junctions of plasmids created in this study were verified using the universal primers supplied by Boehringer Mannheim. All sequencing reactions were performed a minimum of two times on both strands of the template.
Site-directed Mutagenesis of kefC-Plasmid pKC952 carries the entire kefC gene and 152 base pairs of upstream sequence on a HindIII-BamHI fragment cloned into pHG165 (13). The HindIII-BamHI kefCbearing fragment from pKC952 was cloned into the phagemid vector pTZ19U (18) creating the mutagenesis vector pSM7. Site-directed mutagenesis of kefC borne by pSM7 was carried out using the Muta-Gene phagemid in vitro mutagenesis kit, version 2. The supplied instructions were followed with the exception that single-stranded uracil-containing template DNA was prepared by M13K07 infection of CJ236 and transformed with pSM7 (16). The mutagenic primers were supplied with 5Ј phosphorylation: KefCR416S (GGAGAGCAGTAAGGATCCGGTA-ATCTGCCC), KefCS420A (CACCCCGCTAGCGAGCAGTAATCG), KefCS420A2 (CACCCCGCTAGCGAGCAGTAA), and KefCD264A (GGTTCGATAGCGCTCTCCAGCGC). The primers were designed in such a way that a unique restriction site was incorporated at the site of the mutation to allow subsequent identification of mutant plasmids (BamHI for KefCR416S and NheI for KefCS420A and KefCS420A2). Primer KefCD264A was designed to remove an EcoRV site present in wild-type kefC on creation of the desired mutation. Primer KefCS420A2 was used to create the double mutant R416S,S420A (plasmid pSM21) from the single mutant R416S. These primers were employed in separate in vitro reactions to generate the following: pSM9, bearing kefCR416S; pSM10, carrying kefCS420A; pSM21, carrying

RESULTS
Mutations in the kefC locus that affect potassium retention were selected as isolates unable to grow on K 0.1 medium by ampicillin-enrichment of UV-mutagenized cells of E. coli Frag5. The strains grew normally when incubated in K 10 medium (data not shown). We have previously shown that the K 0.1 negative mutants are gain of function alleles and that they can be inactivated by a null mutation at the same site (20). Therefore, the mutants were transduced to tetracycline resistance with P1 phage grown on strains that carried either kefB::Tn10 or kefC::Tn10 and scored for their ability to grow on K 0.1 medium. Of the twelve independent mutants characterized, seven could be converted to K 0.1 ϩ phenotype by insertions of a kefC::Tn10 and were thus designated kefC alleles. The other five mutations were mapped to the kefB locus. 2 The rate and extent of spontaneous K ϩ efflux were measured for each of the kefC mutants after growth in K 120 medium, filtration, washing, and suspension of cells in K 0 media (Fig. 1). When cells were harvested directly from K 120 medium the K ϩ content of the mutants and the parent strain were similar (data not shown), however, each of the mutants displayed a significantly lower K ϩ pool than the parent at the first time sample (Table II) indicating rapid loss over the first 60 s. K ϩ efflux continued in the mutants for 10 -20 min, whereas the parent retained more than 80% of its K ϩ pool over this period. Five strains exhibited a unique efflux profile (strains TK104 and TK121 and TK116 and TK119 were indistinguishable, and these pairs of strains subsequently proved to be isoallelic; see below).
The location of the mutations was determined by direct sequencing of 300 -400-base pair PCR products of the kefC gene. Only a single base change was observed in each of the mutants with the exception of strain TK120 (kefC120), which carries two changes R416S and S420A (Table II). Mutations were found in both the amino-terminal membrane protein domain (E262K and D264A) and in the carboxyl-terminal domain (R416SϩS420A, V427A, and R516C), which carries the Rossman fold. Strains TK104 and TK121 and strains TK116 and TK119 were found to be isoallelic. yabF is an open reading frame that overlaps the kefC gene and is required for full KefC activity (10). The sequence of yabF was determined in each of the mutants and was found not be changed from that of the parent (data not shown).
Strain TK120 carries two mutations located very close together, R416S and S420A. Site-directed mutagenesis was used to separate the mutations, and activity was studied using a construct that gives reduced KefC expression, pkC952 (13). Such a construct was desirable since high level expression of mutant proteins that cause a fast K ϩ leak on the cell could pose a severe growth defect leading to selection for suppressor mutations. Subcloning of the 2.15-kilobase HindIII-BamHI fragment from pkC952 into the mutagenesis vector pTZ19U created a construct, pSM7, that yielded no functional KefC activity (data not shown). This enabled mutations to be introduced into this very high copy number vector without the concern that expression would be toxic to the cell. Mutagenesis was carried out as described under "Materials and Methods," and the resulting clones were used to create separate plasmids carrying the separate R416S and S420A mutations and the two mutations in combination. The DNA was then subcloned into pHG165 to create pSM12 (wild type), pSM14 (R416S), pSM15 (S420A), and pSM30 (R416S,S420A). Plasmids were transformed into strain MJF276 (KefB Ϫ KefC Ϫ ), and the rate of spontaneous K ϩ loss was determined (Fig. 2). As expected, plasmids pSM12 and pkC952, which carry the same sequence, gave similar low rates of K ϩ loss that were slightly faster than those observed in the nontransformed MJF276. Plasmid pSM15, which carries the S420A mutation, introduced a slow leak that was reproducibly faster than the MJF276/pSM12. However, rapid, spontaneous K ϩ efflux was only observed from strains carrying the R416S mutation (MJF276/pSM14 (R416S) and MJF276/pSM30 (R416S,S420A)) (Fig. 2). The rate of K ϩ efflux seen with the combined mutations was greater than that seen with R416S alone, and this is consistent with the slightly enhanced leak seen with S420A alone, i.e. the R416S and S420A mutations are additive with the R416S mutation dominant. The efflux profile for strain TK120 (Fig. 1), which carries the chromosomal R416S ϩ S420A mutations, was similar to that obtained with MJF276/pSM30 (Fig. 2), confirming that the cloned lesions were responsible for the K ϩ leak. Slight differences between the strains may arise due to a slight difference in the genetic background of MJF276 and TK120 and also by the separation in the expression of yabF and kefC in MJF276/ pSM30. This suggestion was supported by analysis of MJF276/ pSM26 (kefCD264A), which carries the D264A mutation in the same plasmid context as the R416S and S420A mutations. The  The missense mutations in the strains were identified by PCR amplification of overlapping regions of kefC and direct sequencing of the products. The parent strain Frag5 was used as a positive control in the PCR-based analysis and has the same kefC sequence as that reported previously (11). The mutations were the result of single base pair mutations that were found on both strands of the template. The resulting codon change is indicated, together with the encoded amino acid alteration. Strain DNA mutation (base) a Codon change Amino acid mutation a Base number 1 is the adenine of the first codon of kefC (ATG). b The K ϩ concentration is that observed at the first time point after suspension of the cells in K 0 medium. spontaneous K ϩ leak seen in MJF276/pSM26 was slower than that seen in TK104 (kefCD264A) (compare Fig. 1 with Fig. 2). Notwithstanding these differences, we conclude that the most important mutation in strain TK120 is the R416S mutation.
YabF is the putative product of the open reading frame 5Ј to kefC and is required in trans for the activity of KefC (10). Since the kefC mutants were identified by their loss of regulation of K ϩ efflux it was possible that the mutations affected the control by YabF. Thus, the K ϩ leak was determined in strains MJF374 (YabF Ϫ ,KefC Ϫ ) 2 and MJF276 (YabF ϩ ,KefC Ϫ ) transformed with pSM12 and pSM14(R416S) (Fig. 3). No significant K ϩ efflux was observed from MJF374/pSM12 or MJF374/pSM14, but normal efflux was observed in the transformed derivatives of MJF276, which possesses YabF activity. Thus, YabF is required for the activity of both the wild-type and the mutated KefC proteins.
We have previously shown that the mutation in TK121 can be partially suppressed by co-expression of the wild-type gene from pkC592 (13), which is equivalent to pSM12 (see "Materials and Methods"). Since the R416S mutation lies in a different domain to TK121 mutation, D264A, we sought to determine whether co-expression of the two KefC proteins would allow either co-dominance or suppression. Plasmids pSM14 (R416S) and pSM12 (wild type) were transformed separately into MJF104 (kefC104, kefB::Tn10), and the spontaneous K ϩ efflux was measured (Fig. 4). Expression of the wild-type kefC gene from a multicopy plasmid, pkC11, or at a reduced expression level from pSM12 partially suppressed the K ϩ loss caused by the kefC104 mutation (D264A). Similarly, transformation of TK120 (kefC120) separately with pSM12, pSM15, or pkC11 suppressed the leak caused by the mutation R416S (data not shown). Thus, both the mutated subunit carrying the D264A mutation and the subunit carrying the combined R416S,S420A mutations can be suppressed by the wild-type gene expressed in either high copy number (pkC11) or low copy number (pSM12). The ability of the KefCS420A subunit to suppress the leak from TK120 in strain TK120/pSM15 suggests that this mutation does not have a major effect on the configuration of the KefC protein, which is consistent with the presence of only a slow spontaneous leak from strain MJF276/pSM15 (Fig. 2). In contrast, the K ϩ leak from MJF104/pSM14, which introduced the R416S mutation, was faster than for either MJF104 or MJF104/pSM12 (Fig. 4), and thus, KefCR416S failed to suppress the D264A lesion. These data indicate that suppres-sion requires wild-type KefC sequences in both the membrane and the carboxyl-terminal domains.
The KefC K ϩ efflux system is maintained in a closed state by GSH or its non-sulfhydryl analogue, ophthalmic acid (6,7). Cells that lack GSH leak K ϩ via KefB and KefC, and this phenotype is reminiscent of those observed with the mutants described here, which raises the possibility that the mutations affect the GSH-binding site. Thus, we sought to determine whether the K ϩ leak observed with the mutations was exacerbated in a GSH-deficient mutant. Strain MJF335 (KefB Ϫ ,KefC Ϫ ,GshA Ϫ ) was transformed separately with pSM14 (R416S), pSM26 (D264A), pSM30 (R416S,S420A), or pSM12 (wild type), and the spontaneous K ϩ leak was determined (Fig. 5, A-C). The rate of K ϩ efflux from MJF335/pSM12 was enhanced over that seen in the isogenic GSH-sufficient strain MJF276/pSM12 and could be inhibited by inclusion of either GSH or ophthalmic acid in the growth medium used to prepare the cells (Fig. 5A). Similarly, when plasmid pSM26 (D264A) was introduced into strain MJF335 the leak was increased compared with that seen in MJF276/pSM26 to the extent that over 60% of the K ϩ pool was released in the first 60 s after suspension of the cells into K 0 medium (Fig. 5B).
GSH or ophthalmic acid reduced the K ϩ leak to that seen in MJF276/pSM26 (Fig. 5B). Thus, the leak caused by the absence of glutathione was additional to the spontaneous leak caused by the D264A mutation. In contrast it was observed that the leak of K ϩ was slower from MJF335/pSM14 (R416S) than from MJF276/pSM14 and was enhanced by growth in the presence of GSH (Fig. 5C). Ophthalmic acid also restored spontaneous K ϩ efflux to strain MJF335/pSM14. Similar data were obtained with MJF335/pSM30 (R416S,S420A) (data not shown). These data clearly show that the spontaneous activity of KefCR416S is dependent upon the presence of GSH or its analogue, which is an inversion of the normal phenotype.
The above analysis was confirmed and extended by analysis of the chromosomal mutations affecting KefC. Each of the strains was transduced to kanamycin resistance using a gshA::Tn10(Kan) insertion mutant and the K ϩ leak determined in the presence and absence of GSH (Table III). For all of the mutants except TK120 (R416S,S420A) the rate of K ϩ was faster in a GSH-deficient derivative than in the GSH ϩ parent, and the leak could be reduced by growth of the cells with GSH (Table III). As observed above for MJF335/pSM14 and MJF335/ pSM30, the GSH-deficient derivative of strain TK120 (R416S,S420A) exhibited a slower K ϩ leak than did TK120 itself (Table III), and the fast leak could be restored by growing the strain in the presence of GSH (data not shown). Thus, the reversal of the normal GSH-gating is specific to the R416S mutation.
GSH adducts activate the KefC system (7). Since the phenotype of the KefC R416S derivative is an altered interaction with GSH the effect of the mutation on activation by the GSH adduct, N-ethylsuccinimidyl-S-GSH, formed by the reaction of N-ethylmaleimide with GSH (7) was investigated. The rate of N-ethylmaleimide-elicited efflux were similar for MJF276/ pSM12, MJF276/pSM14, MJF276/pSM26, and MJF276/pSM30 (data not shown). It is clear that the mutation does not substantially affect the activation of KefC by GSH adducts. DISCUSSION The data presented here identify residues within KefC that when mutated alter the regulation of the efflux system such that it exhibits a spontaneous K ϩ leak. The mutations map to two regions of the protein: a well-defined region in the aminoterminal hydrophobic domain, the HALESDIE sequence; and the carboxyl-terminal hydrophilic domain. All of the residues that cause a spontaneous K ϩ leak are highly conserved across the four members of the KefB/C family of proteins identified to date but are not as well conserved in the other members of the wider family that includes the NapA Na ϩ /H ϩ antiport family and the RosB regulatory protein. 2 In addition we show that one of the mutations, R416S, which is in the carboxyl-terminal domain, specifically alters the regulation of the system by GSH. In this mutant, K ϩ efflux is entirely dependent upon the presence of GSH or its analogue, ophthalmic acid. In the presence of either peptide the efflux system is active, but in its absence the K ϩ leak is much reduced. Neither mutations in the HALESDIE sequence nor other mutations in the carboxylterminal domain have this effect on the phenotype since the absence of GSH increases the K ϩ leak in these strains.
The first implication of these results is that the carboxylterminal domain plays a significant role in regulating the closed state of the KefC system. An obvious hypothesis would be that the two regions identified in this study interact directly to effect the closure of KefC, in a way analogous to the interaction of ␤ subunits of Shaker channels with the S4-S5 linker peptide (12,21). The prediction from positive charge distribu- a Strains deficient in synthesis of GSH were created for each strain carrying a kefC mutation by transduction to kanamycin resistance using Frag56 (gshA::kan) as donor.
b Supplementation of the growth medium with 1 mM GSH ensured that the cells were GSH-replete prior to harvest for measurement of spontaneous K loss.
c Potassium loss was measured for 20 min, and the pattern observed at 3 min was maintained throughout this period of incubation. tion and hydropathy plots is that the HALESDIE sequence of KefC is located at the cytoplasmic face of the membrane as is the carboxyl-terminal domain, which would place the two in the right location to interact. The HALESDIE sequence is part of a strongly conserved region of the KefB/C family of proteins. 2 The sequence can be represented as RHELEXDIEPFK (where X ϭ Ala, Ser, or Thr) and therefore, has a strongly acidic nature although the D264 is not conserved in KefB (A262) and E266 is substituted in KefX (A263) of Myxococcus xanthus. However, in all four members of the KefB/C family there are always three negatively charged amino acids in this sequence that is flanked by conserved positively charged groups. Thus, the mutant analysis has clearly defined a region of KefC that is important for regulation of activity.
The R416S, V427A, and R516C mutations of the carboxylterminal domain affect conserved residues but have different effects on KefC activity. Although all confer a K ϩ leak upon the KefC protein, implying loss of regulation, the effect of removing GSH is to convert the protein to the closed state in the case of R416S and to a more active state with R416C and V427A. R416 falls immediately after the putative ␤-␣-␤ (Rossman) fold of the carboxyl-terminal domain and is conserved in glyceraldehyde-3-phosphate dehydrogenase and in the carboxyl-terminal Rossman fold of TrkA, the regulator of K ϩ uptake in E. coli (22). Analysis of the sequences of the carboxyl-terminal domain in the four members of the KefB/C family shows that only the first 120 amino acids is conserved and therefore, that this region may be important for regulation of activity. The difference in the phenotype between the R416S and the V427A and R516C mutations suggests that the region of the ␤-␣-␤ fold may be critical for controlling the activity of this domain as a regulator of KefC opening and closing. Some support for this hypothesis is the recent observation that GSH adducts bind in the Rossman fold of glyceraldehyde-3-phosphate dehydrogenase from human erythrocytes (23). Membrane-associated glyceraldehyde-3-phosphate dehydrogenase bound various GSH adducts and was photoaffinity-labeled by S-(p-nitrobenzyl)-GSH-125 I-4azidosalicylic acid, and this could be competitively inhibited by low concentrations of NAD ϩ and ATP (23). The residues identified by covalent linking were not in the GXGXXG motif that forms the core of the Rossman fold but at a slightly more distant site within the predicted binding site for NAD ϩ (23,24). The Rossman fold domain of glyceraldehyde-3-phosphate dehydrogenase has also been implicated in the binding of RNA (9,25), and it has been suggested that the fold is particularly suited to binding of both small and large molecules. These observations would be consistent with an important role for the carboxyl-terminal domain of KefC in regulating channel activity and suggest that this domain might contain the binding site for GSH adducts.


extfieldsofstudy: Biology Medicine

text:
Phosphotransferases Associated with the Regulation of Kinesin Motor Activity*

Kinesin, a plus-end-directed microtubule motor protein, functions in concert with accessory factors that have been shown to regulate enzyme activity and may also provide cargo specificity. This report identifies the 79-kDa kinesin-associated phosphoprotein as a phosphoisoform of kinesin light chain. Increased phosphorylation of this light chain isoform is sufficient to account for the increase in kinesin-mediated microtubule-gliding activity. Additionally, it was found that the degree of phosphorylation of this isoform is regulated by a 100-kDa kinase and 150-kDa type 1 phosphatase. Both the kinesin light chain kinase and phosphatase co-purify with the kinesin heavy chain, suggesting that kinesin exists in a large complex capable of self-regulation.

proteins (7), it is also expected to play a role in the regulation of the MT motor proteins. Indeed, a growing body of evidence suggests that this is the case. Kinesin heavy and light chains can be phosphorylated in vitro by purified protein kinase C and protein kinase A (8,9), but these kinases are not likely to phosphorylate kinesin in vivo (10). Cytoplasmic dynein and kinesin have both been shown to exist as phosphoisoforms in vivo (11,12), with both kinesin heavy chain (KHC) and kinesin light chain (KLC) shown to be phosphorylated on multiple sites (12). Increased phosphorylation of cytoplasmic dynein correlates with dissociation from lysosomal vesicles (11). In contrast, the membrane-bound form of kinesin has a higher state of phosphorylation than soluble kinesin (12). In vivo studies by Hamm-Alvarez et al. (13) have shown that treatment of serumstarved CV-1 cells in culture with the phosphatase (PPase) inhibitor okadaic acid (OA) resulted in an increase in the number of vesicle movements within the cells. McIlvain et al. (14) found that treatment of the MT-depleted cytosol with OA before purification of the motor proteins resulted in a 2-fold increase in kinesin motor activity in an in vitro MT-gliding assay. Further, they were able to show that this treatment resulted in increased phosphorylation of three kinesin-associated proteins, a 150-, 79-, and 73-kDa kinesin-associated phosphoprotein (KAPP). These data provided the first direct correlation between phosphorylation of particular proteins and kinesin motor activity.
Here we demonstrate that the 79-kDa KAPP is a kinesin light chain isoform (KLC79) and that phosphorylation of KLC alone is sufficient to reproduce the increased activity described previously. We find that the phosphorylation state of KLC is mediated by a 100-kDa kinase and a 150-kDa type 1 PPase, both of which co-purify with KHC. Together with the other data, the present results suggest that kinesin exists as a complex consisting of at least KHC, KLC, KLC kinase, and KLC PPase.

EXPERIMENTAL PROCEDURES
Cell Culture-The murine T hybridoma cell line 2B23. 18 was grown in Dulbecco's modified Eagle's medium with high glucose and 5% fetal calf serum. The human myelocytic leukemia cell line HL-60 was cultured in RPMI 1640 and 10% fetal calf serum. Chick embryo brains were collected from 12-day-old chick embryos. All media were supplemented with 50 units/ml penicillin and 50 g/ml streptomycin. All cell culture reagents were purchased from Life Technologies, Inc.
Preparation of Cytosol-Cytosolic fractions were prepared as described previously for cell lines (14) and chick embryo brains (15), except endogenous MTs were resuspended in 1 M KCl/20 M taxol, diluted to 0.5 M KCl with PMEE/20 M taxol and pelleted at 100,000 ϫ g, 15 min, 21°C before being used for MT affinity.
Immunoprecipitation-The MT-depleted cytosol was incubated with protein A-agarose beads (Pierce) for 1 h before addition of antibody. After removing the preclearing beads, antibody was added and the samples left on ice for 1 h. Fresh protein A-agarose beads were added, and the samples left on a rotator at 4°C overnight. The beads were then washed repeatedly in PMEE before being used in an assay. The beadcoupled immune complexes were then treated with either buffer or * This work was supported by Grants from the National Institutes of Health and the American Cancer Society. 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.
¶ To whom correspondence should be addressed: inhibitors before the addition of [␥-32 P]ATP. After the phosphorylation reactions were complete, the beads were washed, and the immune complexes released by boiling in SDS-PAGE sample buffer. The kinesin-associated proteins were separated by SDS-PAGE, and the phosphorylated proteins were visualized by autoradiography.
Treatment with Okadaic Acid, Inhibitor-2, Staurosporine, and [␥-32 P]ATP-Samples were treated with either of the PPase inhibitors, OA (LC Services, Woburn, MA) or inhibitor-2 (I-2) (generously provided by David Brautigan, University of Virginia, Charlottesville), or the kinase inhibitor staurosporine (LC Services) for at least 15 min before the phosphorylation reactions were initiated by the addition of [␥-32 P]ATP. The reactions were incubated at 37°C with 3,000 Ci/mmol [␥-32 P]ATP (Amersham Corp.) in the presence or absence of inhibitors. Motor fraction phospholabeling was at a concentration of 200 Ci/ml. Bead-bound KHC immunoprecipitates were adjusted to a volume equal to the preimmunoprecipitation cytosol with PMEE and incubated with 10 Ci/ml [␥-32 P]ATP.
Purification of Microtubule Motor Proteins-MT motors were purified by a MT affinity/ATP elution protocol as described before (14) with an additional step. Actin and myosin were removed by the addition of 9 mg/ml glucose and 10 units/ml hexokinase (Boehringer Mannheim) to the MT-depleted cytosol. The samples were incubated at room temperature until a white flocculate precipitate developed and then centrifuged at 100,000 ϫ g, for 30 min at 21°C to pellet the complex before proceeding with the MT affinity protocol.
MT-gliding Assay-MT-gliding assays were performed as described previously (14). Briefly, microtubules were polymerized by diluting 4 volumes of 3-3.5 mg/ml purified porcine brain tubulin with 1 volume of PMEE containing 5 mM GTP and 100 M taxol and incubated at 37°C for 15 min. A flow chamber was constructed using acid-washed coverslips overlaid on two strips of double stick tape on a standard microscope slide. Tape strips were spaced 2-3 mm apart, forming a chamber of 5-10 l. The chamber was filled with 1 volume of motor fraction, incubated for 5 min at 24°C, and washed with 3 volumes of 150 g/ml casein (60% ␣, 40% ␤, from bovine milk (Sigma)) in PMEE. Where indicated, the motor fraction was preincubated with buffer or 125 nM OA before addition to the slide chamber, as described previously (14). Two volumes of microtubule/nucleotide mixture containing 20 g/ml of polymerized microtubule stock (sheared by trituration to give an average microtubule length of 3-5 m), 10 mM ATP, 150 g/ml casein, 10 mM creatine phosphate (Boehringer Mannheim), and 80 g/ml creatine kinase (Boehringer Mannheim) in PMEE were then flowed into the chamber and incubated for 5 min before visualization by video-enhanced differential interference microscopy. The gliding assays were done using limiting dilutions of the motor fraction in the presence of excess microtubules and analyzed as described previously (14).
Embedded Kinase Renaturation Assay-The kinase was identified by its ability to autophosphorylate under renaturing conditions while embedded within an SDS-PAGE gel matrix. MT affinity-purified motors were separated by SDS-PAGE, and the gels washed in 20% isopropanol in 0.05 M Tris, pH 8.0, 4 mM dithiothreitol (buffer A), then in buffer A alone, and finally in 6 M guanidine hydrochloride (Sigma) in buffer A for 0.5 h with two changes of each buffer. After washing, the proteins were allowed to slowly renature in buffer A plus 0.1% Tween 20 overnight at 4°C with several changes of buffer. The gel was then washed for 30 min in PMEE, 300 M NaVO 4 (buffer B) before incubation for 1 h in buffer B plus 25 M ATP and 4 Ci/ml [␥-32 P]ATP. The phosphorylation reactions were stopped, and the proteins precipitated within the gel by repeated washes with 5% trichloroacetic acid, 1% sodium pyrophosphate. The gels were then stained with Coomassie Brilliant Blue R-250 and autoradiographed.
Velocity Sedimentation-MT affinity-purified motor proteins were separated by velocity sedimentation through a 5-20% linear sucrose gradient. Gradients were typically 13 ml and centrifuged at 100,000 ϫ g for 16 -18 h, 4°C.
Measurement of PPase Activity-PPase activity was determined by the ability of a sample to dephosphorylate [ 32 P]phosphorylase a (Life Technologies, Inc.). The substrate was prepared as the manufacturer recommended. Samples were incubated with the substrate at 30°C, and the dephosphorylation reaction stopped by the addition of 20% trichloroacetic acid. The 32 P freed from [ 32 P]phosphorylase a by PPase activity remained acid soluble while the proteins precipitated out of solution. The amount of freed 32 P was quantitated by measuring the counts per minute (cpm) in the trichloroacetic acid-precipitation supernatant. The resulting cpm were normalized to the peak value for each experiment and reported as the PPase activity.
Sizing of the PPase-To determine the size of the PPase, MT affinitypurified motor proteins were applied to a Superose 6 gel filtration column (Pharmacia Biotech Inc.). The column was developed at 0.5 ml/min with 30 ml PMEE, 0.1% 2-mercaptoethanol, 5% glycerol, and 0.5 ml fractions collected. An elution volume standard curve was constructed using proteins of known molecular weight, and the molecular weight of the PPase determined by comparison of the elution volume of the peak of PPase activity with this curve.
Analysis of Phosphorimages-Phospholabeling was analyzed using a Fuji Fujix BAS1000 phosphorimaging system (Fuji, Stamford, CT). A Fuji imaging plate was exposed to a dried radiolabeled gel inside an Amersham film cassette. After exposure, the imaging plate was scanned by the BAS1000 and converted into digital form. Analysis of the image was done using Fuji Fujix MacBas software for the BAS1000.

RESULTS
The 79-kDa KAPP Is Kinesin Light Chain-Our previously reported correlation between increased phosphorylation of the KAPPs and increased kinesin motor activity suggested that phosphorylation of these proteins plays an important regulatory role. Therefore, the identification of these proteins would lead to a better understanding of kinesin function. Since the 79-kDa KAPP falls within the range of predicted molecular weights for chick KLC, we began by evaluating the isoforms of KLC found in chick embryo brains. The MT-depleted cytosol was immunoprecipitated with the anti-KHC monoclonal antibody SUK4 (16) and the bead-coupled immune complex incubated with [␥-32 P]ATP. As expected, the major components of the SUK4 immune complex were KHC and KLC (Fig. 1). Immunoblotting with a series of antibodies was then used to identify the components of the immune complex. The monoclonal anti-KLC antibodies CKLC.3.9C1 (12,17) and CKLC.2.7F8 (15) were previously produced in our laboratory. Using CKLC.3.9C1 only the 70-kDa predominate isoform of KLC, as seen by Coomassie staining of the gel, was detected. However, CKLC.2.7F8 cross-reacted with the predominant 70-kDa KLC and an additional isoform of KLC with an apparent molecular mass of 79 kDa. Autoradiography of the blots showed that this 79-kDa KLC was the only phosphoprotein within the KHC immune complex. This conclusion is supported by studies that used bovine anti-KLC antibodies. 2 These results identify the 79-kDa KAPP as a phosphoisoform of KLC.
Hyperphosphorylation of KLC Increases Kinesin Motor Activity-Several KAPPs are present in the purified motor fraction from 2B23.18 cells (14). To determine the effect of phosphorylation of KLC79 on kinesin activity, motor proteins were prepared from 2B23.18 cells by a MT affinity protocol and incubated with [␥-32 P]ATP in the presence or absence of OA. The 79-kDa KLC was the only protein found to be consistently phosphorylated under these conditions (Fig. 2A)  phosphorylation of KLC alone was sufficient to stimulate kinesin motor activity, the purified and phosphorylated motor fraction from 2B23.18 cells was assayed for the ability to support MT gliding in vitro. Compared with untreated samples, twice as many microtubules were observed to be bound or gliding in the samples treated with OA (Fig. 2B). Since the same relation between enhanced phosphorylation of KLC79 and kinesin motor activity was seen as when the 150-and 79-kDa KAPPs were hyperphosphorylated (14), we conclude that phosphorylation of KLC79 alone is sufficient for regulating kinesin motor activity as measured in the MT-gliding assay.
A 100-kDa Kinase Activity Associates with Purified Kinesin-The response of KLC79 in the purified motor fraction ( Fig. 2A) suggested that the KLC kinase and PPase remain associated with the motors during MT affinity purification. To further investigate the KLC kinase activity, proteins of purified motor fractions were separated by SDS-PAGE and assayed for their ability to renature and autophosphorylate within an acrylamide gel matrix. Under these conditions, one phosphoprotein was seen at 100 kDa. To verify that this 100-kDa phosphoprotein represented an autophosphorylating kinase and not an ATPbinding protein, the renatured gel was pretreated with the kinase inhibitor staurosporine at 100 nM before the addition of [␥-32 P]ATP. The observed inhibition of phosphorylation confirms that the protein is a kinase (Fig. 3) and makes it a likely candidate for the KLC kinase. Using this technique, a similar 100-kDa putative kinase was found in different species and cell types, including the mouse hybridoma 2B23.18 and the human myelocytic leukemia line HL-60 (data not shown) as well as chick embryo brains (Fig. 3).
A 150-kDa PPase Associates with Purified Kinesin-The enhanced phosphorylation of KLC79 upon OA treatment of the MT affinity-purified motors ( Fig. 2A) indicates that either a type 1 or type 2A Ser/Thr PPase dephosphorylates the protein.
PP1 and PP2A activities can be distinguished from each other based on their sensitivity to OA. The PP2A family is inhibited at 1-10 nanomolar concentrations of OA, whereas the PP1 family remains active at concentrations 100-fold higher (18). To determine if a PP1 or PP2A was responsible for dephosphorylation of KLC79, the MT affinity-purified motors from 2B23.18 and HL-60 cells were used to dephosphorylate [ 32 P]phosphorylase a, a well characterized substrate for PP1 and PP2A PPases. When compared with the buffer controls, little change in the amount of PPase activity was observed upon addition of 5 nM OA to the reaction mixture, whereas the PPase activity was significantly inhibited in the presence of 500 nM OA. This result indicates that a PP1 phosphatase was present in the motor fraction (Fig. 4A).
To further characterize the PPase, the MT affinity-purified motors were fractionated by a Superose 6 gel filtration column (Fig. 4B), and the resulting fractions were assayed for the ability to dephosphorylate [ 32 P]phosphorylase a. Two peaks of PPase activity were identified, corresponding to molecular masses of approximately 150-and 80-kDa. The first peak is the PPase holoenzyme, while the second peak co-eluted with a commercial PP1 catalytic subunit, representing a dimer of the PPase catalytic subunit freed during enzyme purification (19). Similar peaks of activity were also identified from gel filtration analysis of motor proteins purified from HL-60 cells (data not shown).
To confirm that the single PPase identified in the bulk motor fraction was a PP1 enzyme, biochemical classification studies were done on the PPase holoenzyme and catalytic subunit isolated from the 2B23.18 cells. MT affinity-purified motors were separated by velocity sedimentation through a 5-20% linear sucrose gradient, and each fraction was assayed for its ability to dephosphorylate [ 32 P]phosphorylase a (Fig. 5A). The fractions corresponding to the holoenzyme (which co-sedimented with kinesin) and the catalytic subunit were incubated with OA (Fig. 5B) or I-2 (Fig. 5C) before being analyzed for PPase activity. I-2 is a PP1-specific inhibitor. It does not inhibit PP2A even at micromolar concentrations. The PPase holoenzyme was not affected by 5 nM OA, showed little activity after incubation with 125 nM OA, and was inhibited by I-2 at nanomolar concentrations. The PPase catalytic subunit was slightly more sensitive to the inhibitors, but exhibited the same overall characteristics as the holoenzyme. In contrast, a commercial PP2A catalytic subunit was completely inhibited at 5 nM OA but was not effected by 200 nM I-2 (data not shown), which confirms that the 150-kDa motor-associated PPase is a member of the PP1 family.
Kinesin Exists in a Large Complex Containing KHC, KLC,

FIG. 2. Hyperphosphorylation of KLC increases kinesin motor activity.
A, MT affinity-purified motors were treated with a buffer or 125 nM OA before the addition of [␥-32 P]ATP to initiate the phosphorylation reaction. The proteins were then separated by SDS-PAGE, and the phosphorylated proteins were visualized by autoradiography. B, motor fractions were treated with buffer or 125 nM okadaic acid before being absorbed onto acid-washed coverslips. After absorption, taxolstabilized microtubules, and 10 mM ATP were flowed into the slide chamber. The number of microtubules bound and gliding was visualized by video-enhanced differential interference microscopy. The error bars represent the S.E .   FIG. 3. A 100-kDa kinase co-purifies with the motor proteins. Kinesin purified by MT affinity was further isolated by sucrose gradient density sedimentation, and the proteins were separated by SDS-PAGE. The kinesin-associated proteins were assayed for ability to autophosphorylate under renaturing conditions while embedded within an acrylamide gel matrix in the presence or absence of the kinase inhibitor staurosporine (STR).
KLC kinase, and KLC PPase-The antibody SUK4 binds kinesin heavy chain near the motor domain (16) and immunoprecipitates a complex of proteins including KHC, KLC, and other accessory factors needed for kinesin motility in vitro (20). To determine if the KLC kinase and PPase are among these accessory proteins, the SUK4 immune complex from 2B23.18 cells was incubated with [␥-32 P]ATP in the presence or absence of phosphotransferase inhibitors. Without treatment with inhibitors, the two major proteins that were consistently phosphorylated within the kinesin heavy chain immune complex of these cells were the 150-kDa KAPP and KLC79 (Fig. 6) (as previously reported (14)), which confirms that the KLC kinase is kinesin associated.
To evaluate the effect of kinase inhibition on the phosphorylation of KLC79, the SUK4 immune complex was pretreated with 100 nM staurosporine before incubation with [␥-32 P]ATP. Under these conditions phosphorylation of KLC79 was eliminated (Fig. 6A). These data are in agreement with the autophosphorylation inhibition of the kinase shown in Fig. 3 and further suggest that the 100-kDa kinase present in the MT affinity-purified motor fraction phosphorylates KLC within the kinesin complex.
In addition, pretreatment of the kinesin heavy chain immune complex with 500 nM OA before incubation with [␥-32 P]ATP increased phosphate incorporation into KLC (Fig.  6B) and demonstrated that the KLC PPase is active within the kinesin complex.

DISCUSSION
Identification of the kinesin-associated phosphoproteins and the phosphotransferases that regulate them may elucidate the control of vesicle motility mediated by the motor proteins. Our original work describing the KAPPs (14) and later work on KLC (12) suggested that the 79-kDa KAPP was not KLC. This conclusion was based primarily on results in both laboratories from Western blots using the CKLC.3.9C1 antibody. However, our current work characterizes CKLC.3.9C1 as cross-reactive with only the predominant isoform of KLC and explains why Lee and Hollenbeck (12) did not observe cross-reactivity of the 70 -80-kDa KAPPs with CKLC.3.9C1 in their studies. An alternative anti-KLC monoclonal antibody (CKLC.2.7F8) made this clarification possible because it cross-reacted with multiple isoforms, including the 79-kDa KAPP of KLC in multiple species, making it a particularly useful tool in our studies on phosphoisoforms of KLC.
KLCs are suspected to be involved in cargo selection. They are associated with the KHC fan-shaped tail and therefore are located at the proposed vesicle docking site (5). This strongly suggests that the multiple isoforms of KLC provide cargo specificity. An OA-induced increased phosphorylation state of the KLC, represented by KLC79, may resemble the membranebound form of KLC and thus activate kinesin motor activity. This idea is consistent with data showing that the more highly phosphorylated forms of kinesin are vesicle-bound, whereas less phosphorylated forms are soluble (12).
The experiments that led to the identification of the KAPPs and the correlation between KAPP phosphorylation and kinesin activity (14) relied on cytosolic phosphorylation of the activating proteins. Here we demonstrate that further purification did not result in a loss of hyperphosphorylation activity. In fact, we were still able to obtain the same level of activation as observed in the previous report under conditions where only KLC was consistently phosphorylated.
The level of phosphorylation of KLC is controlled by two phosphotransferases that can be found in association with the kinesin molecule. The kinase is at present uncharacterized, but its size and sensitivity to specific kinase inhibitors indicates that it may be a novel enzyme. The phosphatase has been characterized to be a type 1 Ser/Thr PPase. Other type 1 PPases have been implicated in the regulation of both cytoplasmic dynein (21) and the nonmuscle myosin motors (7). Although the apparent molecular weight of the motor-associated PP1 is similar to the 150-kDa KAPP, they are different proteins, since the PP1 size was estimated for the native holoenzyme, whereas the KAPP size was derived from SDS-PAGE analysis. Work is underway to define which of the three subsets of PP1 is the relevant KLC phosphatase.
The present data show that phosphorylation of the 150-kDa KAPP is not essential for the enhancement of kinesin activity in the MT-gliding assay. However, it is associated with kinesin through various purification steps in several cell lines that we have examined and thus is likely to play a regulatory role that is not measured in the in vitro MT-gliding assay. For example, the 150-kDa KAPP may be important in maintaining the kinesin motor complex together in membrane association of the kinesin complex or in another step of in vivo vesicle motility, since highly purified kinesin is unable to transport vesicles in vitro (22). We are currently investigating the parameters that define the association of the 150-kDa KAPP with kinesin as well as regulation of its phosphorylation state in different kinesin preparations.
The 100-kDa kinase was the only kinase activity detected in the MT affinity-purified motor preparation, and the KLC was the only phosphoprotein detected when the MT affinity-purified motors were incubated with [␥-32 P]ATP. This suggests that the 100-kDa kinase is a KLC kinase. This hypothesis is supported by similar results obtained when a different method, immunoprecipitation, was used in the purification. These data suggest that autophosphorylation activates the 100-kDa kinase, which then results in phosphorylation of the KLC and subsequent activation of kinesin motor activity. To unequivocally identify the 100-kDa kinase and 150-kDa PPase as KLCspecific, all of the components will have to be purified to homogeneity. Nonetheless, the work presented here and in a previous paper (14) suggests that the active kinesin motor is an assembly of a number of proteins, including kinesin heavy and light chains, a kinase, and a phosphatase.
We consider it likely that phosphorylation of kinesin accessory factors is one step in a cascade of signal transduction that regulates kinesin activity and anterograde organelle movement. Our data support this by demonstrating that the phosphorylation level of KLC is modulated by a kinesin-associated kinase and PPase, and the level of KLC phosphorylation directly correlates with activation of kinesin motor activity. Future studies will address how the activities of the KLC kinase and PPase are controlled in turn.


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Polyoma middle T-induced vascular tumor formation: the role of the plasminogen activator/plasmin system.

The middle T antigen of murine Polyomavirus (PymT) rapidly transforms endothelial cells, leading to the formation of vascular tumors in newborn mice. Transformed endothelial (End.) cell lines established from such tumors exhibit altered proteolytic activity as a result of increased expression of urokinase-type plasminogen activator (uPA) and are capable of inducing vascular tumors efficiently when injected into adult mice. In this study we have used mice lacking components of the PA/plasmin system to analyze the role of this system in the transformation process and in tumor growth. We found that the proteolytic status of the host is not a critical determinant for PymT-induced vascular tumor formation. In addition, the lack of either uPA or tissue-type PA (tPA) activity is not limiting for the establishment and proliferation of End. cells in vitro, although the combined loss of both PA activities leads to a marked reduction in proliferation rates. Furthermore, the in vitro morphogenetic properties of mutant End. cells in fibrin gels could only be correlated with an altered proteolytic status in cells lacking both uPA and tPA. However, in contrast with tumors induced by PymT itself, the tumorigenic potential of mutant and wild-type End. cell lines was found to be highly dependent on the proteolytic status of both the tumor cells and the host. Thus, genetic alterations in the PA/plasmin system affect vascular tumor development, indicating that this system is a causal component in PymTmediated oncogenesis.

Abstract.
The middle T antigen of murine Polyomavirus (PymT) rapidly transforms endothelial cells, leading to the formation of vascular tumors in newborn mice. Transformed endothelial (End.) cell lines established from such tumors exhibit altered proteolytic activity as a result of increased expression of urokinase-type plasminogen activator (uPA) and are capable of inducing vascular tumors efficiently when injected into adult mice. In this study we have used mice lacking components of the PA/plasmin system to analyze the role of this system in the transformation process and in tumor growth. We found that the proteolytic status of the host is not a critical determinant for PymT-induced vascular tumor formation. In addition, the lack of either uPA or tissue-type PA (tPA) activity is not limiting for the es-tablishment and proliferation of End. cells in vitro, although the combined loss of both PA activities leads to a marked reduction in proliferation rates. Furthermore, the in vitro morphogenetic properties of mutant End. cells in fibrin gels could only be correlated with an altered proteolytic status in cells lacking both uPA and tPA. However, in contrast with tumors induced by PymT itself, the tumorigenic potential of mutant and wild-type End. cell lines was found to be highly dependent on the proteolytic status of both the tumor cells and the host. Thus, genetic alterations in the PA/plasmin system affect vascular tumor development, indicating that this system is a causal component in PymTmediated oncogenesis.
1. Abbreviations used in this paper : End., endothelioma; MMP, matrix metalloproteinase; PA, plasminogen activator; uPA, urokinase-type PA; uPAR, uPA receptor; tPA, tissue-type PA; PAI, PA inhibitor; Plg, plasminogen; PTAH, phosphotungstic acid-hemotoxylin; PymT, Polyoma middle T antigen. O ncogenesis is a multistep process that requires a long latency period between the initiating event and the appearance of the tumor. However, a few viral oncogenes are known to rapidly transform target tissues without the need for additional genetic events; one such viral oncogene is the Polyoma middle T antigen (PymT). 1 The PymT antigen specifically transforms proliferating endothelial cells apparently in a single-step manner, resulting in the formation of hemorrhagic cystic tumors in embryonic and neonatal mice, but not in adult mice where endothelial cell proliferation has ceased (for review see Pepper et al., 1997). The potency with which PymT transforms endothelial cells is thought to be a consequence of its interaction with proteins of the signal transduction machinery (for review see Brizuela et al., 1994;Kiefer et al., 1994 a ). These tumors are functionally reminiscent of human vascular tumors in that they are endothelial specific and organ nonspecific (for review see Wagner and Risau, 1994;Pepper et al., 1997), and that tumor-bearing mice develop features of the Kasabach-Merritt syndrome, i.e., thrombocytopenia, anemia, and associated splenomegaly (Dubois-Stringfellow et al., 1994 a ).
Tumor formation and subsequent invasion involve the disruption of anatomical barriers including basement membranes and penetration of tumor cells into normal adjacent tissues. Several lines of evidence indicate that enhanced invasive and metastatic potential is correlated with increased protease activity. It has been shown that both tumor and stromal cells express enzymes that degrade components of the extracellular matrix. Many of the relevant enzymes belong to one of two families: the serine proteases, in particular the plasminogen activator (PA)/plasmin system, and the matrix metalloproteinases (MMPs). Urokinase and tissue-type PAs (uPA and tPA) are the principal activators of plasminogen (Plg), the zymogen from which plasmin is derived. uPA is secreted in the form of an inactive precursor that binds with high affinity to a specific glycosylphosphatidylinositol-anchored cell surface receptor. Plasmin is a protease of tryptic specificity that either directly, or indirectly through the activation of latent MMPs, hydrolyzes extracellular proteins. The existence of multiple specific physiological inhibitors of both plasmin, i.e., ␣ 2antiplasmin, and PAs, i.e., PA inhibitors 1 and 2 (PAI-1 and PAI-2), provides additional points of regulation along this protease cascade (for review see Danø et al., 1985;Vassalli et al., 1991;Pepper et al., 1996).
The recent advent of mice lacking uPA, uPA receptor (uPAR), tPA, PAI-1, and Plg has questioned much early dogma concerning the role of the PA/plasmin system in processes requiring extracellular matrix breakdown. Thus, uPA Ϫ / Ϫ , tPA Ϫ / Ϫ , and uPA Ϫ / Ϫ and tPA Ϫ / Ϫ (utPA Ϫ / Ϫ ) mice, as well as Plg Ϫ / Ϫ mice, develop normally in utero and are fertile. However, utPA Ϫ / Ϫ mice develop multiple organ failure starting at 2-3 mo of age as a result of extensive extravascular fibrin accumulation as well as generalized microvascular thrombosis (Carmeliet et al., 1994). utPA Ϫ / Ϫ mice thus have retarded postnatal growth, exhibit reduced fertility, and have a shortened life span. A similar phenotype is observed in Plg-deficient mice (Bugge et al., 1995 a ;Ploplis et al., 1995). The phenotype of both utPA Ϫ / Ϫ and Plg-deficient mice therefore appears to be due to a systemic defect in fibrinolysis. Although no overt phenotypic alterations have been observed in uPAR Ϫ / Ϫ mice with respect to development, fertility, and homeostasis (Bugge et al., 1995 b ), it has recently been reported that adult mice with a combined deficiency in tPA and uPAR display hepatic sinusoidal fibrin deposits, demonstrating that uPAR plays a role in physiological fibrinolysis (Bugge et al., 1996). Finally, development and fertility are unaffected in PAI-1-deficient mice, although the absence of PAI-1 induces a mild hyperfibrinolytic state (Carmeliet et al., 1993 a,b ). Therefore, neither PAs nor Plg are essential for mouse development, postnatal growth, or fertility, suggesting that the primary role for the PA/plasmin system in the mouse is fibrinolysis.
In previous studies with PymT-transformed endothelial (End.) cell lines in a fibrin gel system, we observed that End. cells formed cystlike structures rather than capillarylike tubes, which correlated with their increased proteolytic activity . Reduction of protease activity by the addition of broad spectrum serine protease inhibitors led to the formation of capillary-like tubes, resulting in the hypothesis that tightly controlled extracellular proteolysis is essential for normal capillary morphogenesis . Further support for the role of the PA/plasmin system in aberrant morphogenesis in vitro came from experiments in which depletion of Plg from serum or addition of neutralizing antibodies to murine uPA inhibited cyst formation (Dubois-Stringfellow et al., 1994 b ). These findings implicated increased proteolysis, and in particular increased uPA activity, in aberrant vascular morphogenesis in vitro and further suggested a causal role for PymT in the elevated proteolytic activity of End. cells. In this study, mice lacking uPA, tPA, utPA, PAI-1, or Plg were used to investigate whether the PA/plasmin system might be important for PymT-or End. cell-induced tumor formation.

Virus Induced Tumor Induction in Neonatal Mice
Helper-free stocks of the N-TKmT virus were obtained from the viral producer cell line GP+E subclone 108.4.2 as described previously (Williams et al., 1988). Neonatal mice of various genotypes were injected intraperitoneally with 1-2.5 ϫ 10 5 neo R -transferring particles. The mice were observed carefully and analyzed as soon as the first mouse of an injected litter showed signs of anemia.

Derivation and Maintenance of End. Cells
Macroscopically visible tumors from mice injected with PymT viral particles were explanted, and End. cell lines were established as previously described (Kiefer et al., 1994 b ). End. cells could be established with relative ease from heterogeneously growing primary cultures, since they outgrow contaminating fibroblasts. Within 5-10 passages, a homogeneously growing, mostly clonal population of End. cells was obtained, which was used for the experiments described. Cell lines were maintained in DME supplemented with 5% FCS, glutamine, and nonessential amino acids and subcultured at confluence every 4-8 d. The origin and nomenclature of these cell lines are summarized in Table I.

Determination of Proliferation Rates of End. Cells
End. cells were plated at a density of 4 ϫ 10 5 -1 ϫ 10 6 per 25-cm 2 flask, counted, and replated at the same density every 6-8 d. Total cumulative cell numbers are represented over 23 d in culture.

Tumor Induction by Injection of End. Cells in Adult Mice
For the induction of End. cell-induced tumors, C57Bl6/129 wild-type and mutant mice, derived from brother-sister matings and interbred for more than six generations (10-14 wk old), and CD1 nude mice (8-10 wk old) were injected subcutaneously into the flanks of the right leg with 1.5-2 ϫ 10 6 cells, and then scored for tumor formation. The times taken for the onset of tumors and the latency period to form tumors of 125 mm 2 (maximum size) were recorded. All mice were killed when they had 125-mm 2 tumors. The observation period was until 60 d after injection.

Northern Blot Hybridization
RNA analyses were carried out using standard Northern techniques (Kiefer et al., 1994 b ). The probes were kindly provided by D. Belin (University of Geneva, Switzerland) (uPA), S. Strickland (State University of New York, Stony Brook) (tPA), and R. Bravo (Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ) (PAI-1).

Immunohistochemical Analysis and Immunocytochemistry
Tissue specimens were explanted, dissected, fixed overnight in 4% paraformaldehyde in PBS, and embedded in paraffin; 5m-thick sections were stained with hematoxylin and eosin. Adjacent sections were used for immunocytochemical reactions with antibody against factor VIII-related antigen/von Willebrand factor (vWF) (Dakopatts, Copenhagen, Den-mark). Some sections were processed using special staining techniques for fibrin. These included the Lendrum (Lendrum et al., 1962) and Mallory's phosphotungstic acid-hematoxylin (PTAH) techniques.

Zymography and Reverse Zymography
Confluent monolayers of End. cells in 35-mm tissue-culture dishes were washed twice with serum-free DME, and 1.5 ml serum-free DME containing 200 KIU/ml Trasylol (Bayer AG, Zurich, Switzerland) was added. 15 h later, cell extracts and culture supernatants were prepared and analyzed by zymography and reverse zymography as previously described (Vassalli et al., 1984;Montesano et al., 1990). Cell number was determined in a second set of dishes processed in parallel, and cell extract and culture supernatant samples were analyzed on the basis of cell equivalents.

In Vitro Fibrin Gel Assay
End. cells were seeded in suspension into 500-l fibrin gels at 1 ϫ 10 4 cells per gel. 500 l DME containing 10% FCS was added to each well above the fibrin gels. Fibrin gels were prepared as previously described . All experiments were performed in the absence or presence of 200 KIU/ml Trasylol, which was added both to the gel and to the medium at the time of embedding. Medium ( Ϯ Trasylol) was renewed every 2-3 d. Between 4 and 16 d after seeding, cultures were fixed in situ overnight in 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4, and photographed using a Phase Contrast-2 microscope (Nikon Inc., Garden City, NY).

PymT Efficiently Induces Vascular Tumors in the Absence of Individual Components of the PA/Plasmin System
To determine if PA/plasmin-dependent proteolytic activity is an essential requirement for the formation of vascular tumors by PymT, we inoculated newborn mice lacking uPA, tPA, utPA, PAI-1, or Plg with the PymT transducing retrovirus N-TkmT (Williams et al., 1988). The frequency of tumor formation was almost 100% in all mutant mice tested, and the time required for the development of lethal tumors (latency) varied only slightly (Table I). The high penetrance in Plg Ϫ / Ϫ mice was unexpected and no delay or inhibition of tumor formation was observed. Most mice were terminally ill within 8-10 d, and only the doublemutant pups lacking uPA and tPA succumbed after a slightly longer period of 12-14 d (Table I). These utPA Ϫ / Ϫ tumors retained the characteristic morphologic properties of PymT-induced tumors (Fig. 1, A and B ). All tumors appeared as blood-filled/hemorrhagic cysts lined by endothelial cells and were not different from similar tumors induced by PymT in control mice (Kiefer et al., 1994 b ). Representative sections through a typical lesion that formed in a double mutant (utPA Ϫ / Ϫ ) mouse as well as in a Plg Ϫ / Ϫ mouse are shown in Fig. 1. Staining with anti-vWF antibody shows endothelial cells lining these cysts (Fig. 1 B ). These results indicate that the PA/plasmin-dependent proteolytic status of the host is apparently not a critical determinant in the transformation of endothelial cells by PymT and in the formation of vascular tumors in newborn mice.

Derivation of End. Cell Lines Lacking uPA, tPA, utPA, and PAI-1
We have previously established transformed End. cell lines from tumor-bearing wild-type and various Src-kinase mutant mice, as well as by infection of primary endothelial cells by PymT in vitro (for review see Wagner and Risau, 1994;Pepper et al., 1997). To test whether the absence of components of the PA/plasmin system affects the capacity to establish permanent End. cell lines in vitro, we derived a number of independent cell lines from tumors induced in tPA-, uPA-, utPA-, and PAI-1-deficient mice ( Table I).
Each of these cell lines displayed the characteristic spindle-shaped, highly refractile morphology that is typical of endothelial cells (data not shown). The time required to establish the different mutant cell lines as homogeneously growing, characteristic End. cells was not significantly different using single knockout mice. However, the double mutant utPAϪ/Ϫ End. cells took three times as long to establish when compared with controls (data not shown). With the exception of ut1 and ut2, all End. cell lines exhibited roughly equivalent growth parameters in vitro as measured by cell doubling times after 01ف passages (Fig. 2). The proliferation rate of ut1 and ut2 cell lines was Ͻ50% of controls. All End. cell lines obtained expressed the endothelial-specific tyrosine kinase receptor Flk-1 (Fig. 3 A) and CD31/PECAM-1 (data not shown), confirming their endothelial origin. Most of these cell lines were clonal, since only one integration site for the provirus was detected by Southern blotting (data not shown).

Morphogenetic Behavior of Mutant End. Cells in Fibrin Gels
We have previously reported that End. cells form large cystlike structures lined by a monolayer of endothelial cells when embedded in three-dimensional fibrin gels in vitro . In the present study we have observed that although all six wild-type cell lines have the capacity to form cysts in fibrin gels, there is heterogeneity with respect to the size of the cysts and the frequency with which they form. In addition to cysts, the presence of narrow or ectatic tubes was observed in four out of six lines ( Fig. 4 and Table II). When grown in fibrin gels in the presence of Trasylol, a broad-spectrum serine protease inhibitor, cyst formation was inhibited and tubelike structures were observed in most wild-type End. cell lines ( Fig. 4 and data not shown).
Both uPAϪ/Ϫ cell lines (u23 and u24) retained their capacity to form medium to small cysts ( Fig. 4 and Table II). Cyst formation was completely inhibited in the presence of Trasylol, demonstrating the requirement for serine protease activity (data not shown). However, in contrast with wild-type cells, tube formation was never observed in the presence of Trasylol in either uPAϪ/Ϫ cell line; instead these cells either formed multicellular aggregates or remained as single isolated cells (data not shown). The two tPAϪ/Ϫ cell lines (t37 and t38) formed small cysts and occasional tube-like structures ( Fig. 4 and Table II). Cyst and tube formation could be prevented by addition of Trasylol to t37 but not to t38 cells (data not shown). In striking contrast with most of the cell lines described thus far, both utPAϪ/Ϫ cell lines (ut1 and ut2) completely lost their capacity to form cysts ( Fig. 4 and Table II). In addition, two clearly distinguishable cell populations could be identified: cells either organized into a network of cell cords or remained isolated as single rounded cells ( Fig. 4 and Table  II). Finally, PAI-1-deficient cells formed cysts, although in later passage p1 cells, rapid and massive fibrinolysis precluded the formation of either cysts or tubes ( Fig. 4 and Table II). p3 cells appeared to be heterogeneous: in early passage cells, two cell populations that either formed cysts or cell cords could be identified. Cyst formation (in p1 and p3 cells) and fibrinolysis (in late passage p1 cells) could be inhibited by Trasylol. Cell cord formation by ut1, ut2, and p3 cells was unaffected by addition of Trasylol (data not shown). These findings show that, contrary to what might have been expected, uPAϪ/Ϫ and PAI-1Ϫ/Ϫ cells do not display a decreased or increased capacity to form cysts. The observation that the capacity to form cysts was re-duced to a greater extent in tPAϪ/Ϫ cells than in uPAϪ/Ϫ cells suggests that, although both enzymes are capable of fibrinolysis in this system, tPA is more efficient.

Tumorigenic Potential of uPA-, tPA-, and utPA-deficient End. Cells in Adult Wild-Type Mice
To define a causal role for the PAs in End. cell-induced vascular tumors, we have used cells lacking uPA, tPA, and both uPA and tPA (utPA) for tumor formation studies in adult wild-type mice. We have used immunologically competent adult mice in these studies to avoid variability that may arise from the use of newborn mice with a developing immune system. Tumors were visible as small outgrowths in all mice injected with wild-type End. cells between day 4 and 7 and grew in size to 125 mm 2 by 11-18 d (Fig. 5 A). Histologically, these tumors consisted of a central hemorrhagic/necrotic core surrounded by an intense inflammatory cell infiltrate with neovascularization and very little fibrin deposition (Fig. 6, A and B). Similar results were obtained in mice injected with tPA-deficient End. cells, and there was no significant delay in either the onset or growth rates of these tumors. In contrast, tumor induction with uPAϪ/Ϫ End. cells showed incomplete penetrance and a delay in formation (Fig. 5 A). Only 50% of the mice developed tumors by days 11-14, and it took between 18 and 25 d for these tumors to reach maximum size. The remaining mice did not develop tumors for the entire 60-d observation period. Similarly, about two-thirds of the mice injected with End. cells deficient in both uPA and tPA developed tumors at an extremely slow rate (Fig. 5 A). Tumors were visible between 19 and 24 d after injection, and it took between 40 and 60 d to form tumors of maximum size. The remaining mice did not develop tumors. Histologically, these tumors were similar to those induced by wild-type cells, with the notable exception that extensive fibrin deposition was observed with the Lendrum and PTAH staining techniques (Fig. 6, E and F; data not shown). These results indicate that End. cell lines lacking only tPA activity are not inhibited in their capacity to form tumors. However, cell lines lacking uPA activity (both uPAϪ/Ϫ and utPAϪ/Ϫ) display a reduced efficiency in tumor formation in wild-type mice; when tumors do arise, their growth rate is significantly retarded, indicating that uPA activity of tumor cells is critical for efficient tumor growth.
To rule out the possibility that the observed differences in tumor formation rates were due to various degrees of histoincompatibility, we performed the tumor formation studies in immunoincompetent nude mice. After injection of wild-type and tPAϪ/ϪEnd. cells, tumors were visible around day 5 and grew in size to 125 mm 2 by 10-11 d (Fig.  5 A). In contrast, tumors were only visible between 7 and 14 d after injection with uPAϪ/Ϫ End. cells, and these tumors grew in size to 125 mm 2 by 18-22 d (Fig. 5 A). Similar results were obtained when utPAϪ/Ϫ End. cells were used, with tumors being visible between 10 and 14 d and reaching maximum size by 22-26 d (Fig. 5 A). These results in immunoincompetent mice confirm that the lack of tPA activity in End. cell lines does not affect their tumorigenic potential, whereas lack of uPA activity retards the tumorigenic potential of both uPAϪ/Ϫ and utPAϪ/Ϫ End. cell lines.

Tumor Induction by Wild-Type and Mutant End. Cells in Adult uPA-, tPA-, and utPA-deficient Mice
We next investigated if adult mice lacking uPA, tPA, or both utPA are susceptible to tumor induction by wild-type End. cells. Cells were inoculated into the various mutant mice and tumor growth rates were scored (Fig. 5 B). No significant delay was observed in the onset of tumor development in tPA-deficient mice when compared with wildtype controls, although the time taken to reach maximumsized tumors was slightly delayed (between 14 and 20 d in tPA-deficient mice as compared with 11-18 d in wild-type mice). However, tumor growth was delayed and variation Figure 5. Incidence and latency of tumor formation. End. cellinduced tumor formation was investigated using mutant End. cells in wild-type and nude mice (A), wild-type End. cells in mutant mice (B), and utPAϪ/Ϫ End. cells in mutant mice (C). All mice were inbred for more than six generations and injected with the following cell lines; the time taken for the onset of tumors (days after injection) and the latency period for the formation of 125-mm 2 tumors was recorded: wild-type, s1 (p35 and 40) and s2 (p23, 34 and 40); uPAϪ/Ϫ, u23 (p33) and u24 (p23); tPAϪ/Ϫ, t37 (p25 and 35) and t38 (p6 and 25); and utPAϪ/Ϫ, ut1(p20) and ut2 (p28 and 40). Observation period was for up to 60 d after injection. n, number of mice from at least two independent experiments. Figure 6. Morphology of End. cell-induced tumors in wild-type and mutant adult mice. (A) s1-induced subcutaneous tumor in a wildtype mouse after 3 d; B is the consecutive section stained with the Lendrum technique: note the absence of fibrin deposition, (C) b3induced subcutaneous tumor in a tPAϪ/Ϫ mouse, and (D) s2-induced tumor in a uPAϪ/Ϫ mouse after 3 d. Note the presence of a cen-in tumor formation rates was observed in uPA-deficient mice as follows: (a) only 3 out of 11 uPA-deficient mice developed tumors 12 to 14 d after injection, which grew to maximum size by day 27; (b) tumors were also visible in two other uPA-deficient mice between 12 and 14 d, but these tumors subsequently regressed; (c) the remaining six mice did not develop tumors throughout the entire 60-d observation period. Of the utPA-deficient mice injected with wild-type cells, only two out of four had visible tumors 18-20 d after injection, which then progressed to 125mm 2 tumors by days 40-50. The remaining two mice did not develop tumors for the entire observation period. Only four utPAϪ/Ϫ mice could be used in this study since most of these mutant mice, even uninjected, became ill as they grew older (Carmeliet et al., 1994). Histologically, tumors induced by wild-type cells in mutant mice (Fig. 6, C and D) were similar to those induced in wild-type mice (Fig. 6, A and B), both at early (3 d; Fig. 6, C and D) and late (27 d; Fig. 6 G) time points. However, tumors induced by wild-type cells in utPAϪ/Ϫ mice were consistently associated with extensive fibrin deposition, as seen using the Lendrum and PTAH staining techniques ( Fig. 6 H and data not shown). These results indicate that the host's PA status, and in particular the presence of uPA activity, plays a critical role in determining if proteolytically competent tumor cells can proliferate in vivo.
Since lack of uPA activity in both the host and the injected tumor cells affected the efficient formation of tumors, we next investigated the potential of utPAϪ/Ϫ End. cells to form tumors in mice lacking either uPA or tPA. Tumor formation by utPAϪ/Ϫ End. cells was inhibited in tPAϪ/Ϫ mice, since only three of the seven tPAϪ/Ϫ mice initially developed tumors between days 16 and 21; however, these tumors regressed by day 40 in all cases (Fig.  5 C). The remaining mice remained tumor free. utPAϪ/Ϫ End. cells did not form tumors in any of the uPAϪ/Ϫ mice during the entire observation period (Fig. 5 C). These results suggest that, although the lack of tPA alone has no significant effect on tumor formation, the uPA status of both the host and invading tumor cell has an important role in determining the progression of End. cell tumors.

Discussion
The specific formation of vascular tumors by PymT antigen provides an excellent system to study the molecular mechanisms underlying endothelial cell transformation. One of the components of this process is extracellular matrix degradation and invasion of surrounding tissues, which in the case of PymT results in a profound subversion of normal vasculature. In an attempt to analyze the role of the PA/plasmin system in PymT-mediated oncogenesis, we have investigated its ability to transform endothelial cells in mice lacking uPA, tPA, both uPA and tPA (utPA), or Plg. We found that the proteolytic status (PA/plasmin system) is not a critical determinant, and hence is not limiting for PymT-induced tumor formation. Moreover, the lack of either uPA or tPA activity does not affect the establishment of End. cells in vitro, although the combined loss of both PA activities leads to a reduced proliferation rate. Cyst formation by End. cells in fibrin gels in vitro could be correlated with altered proteolytic activity only in cells lacking uPA and tPA. However, End. cell-induced tumor formation in vivo, as opposed to tumors induced by PymT itself, is dependent on the proteolytic status (PA/plasmin system) of both the tumor cells and the host.
The finding that PymT was able to induce vascular tumors efficiently in the absence of uPA, tPA, utPA, and Plg indicates that the primary transformation process is independent of the PA/plasmin proteolytic status of the endothelial cells. This suggests that PymT-induced tumors can only be used as an in vivo assay for determining the role of this oncogene and associated signaling components in the initial transformation process. To date, the only signaling molecule thus identified is the Src-like kinase, Yes, since PymT could not efficiently transform endothelial cells in Yes-deficient mice in which the latency for tumor formation was also increased (Kiefer et al, 1994b).
Mutant End. cells lacking either uPA or tPA show no proliferation defect in culture. However, End. cell lines lacking both uPA and tPA require a longer time for establishment (three times longer compared with wild-type End. cells) and, when established, exhibit a marked reduction in cell proliferation in vitro. This proliferation defect is also exemplified in End. cell-induced tumor formation in vivo where cells lacking both PAs do not form tumors efficiently, and, when they do, the time taken to reach maximum size is about three times that seen with wild-type End. cells.
Assessment of the morphogenetic behavior of mutant End. cells in fibrin gels in vitro revealed that, although tPA appeared to be more efficient, the absence of either uPA or tPA alone did not render End. cells incapable of forming cysts, indicating that one PA is sufficient. However, combined loss of both uPA and tPA prevented cyst formation, clearly indicating that PA activity is required. The previous prediction that the "proteolytic balance," as represented by the ratio of PA:PAI-1, would predict the behavior of End. cells in fibrin gels ) therefore appears to hold true only when both PAs are absent. These findings indicate that the in vitro system only partially mimics the in vivo situation, and that the behavior of End. cells in fibrin gels is a poor predictor of End. cell tumorigenesis.
The incidence and rate of tumor formation in wild-type mice using End. cells lacking tPA activity were not signifi- cantly affected. However, there was a marked decrease in the number of mice that developed tumors when injected with uPAϪ/Ϫ and utPAϪ/Ϫ End. cells. When tumors did form in a subset of these mice, the onset was delayed and the time taken to develop to maximum size was increased. In some cases, mice developed tumors initially, but these subsequently regressed completely. The data from tumor formation studies with nude mice ruled out the possibility that various degrees of histoincompatibility may have arisen during the establishment or the maintenance of mouse strains and tumor cell lines, thus affecting the experimental outcome. While wild-type and tPAϪ/Ϫ cell lines still formed tumors with 100% penetrance in nude mice, both uPAϪ/Ϫ and utPAϪ/Ϫ End. cell lines showed a delay in the onset of tumor formation as well as an increase in the time taken to form 125-mm 2 tumors. Furthermore, we have previously generated numerous End. cell lines lacking functional c-src, fyn, and yes genes in the same genetic background, C57Bl/6 ϫ 129. These cell lines have been used in similar tumorigenicity studies, and no variations in the tumorigenic profile were found (Kiefer et al., 1994). Thus, the observed delay in End. cell-induced tumor formation and the consequent low incidence of tumor-bearing mice are not likely to result from genetic vari-ations but could be from inefficient matrix degradation and a reduction in migratory/invasive properties of the tumor cells, which in turn lead to inefficient tumor expansion. This observation that End. cell-induced tumors cannot be efficiently formed when endogenous uPA is lacking but are independent of tPA may reflect the longstanding notion that uPA is required in situations of cell migration, morphogenesis, and tissue remodeling, while tPA is primarily implicated in intravascular thrombolysis.
The finding that mice lacking uPA activity (both uPA and utPA null mice) do not display efficient formation of wild-type End. cell-induced tumors was surprising. There was a marked decrease in the formation of tumors, and, when formed, there was a delay in the onset and latency. This indicates that the host's proteolytic status is also critical for the efficient formation of these tumors. Although the exact role of the host's uPA activity in tumor formation is presently unclear, it could be envisaged that the uPA proteolytic mechanism has to involve concerted interactions between both the invading tumor cells and the host's matrix for efficient fibrinolysis and subsequent matrix degradation. Thus, the lack of uPA activity in either would result in inefficient colonization by the tumor cells, as was shown here.
In conclusion, our studies have revealed that the pathogenesis of PymT-induced and End. cell-induced tumors is inherently different. In the former, the PymT virus infects endothelial cells in an existing vascular tree without the requirement for the PA/plasmin system in the initial transformation process (Fig. 7 A). Therefore, PymT-mediated vascular tumor formation appears to be PA/plasmin independent. On the other hand, in End. cell-induced tumors, the injected cells first have to invade host tissues and the basement membrane of existing blood vessels to gain access to the circulation to integrate within host vessels. This invasive process, which is reminiscent of tumor cell intravasation into the vascular tree, is dependent on both the host's and invading tumor cell's PA/plasmin status (Fig.  7 B). In addition, proliferation is impaired in cells entirely lacking PA activity. Defects in both invasion and proliferation, both of which appear to be PA dependent, are therefore likely responsible for the reduction in End. cell-induced tumor growth. Whether other proteases such as the MMPs, which have been shown to be involved in vascular tumor formation in vivo (Taraboletti et al., 1995), may play a role in this process has to be investigated in future experiments.