extfieldsofstudy: Chemistry Medicine

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A New Tyrosine-phosphorylated 97-kDa Adaptor Protein Mediates Interleukin-2-induced Association of SHP-2 with p85-Phosphatidylinositol 3-Kinase in Human T Lymphocytes*

Interleukin (IL)-2 is a major cytokine that controls differentiation and proliferation of T lymphocytes. In this report we characterize an as yet unidentified 97-kDa protein that is a major tyrosine kinase substrate in IL-2-stimulated cells. pp97 was found to associate with the p85·p110 phosphatidylinositol 3-kinase complex, the Src homology 2 (SH2) domain-containing tyrosine phosphatase SHP-2, and the adaptor molecules CrkL and Grb2. We demonstrate that these interactions are directly mediated through the SH2 domains of CrkL, p85, and SHP-2 and through the SH3 domains of Grb2. pp97 was found to mediate the IL-2-induced interaction between p85 and both a phosphorylated and a non-phosphorylated form of SHP-2. In this study we show that pp97 behaves as a docking protein and associates with at least CrkL, p85, and SHP-2 in the same multimolecular complex. We thus characterized pp97 as a new tyrosine kinase substrate in human T lymphocytes which might play a central role in the regulation of several pathways activated by IL-2.

Interleukin (IL) 1 -2 is a major cytokine that controls the transition from G 1 to the S phase of the cell cycle and thus the proliferation of antigen-activated T lymphocytes. The high affinity IL-2 receptor (IL-2R) is composed of three subunits, ␣, ␤, and ␥, the latter being shared with the IL-4, -7, -9, and -15 receptors (1,2). Only the ␤ and ␥ chains have been demonstrated to contain a long enough cytoplasmic region to transduce signals, the ␣ subunit solely functioning as an affinity converter. Although the IL-2R does not contain intrinsic enzymatic activity, cell stimulation by IL-2 leads to an important and rapid increase of tyrosine phosphorylation mediated by non-receptor tyrosine kinases that are physically associated with the IL-2R␤ and -␥ chains. It has been shown that the first steps of IL-2 signaling depend upon the activation of several tyrosine kinases of the Jak, Syk, and Src families (3)(4)(5)(6). The IL-2 receptor itself becomes phosphorylated on tyrosine residues, which creates docking sites for a number of SH2 or phosphotyrosine binding domains containing signaling molecules (7,8). Among these, the adaptor protein Shc is recruited on a tyrosine residue located at position 338 of IL-2R␤ (9). Shc is then tyrosine-phosphorylated leading to the recruitment of the Grb2⅐Sos complex responsible for the activation of the mitogen-activated protein kinase pathway via Ras (10). Grb2 is a signaling adaptor molecule composed of one SH2 and two SH3 domains, the latter mediating the constitutive association with Sos, the guanosine nucleotide exchange factor for Ras (11).
IL-2 has also been reported to increase the activity of phosphatidylinositol 3-kinase (PI3K) (12)(13)(14)(15). PI3K activated in response to IL-2 is a type I PI3K composed of two subunits, a p85 regulatory subunit (p85) and a p110 catalytic subunit. p85 contains one SH3 domain, two proline-rich motifs, and two SH2 domains (16,17). Through its multiple possibilities of proteinprotein interactions, p85 is responsible for the recruitment of p110 to the membrane and its activation. The lipid kinase activity of PI3K leads to the phosphorylation of phosphatidylinositol on the D3 position of the inositol ring. PI3K was found to be critical for the transduction of anti-apoptotic and proliferative signals as it regulates Bcl-2 expression and c-Myc activation, possibly through a pathway involving Akt/PKB and p70S6 kinases (18,19). PI3K activation appears to result from conformational changes induced either by the phosphorylation of p85 (20) or by association of the p85-SH2 domains with tyrosine residues located in a YXXM (Y indicates tyrosine, X indicates any amino acid, and M indicates methionine) environment (21)(22)(23). This type of association recruits PI3K to the membrane receptor complex, a required and sufficient event to mediate activation, as recently emphasized by the observation that direct targeting of p110 to the cell membrane results in constitutive activation of PI3K (24). However, the mechanism of activation of PI3K in response to IL-2 remains to be determined since p85 tyrosine phosphorylation is not seen in most cells, in particular in the Kit 225 cell line used in this study, and since the IL-2 receptor does not contain a YXXM motif suggesting the involvement of intermediary molecules.
Adachi et al. (25) reported the tyrosine phosphorylation of the SH2 domain-containing tyrosine phosphatase SHP-2 in response to IL-2. SHP-2 (also called Syp, PTP1D, PTP2C, or SHPTP2) is a ubiquitously expressed 70 -72-kDa cytosolic phosphatase that contains two SH2 domains in tandem (26 -28). The mechanisms of SHP-2 activation may be quite similar to those described for PI3K as they seem to involve either tyrosine phosphorylation (29) or SHP-2 association with other tyrosine-phosphorylated proteins (30,31). SHP-2 has been described to transduce positive signals by binding directly to various receptor protein tyrosine kinases such as PDGF-, EGF-, or insulin receptors or to adaptor molecules such as IRS-1 or Grb2 leading to activation of Ras (32,33). IL-2-induced activation of SHP-2 was shown to be a Jak-independent mechanism probably mediated through Src kinases or a new type of kinase and is strictly dependent upon the expression of the IL-2R␤ and -␥ chains (25,34). The identification of SHP-2interacting proteins will be of interest in order to understand its function in IL-2 signaling and to identify potential downstream pathways.
We recently reported that IL-2 induces the tyrosine phosphorylation of the proto-oncogene product CrkL and regulates its association with p120Cbl and with the p85 regulatory subunit of PI3K (35). CrkL is a member of the Crk family, a group of SH2 and SH3 domains containing adaptor proteins, comprising the two splice products Crk-I and Crk-II and the product of a separate but related gene, CrkL (36 -38). During the course of this previous study we observed a p97-100-kDa protein associated with CrkL and phosphorylated on tyrosine residues in response to IL-2. In this report, we demonstrate that pp97 phosphoprotein participates in the formation of a multimolecular complex containing the signaling molecules CrkL, Grb2, SHP-2, and the PI3K regulatory p85 subunit. pp97 is one of the major early tyrosine kinase substrates in IL-2 signaling, and it directly associates with CrkL, p85, and SHP-2 through their respective SH2 domains. We also provide evidence that pp97 mediates the interaction between p85 and SHP-2 that occurs in response to IL-2. Given its characteristic features, as described here, it is possible that pp97 may be related to phosphoproteins that display similar molecular mass and migration properties and that have been recently described. Indeed, three independent reports have described 97-and 100-kDa phosphoproteins in Bcr-Abl-transformed cells or in response to M-CSF or IL-3 (39 -41). Similar to the pp97 described in our study, these phosphoproteins directly associate with SHP-2 and p85. The apparently central role of pp97 in the formation of a large and unique complex containing CrkL, SHP-2, and PI3K suggests that pp97 might exert an important function at the crossroad of three major signaling pathways triggered by IL-2.

MATERIALS AND METHODS
Cell Lines and Culture Conditions-The human T-cell chronic lymphocytic leukemia-derived, IL-2-dependent Kit 225 cell line was kindly provided by Dr. T. Hori (Kyoto University, Japan (42)). Cells were maintained in culture medium (RPMI 1640, 10% fetal calf serum, and antibiotics) supplemented with 0.5 nM recombinant human IL-2 (generously provided by P. Ferrara, Sanofi, France). Kit 225 cells were deprived of IL-2 by washing three times and resuspending the cells in culture medium without IL-2 at 5 ϫ 10 5 /ml for 48 h. For stimulation, IL-2-deprived Kit 225 cells were resuspended in culture medium at 2 ϫ 10 7 /ml and incubated at 37°C without (control) or with 1 nM recombinant IL-2 for various periods as specified. Cells were harvested by centrifugation and lysed at 5 ϫ 10 7 ml in cold 0.5% Triton lysis buffer (50 mM Tris, pH 7.6, 150 mM NaCl, 0.5% Triton X-100, 10 mM NaF, 1 mM EDTA, 1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM vanadate, and 5 g/ml each leupeptin, aprotinin, and pepstatin). Lysis was performed for 30 min on ice. Lysates were clarified by centrifugation at 15,000 ϫ g for 20 min at 4°C.
Human peripheral blood mononuclear cells were obtained from volunteer anonymous healthy donors. Mononuclear cells were separated by Ficoll-Hypaque gradient centrifugation. Non-adherent lymphocytes were stimulated with 1 g/ml phytohemagglutinin for 3-5 days and further expanded for 4 days in presence of 1 nM IL-2. T-cell blasts were then washed 3 times in order to remove IL-2 and incubated 18 h at 37°C in culture medium without IL-2. T-cell blasts were stimulated and lysed as described above for Kit 225 cells.
Reagents and Antibodies-Polyclonal antibodies against Cbl (Sc-170), Grb2 (Sc-422, used for immunoprecipitation), IL-2R␤ (Sc-1240), and CrkL (Sc-319) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal antibodies to Grb2 (G 16720, used in Western blot) were obtained from Transduction Laboratories (Lexington, KY). Anti-p85 antiserum (06-195) and anti-phosphotyrosine antibody 4G10 were obtained from Upstate Biotechnology Inc. (Lake Placid, NY). Anti-SHP2 sera were kind gifts from J. C. Cambier (National Jewish Institute, Denver, CO) and A. Kazlauskas (Schepens Eye Research Institute, Boston, MA). A polyclonal antibody was also raised in the laboratory against a GST fusion protein containing the N-terminal half (amino acid residues 1 to 343) of human p85␣. Isoform-specific antisera directed against p110 were a kind gift from S. Roche (CNRS EP612, Montpellier, France). The monoclonal anti-GST antibody was raised and purified in the laboratory. The Tyr-317 phosphopeptide (PSpYVNVPD) derived from the human Shc protein was provided by C. Garbay (INSERM Unit 266, Paris, France). The Tyr-740 phosphopeptide (GGpYMDMSKDESID) derived from the PDGF receptor was synthesized by Genosys. Endoglycosidase F/N-glycosidase F mix was obtained from Boehringer Mannheim.
Immunoprecipitation and Immunoblotting-Cell lysates (2.5 to 5 ϫ 10 7 cell equivalent) were precleared with protein G-Sepharose beads. 1-10 g of antibodies were added to the lysates. After 2 h of rocking at 4°C, 20 l of protein G-Sepharose beads were added, and incubation was continued for an additional 45 min. Immunoprecipitates were washed 5 times with cold lysis buffer, boiled in 2ϫ Laemmli Sample Buffer, and resolved by SDS-PAGE followed by immunoblotting. Immunodepletion experiments were performed by three successive rounds of immunoprecipitation using protein G-Sepharose beads-adsorbed antibodies, followed by one round with beads alone, and the final immunoprecipitation was performed on the supernatants as described above. For immunoblotting the PVDF membranes (Amersham Pharmacia Biotech) were blocked for 2 h at room temperature with either 5% non-fat dry milk or 3% bovine serum albumin in Tris-buffered saline (TBS), 0.5% Tween (TBS-T). Filters were washed 4 times in TBS-T and incubated for 1.5 h with optimal concentrations of primary antibodies diluted in TBS, 0.1% Tween. Following 4 additional washes in TBS-T, the filters were further incubated for 45 min with horseradish peroxidaseconjugated secondary antibodies (sheep anti-mouse Ig from Amersham Pharmacia Biotech or goat anti-rabbit Ig from Dako). Visualization was performed using Amersham Pharmacia Biotech ECL reagents and autoradiographic films.
GST Fusion Proteins-A plasmid encoding for GST-Grb2 full-length fusion protein was kindly provided by Dr. P.-O. Couraud (ICGM, Paris, France) and was used as a template to amplify the Grb2-SH2 domain in a BamHI-EcoRI-linearized PGEX-4T. For this purpose we used the following pair of primers: AGA CGG ATC CAT GAA ACC ACA TCC GTG GTT T (sense) and AGA CGA ATT CTC AGA CGT ATG TCG GCT GCT GTG G (antisense). GST-CrkL constructs (CrkL full-length, SH2, and SH3N) were a kind gift from B. J. Druker (Oregon Health Sciences University, Portland, OR). Plasmids encoding for the SHP-2 SH2 (Nterminal, C-terminal, and tandem) were kindly provided by J. C. Cambier. GST-Grb3-3, a naturally occurring form of Grb2 containing a nonfunctional SH2 domain, was provided by Rhone Poulenc Rorer. GST-p85 full-length protein and domains were constructed as follows. The cDNA for human p85␣ was oligo(dT) reverse-transcribed and amplified by polymerase chain reaction using the pair of primers CCG GAT CCA TGA GTG CTG AGG GGT A (sense) and CGG AAT TCT CAT CGC CTC TGC TGT GC (antisense) and cloned in the BamHI-EcoRI sites of PGEX-4T. The various p85 subdomains were amplified by polymerase chain reaction, and appropriate fragments were cloned into PGEX vectors (Amersham Pharmacia Biotech) to yield fusion proteins containing the SH3 domain (GST-SH3, amino acid residues 1-83), the BCR homology domain framed by the two proline-rich regions (GST-PBP, residues 73-336), and the two SH2-containing C-terminal half of p85 (GST-SH2(N ϩ C), residues 313-724). The primers and insertion sites are as follows. SH3 was inserted between BamHI-EcoRI sites of PGEX-4T (sense primer CCG GAT CCA TGA GTG CTG AGG GGT A, antisense primer CCG AAT TCA CGA GAT TTT TTT CCT TCC A); the PBP domain was inserted between the BamHI and EcoRI sites of PGEX4T (sense primer TAG CGC GGA TCC TAC GTA GAA TAT ATT GGA AGG, antisense primer CCG AAT TCA TCC CCA GTA CCA TTC AGC A); the SH2 (N ϩ C) was inserted between the BamHI and EcoRI sites of PGEX-2T (sense primer CCG GAT CCA AAC CTA CTA CTG TAG CC, antisense primer CGG AAT TCT CAT CGC CTC TGC TGT GC). All constructs were verified by DNA sequencing. GST fusion proteins were produced and purified as described previously (43).
GST Pull-down Experiments-5-10 g of purified GST fusion proteins were adsorbed onto glutathione-Sepharose beads (Amersham Pharmacia Biotech). Precleared lysates were rocked with coupled beads for 2 h at 4°C. The beads were washed 5 times with lysis buffer, boiled in 2ϫ Laemmli sample buffer, and bound proteins analyzed by Western blotting as described under "Immunoprecipitation and Immunoblotting." Far Western Blotting-Proteins were immunoprecipitated, separated, and transferred to PVDF membrane as described above. A first incubation of the membrane was performed with 5 nM of indicated fusion proteins in TBS, 0.1% Tween, 3% bovine serum albumin for 1.5 h. The membrane was washed 4 times and incubated with a monoclonal anti-GST antibody for 1.5 h. After 4 washes the membrane was further incubated 45 min with an anti-mouse antibody coupled to horseradish peroxidase. Vizualization was performed using Amersham Pharmacia Biotech ECL reagents and autoradiography.

RESULTS
pp97 Associates with p85, SHP-2, CrkL, and Grb2 in Response to IL-2-We have previously described the IL-2-dependent tyrosine phosphorylation of CrkL in T lymphocytes and its association with the proto-oncogene product Cbl and the p85 regulatory subunit of PI3K (35). CrkL also associated with several unidentified tyrosine-phosphorylated proteins including a relatively faint band at 70 -72 kDa, an 85-kDa protein that is constitutively phosphorylated in Kit 225 cells, and a major IL-2-inducible signal at 97-100 kDa. As shown in Fig. 1A pp97 is one of the major phosphoproteins coprecipitated with CrkL and p85 in an IL-2-dependent manner. pp97 is also present in Grb2 immunoprecipitates from IL-2-stimulated cell lysates, albeit at a lower intensity than in p85 immunoprecipitates. The p70 -72-kDa tyrosine-phosphorylated protein detected in p85 and Grb-2 immunoprecipitates (also observed in CrkL immunoprecipitates upon longer exposure) was formally identified as the cytosolic tyrosine phosphatase SHP-2 with a specific anti-SHP-2 antiserum. By using the same antibody we demonstrated that pp97 was also coprecipitated with SHP-2 ( Fig. 1A, lanes 7 and 8). Upon IL-2 stimulation, p85 and Grb2 become detectable in SHP-2 immunoprecipitates (Fig. 1A, lower panels, lanes 7 and 8). As reported recently, and probably due to a very low stoichiometry of the complexes described, CrkL was not detectable in any co-immunoprecipitation experiment (35). Unexpectedly, SHP-2 immunoblotting revealed two specific bands coprecipitated with p85, whereas only the slower migrating species was detected in association with Grb2. Comparison of the anti-phosphotyrosine (4G10) and SHP-2 immunoblots indicated that the slower migrating band corresponded to the tyrosine-phosphorylated form of SHP-2, whereas the faster migrating band was not tyrosine-phosphorylated and represented the major band revealed in SHP-2 immunoprecipitates. It is worth noticing that the amount of phosphorylated SHP-2, as seen by anti-phosphotyrosine Western blotting, is of similar intensity in SHP-2, p85, and Grb2 immunoprecipitates, suggesting that the majority, if not all, of phosphorylated SHP-2 associates with p85 and Grb2. In order to exclude the possibility that pp97 phosphorylation may represent a unique feature in Kit 225 cells we also evidenced the tyrosine phosphorylation of p85-associated pp97 in human T-cell blasts in response to IL-2 (Fig. 1B). Similar results were also obtained in the NKL cell line, a human IL-2-dependent natural killer cell line (not shown). As shown in the various co-immunoprecipitations and specific Western blot experiments performed, IL-2 stimulation thus induces the association of four important signaling molecules, namely p85, SHP-2, Grb2, and CrkL. The presence of pp97 in all these immunoprecipitates led us to investigate a possible adaptor function of this unidentified molecule.
Interactions of pp97 with p85 and SHP-2-We further studied the kinetics of phosphorylation of pp97 associated with p85 and SHP-2 in response to IL-2. As shown in Fig. 2A, pp97 becomes detectable by anti-phosphotyrosine immunoblotting in p85 and SHP-2 immunoprecipitates as soon as 30 s following IL-2 stimulation of Kit 225 cells. After 2 min of stimulation pp97 displays a slower migration profile and is detected as a higher molecular weight protein, whereas its level of tyrosine phosphorylation decreases steadily but remains detectable after 60 min of stimulation. As shown in Fig. 2A, pp97 appears to be the main common tyrosine-phosphorylated protein associated with p85 and SHP-2 in IL-2-stimulated cell lysates. Control blots with anti-p85 or anti-SHP-2 (not shown) confirmed the equal loading of all lanes, and also evidenced, as illustrated above in Fig. 1A, that p85 was associated with both a tyrosinephosphorylated form and a non-phosphorylated form of SHP-2. This result suggests that p85, which is not tyrosine-phosphorylated in IL-2-stimulated human T lymphocytes, may interact indirectly with SHP-2, and the presence of pp97 in both types of immunoprecipitates suggests that pp97 might function as an intermediary molecule between these two proteins.
To analyze further these interactions, GST fusion proteins containing the various domains of p85 and SHP-2 were used in pull-down experiments from IL-2-stimulated cell lysates (Fig.  2B). pp97 was easily detected using the p85(N ϩ C)SH2 con- Immunocomplexes were washed, separated on SDS-9% PAGE, and transferred on PVDF. The membrane was immunoblotted (IB) with anti-phosphotyrosine antibody (4G10), dehybridized, cut into different size regions, and reprobed with the specified antibodies. B, 5 ϫ 10 7 T-cell blasts were untreated (Ϫ) or stimulated 5 min with 1 nM IL-2 (ϩ). Cells were lysed and immunoprecipitations were performed with pre-immune serum (Pre-I) or anti-p85 (p85). Immunocomplexes were washed, separated, and transferred as above.
struct but did not bind to either p85 SH3 or p85 proline-rich region (PBP). It is not clear why in these experiments pp97 binding to the full-length construct appeared rather weak. This may be due to steric hindrance or conformational factors of the full-length protein that would favor interaction with the p85 tandem SH2 in vitro in the absence of the other p85 domains. In addition, another phosphoprotein, with a broad migration between 75 and 80 kDa also bound GST-p85(N ϩ C)SH2 as well as GST-p85 and was formally identified as the IL-2R␤ chain by specific Western blotting (Fig. 2B, left panel). As observed on this figure the presence of IL-2R␤ does not correlate with the level of pp97 indicating that IL-2R␤ is not likely to mediate the interaction between pp97 and p85. Conversely, pp97 probably does not represent the missing adaptor protein that should link p85 to the IL-2R. In agreement with our previously published data, the tyrosine-phosphorylated protein detected in the p85-SH3 pull-down was identified as p120Cbl (not shown). On the other hand, pp97 also associated with SHP-2 SH2 domains, and this interaction appears to require that both domains be present in tandem in the GST protein, as either SH2 domain alone was quite inefficient. Two additional phosphoproteins are detected that bind to SHP-2 SH2 and remain to be identified.
Since it has been suggested that p85 may be present in cells in molar excess to the p110 catalytic subunit, and may thus exist as a free form, we investigated whether pp97 could be associated with p85⅐p110 complexes. To address this question, immunoprecipitation experiments were performed with specific anti-p110␣ and ␤ antibodies (Fig. 3). pp97 was clearly detected in association with both p110␣ and p110␤, which indeed coprecipitated p85. This result demonstrates that pp97 associates with p85⅐p110 complexes and might therefore func-tion in the regulation of PI3K activity by IL-2.
p85-SHP-2 Interaction Is Mediated Through p85-SH2 Domain-The SH2-mediated interactions described above were further characterized by peptide competition assays in either p85 or SHP-2 immunoprecipitates. A phosphorylated tyrosine 740-containing peptide (YMDM) derived from the PDGF receptor, which competes with the p85-SH2 domains, was used in p85 or SHP-2 immunoprecipitates. A phosphorylated tyrosine 317-containing peptide derived from Shc (YVNV), which competes with the Grb2 SH2, provided a specificity control in these experiments. As shown in Fig. 4A, the PDGF-R phosphopeptide displaced pp97 and SHP-2 from a p85 immunoprecipitate. On the other hand, the PDGF-R phosphopeptide efficiently competed with p85 but did not decrease pp97 in SHP-2 immunoprecipitates (Fig. 4B). The apparent simplicity of the antiphosphotyrosine pattern, as compared with Fig. 1A, results from a shorter exposure being shown in Fig. 4 to better evidence the effects of peptide competition on pp97 association. Altogether these results show that p85 interacts with pp97 and SHP-2 through its SH2 domains recognizing tyrosine-phosphorylated residues probably located in the consensus YXXM environment. On the other hand the SHP-2-p85 interaction depends upon this YXXM consensus motif but is independent of the phosphorylation of both partners. Additionally, SHP-2-pp97 interaction is mediated via SHP-2 SH2 domains and is not affected by the phosphopeptides used in these experiments. These observations reinforce the hypothesis that p85 and SHP-2 might interact together through a common tyrosinephosphorylated intermediary molecule.
CrkL Associates with pp97 in Response to IL-2-We recently described the association of CrkL with multiple signaling molecules in response to IL-2 (35 and Fig. 1A). Kinetic experiments shown in Fig. 5 indicate that tyrosine phosphorylation of pp97 is detectable more rapidly in CrkL immunoprecipitates than in p85 or SHP-2 immunoprecipitates (Fig. 2A). It reaches a maximum between 0.5 and 2 min of stimulation, whereas the tyrosine phosphorylation of CrkL was maximum around 10 min. This observation would indicate that the presence of tyrosine-phosphorylated pp97 associated with CrkL is not dependent on the tyrosine phosphorylation of CrkL itself. It is of interest to note that CrkL immunoprecipitates also contain p120Cbl, as described previously, and that pp70 -72 SHP-2 protein associates with CrkL with a kinetics similar to that of pp97 (open arrowheads, Fig. 5A).
To characterize further the interactions between CrkL and pp97, CrkL SH2 and SH3 domains were used as GST fusion proteins in pull-down experiments (Fig. 5B). Multiple tyrosine- phosphorylated proteins are precipitated when using the fulllength GST-CrkL, with pp97 representing a major species that appears in IL-2-stimulated cell lysates. A number of these proteins parted between the SH2 and SH3 domains, but pp97 was detectable on both. The lower intensity of pp97 on the domains, as compared with the full-length CrkL, was observed in multiple experiments and suggests that cooperative interactions might occur between CrkL SH2 and SH3 domains for pp97 association (Fig. 5B). These experiments were also performed in the presence of 50 mM phenyl phosphate which should prevent recognition of phosphotyrosine residues by SH2 domains. Indeed, phenyl phosphate inhibited association of all phosphoproteins to the SH2 domain, including pp97, but unexpectedly also inhibited association to the SH3 domain (Fig. 5B,  lanes 9 and 10). Western blotting of the membrane with p85 was performed as a control and showed that the effect of phenyl phosphate was fairly specific as p85 association with CrkL SH3 domains was only reduced to a level consistent with that of the direct association with p85 proline-rich motifs as described previously (35). These results thus suggest that association of pp97 with CrkL may involve a possibly direct interaction with the SH2 domain, whereas association with the SH3 domain is likely to be indirect and mediated by a phosphotyrosine-dependent interaction with an other protein.
Association of pp97 with Grb2 Involves Grb2 SH3 Domains-As shown in Fig. 1A, pp97 was also observed in association with the adaptor molecule Grb2. pp97 associated with Grb2 was easily recognizable by its pattern of phosphorylation and migration, despite a relatively low signal intensity. The time-dependent phosphorylation of pp97 in Grb2 immunoprecipitates (Fig. 6A) appeared to be superimposable to the kinetics observed with CrkL (Fig. 5A). As shown in Figs. 1A and 6A, multiple phosphoproteins are present in Grb2 immunoprecipitates including p120Cbl, an unidentified pp75 and p52 and p46Shc as described previously. In addition a pp70 -72 band was detectable just below pp75 and was identified as SHP-2, as shown above in Fig. 1A, and by SHP-2 immunoblotting of the membrane (not shown).
Full-length GST-Grb2 was able to bind pp97, but unexpectedly, using GST fusion proteins of the Grb2 domains; the pp97-Grb2 interaction was found to be specifically mediated through the Grb2 SH3 domains (Fig. 6B). Indeed, pp97 was undetectable in pull-down experiments using a Grb2 SH2 GST fusion, whereas a GST-Grb3-3 fusion protein corresponding to a naturally occurring mutant form of Grb2 expressing a deleted, nonfunctional SH2 domain could coprecipitate pp97 in response to IL-2. Consistent with our previous findings, Grb3-3 also bound p120Cbl, seen as a doublet at approximately 115-120 kDa. Furthermore, it was observed that when the experiment was performed in the presence of 50 mM phenyl phosphate, which indeed was efficient in displacing the unidentified pp75 from the SH2 domain, pp97 association with Grb3-3 was unaffected, suggesting that this interaction was independent of tyrosine phosphorylation, and might be direct (Fig. 6B).
pp97 Interacts Directly with p85-, SHP-2-, and CrkL-SH2 Domains-At this stage of the study we have characterized pp97 as a protein that interacts with the SH2 domains of CrkL, p85, and SHP-2 and with the SH3 domains of CrkL and Grb2. We were then interested in determining which of these interactions was direct. For this purpose we turned to the far Western technique which is currently one of the most reliable approaches to evidence direct protein-protein interactions between bacterially expressed and whole cell lysate-derived proteins. As described under "Materials and Methods," these experiments were performed using a constant concentration of 5 nM for each recombinant protein, a concentration determined in preliminary experiments to give negative results with the GST moiety alone. Far Western experiments performed with the p85-SH2 domains fusion protein indicate that p85 binds directly to the tyrosine-phosphorylated pp97 present in p85, SHP-2, CrkL, or Grb2 immunoprecipitates (Fig. 7A). It has to be noticed that the level of the signals obtained in far Western blots followed exactly the level of signals obtained in antiphosphotyrosine blots (compare with Fig. 1A) with Grb2 giving the lowest intensity signal, thus providing an additional indication that pp97 as seen in far Western is probably the same protein that associates with all four molecules. SHP-2 immunoprecipitates contain the largest amount of pp97 and were therefore used to investigate whether GST-SHP2 or GST-CrkL-SH2 recognized pp97 in far Western experiments (Fig. 7, B and  C). The results indicate that both SHP-2 and CrkL could interact directly with pp97 through their SH2 domains. However, the CrkL SH2, which indeed recognized phosphorylated Cbl quite efficiently in Cbl immunoprecipitate from IL-2-stimulated cell lysates (Fig. 7C), gave a lower signal than SHP-2 SH2, possibly due to a lower affinity or to a smaller number of its putative phosphotyrosine target residues in pp97. In parallel experiments no direct interaction could be demonstrated using the Grb2 or CrkL SH3 domains (not shown).
Additionally, results obtained from the experiment shown in Fig. 7A indicated that p85-SH2 domains did not recognize either SHP-2 or CrkL in far Western. This observation is consistent with the hypothesis made from competition peptide experiments and suggests that interactions seen in immunoprecipitations are indirect.
pp97 Simultaneously Associates to p85, SHP-2, and CrkL-To investigate whether p85, SHP-2, and CrkL associate with pp97 in the same complex, we performed immunodepletion experiments using either anti-p85 or anti-SHP2 rabbit sera followed by a final CrkL immunoprecipitation. When p85 was depleted from stimulated lysates, the level of CrkL-associated pp97 was dramatically reduced (Fig. 8A), whereas the phosphorylated Cbl signal, migrating with the 112-kDa marker, was largely unaffected, as described previously (35). These experiments also evidenced that when p85 was depleted, the CrkL-associated SHP-2 became undetectable, suggesting that these three molecules are part of the same complex. Conversely, when SHP-2 immunodepletions were performed, pp97 association with CrkL was also decreased to a similar extent. In addition we have previously reported that CrkL and p85 interacted constitutively via the CrkL SH3 domain and that IL-2 stimulation induced an increased association involving tyrosine-phosphorylated residues. In the experiments shown here, the IL-2-mediated increased association of p85 with CrkL (Fig. 8B, lanes 9 and 10) was no longer detectable (lanes 12 FIG. 6. pp97 associates with the Grb2-SH3 domain. A, 5 ϫ 10 7 Kit 225 cells were stimulated for the indicated times with 1 nM IL-2, lysed, and immunoprecipitated (IP) with anti-Grb2 antibody. Immunoprecipitates were washed, separated on SDS-9% PAGE, transferred to PVDF membrane, and immunoblotted (IB) with anti-phosphotyrosine antibody (4G10). Equal loading of the gel was controlled by a Grb2 immunoblotting. B, lysates from 5 ϫ 10 7 Kit 225 cells stimulated for 5 min with IL-2 were incubated with 5 g of the indicated GST fusion proteins immobilized on glutathione-Sepharose beads. Where indicated (ϩ), the incubations were performed with 50 mM phenyl phosphate (PP). Precipitates were washed, separated, and revealed as described in A. verse 10) in the absence of pp97 that had been removed as a consequence of SHP-2 depletion, whereas the constitutive association remained unaffected (Fig. 8B, lanes 9 and 11). These results led us to conclude that pp97 may mediate the IL-2-dependent association between p85, SHP-2, and CrkL in a large complex.
pp97 Is Not Glycosylated-The typical migration profile displayed by pp97 may be due to post-translational modifications such as glycosylation. To investigate this point, in vitro deglycosylation experiments were undertaken using a mixture of endoglycosidase F/N-glycosidase F. This treatment was shown to be effective in deglycosylating the IL-2R␤. Indeed, the treatment of IL-2R␤ chain immunoprecipitates resulted in a shift of migration, and the immunoprecipitated proteins appeared to migrate as a lower apparent molecular weight species in comparison with untreated immunoprecipitates (Fig. 9). Under the same conditions however, the p85-associated pp97 did not reveal any modification of its migration profile, whereas the efficiency of deglycosylation can be evidenced by the faster migration pattern of deglycosylated immunoglobulin heavy chains. Taken together these results indicate that pp97 is probably not a N-glycosylated protein.

DISCUSSION
Binding of IL-2 to its high affinity receptor leads to a rapid and important increase in tyrosine kinase activity, responsible for the phosphorylation of various substrates which results in the activation of downstream effectors such as PI3K and SHP-2. In this report, we demonstrate that a common 97-kDa protein that is tyrosine-phosphorylated in IL-2-stimulated T lymphocytes coprecipitates with the PI3K p85⅐p110 complex, the tyrosine phosphatase SHP-2, and the SH2-SH3-containing adaptor molecules CrkL and Grb2. As evidenced by pull-down and far Western experiments, pp97 interacts directly with the SH2 domains of p85, SHP-2, and CrkL, suggesting that pp97 may be phosphorylated on multiple tyrosine residues located in the specific environments required for recognition by these SH2 domains. Additionally, pp97 was shown to bind to the SH3 domains of CrkL and Grb2, through interactions which seem to be mediated by different mechanisms as the CrkL-SH3-pp97 association can be disrupted by phenyl phosphate whereas the Grb2-SH3-pp97 interaction cannot. These results indicate that the CrkL SH3 domains do not directly recognize pp97 and that this interaction requires an intermediary molecule that itself associates with pp97 through a phosphotyrosine-dependent mechanism. On the other hand Grb2 interacts with pp97 independently of phosphotyrosine residues. This interaction may be direct, although the presence of an intermediary molecule between Grb2-SH3 domains and pp97 could not be excluded since far Western blotting with the Grb2-SH3 domains did not evidence a direct interaction with pp97. Furthermore, pp97 is only detected through its phosphorylation, and the lack of specific antibodies against pp97 does not allow investigations of a possible constitutive association of non-phosphorylated p97 with SH3 domains containing proteins.
Since the identity of pp97 has not yet been established, the question of its relationship with other known proteins can only be addressed indirectly. In particular, the recently described SIRP/ SHPS SHP-2-associated transmembrane glycoproteins share similar molecular mass and are heavily phosphorylated in response to growth factor stimulation (44 -46). In cell fractionation experiments, however, we found that pp97 is mainly observed in the cytosol and thus is unlikely to be a transmembrane protein (not shown). Furthermore, unlike the SIRP/SHPS-glycosylated transmembrane protein family, the migration profile of pp97 was not modified by treatment with N-glycosidase. Finally, pp97 is not recognized, in Kit 225 cells, by two different antibodies kindly provided by A. Ullrich against murine SIRP (not shown). Gab1, another adaptor protein involved in EGF and insulin signaling has also been recently identified that shares some properties with pp97 (47). In our investigations and in agreement with the data previously published by Holgado-Madruga et al. (47), Gab-1 migrates at a significantly higher apparent molecular mass, closer to 115 kDa, than pp97 does and appears poorly expressed in lymphoid tissues. Additional experiments clearly excluded the possibility that pp97 be one of the already known tyrosine kinase substrates involved in IL-2 signaling such as Vav or STAT5 (not shown).
Between 30 s and 2 min of IL-2 stimulation pp97 is maximally tyrosine-phosphorylated and is detected as a broad band of 90 -95 kDa apparent molecular mass and undergoes a migration shift after 2 min. Taken together with its association with the SH2 domains described above, this characteristic migration pattern is reminiscent of similar molecular weight phosphoproteins recently described in other systems (39 -41). Carlberg and Rohrschneider (39) and Craddock and Welham (41) described the tyrosine phosphorylation of an unidentified p100 protein associated with SHP-2 in response to M-CSF or IL-3, respectively, and Gu et al. (40) characterized a similar p97 phosphoprotein in Bcr-Abl-transformed and in IL-3-stimulated murine hematopoietic cells. It is thus possible that the pp97 described here, in human IL-2-stimulated cells, may be related to these. However, an answer to this question will have to wait until pp97 is identified or specific antibodies obtained.
As shown in several figures in this report, the tyrosine phosphorylation of pp97, as seen in anti-phosphotyrosine Western blotting, is maximum around the 2-min time point, then declines progressively. In parallel experiments we have also observed that when cells were stimulated by IL-2 in the presence of the phosphatase inhibitor pervanadate, the phosphorylation of pp97 was greatly increased (not shown). Although we did not address this question specifically, it is tempting to speculate that SHP-2, which associates with pp97, might be responsible for its dephosphorylation. Another characteristic feature of pp97 is a migration shift detectable after 2 min of stimulation. This shift is unlikely to be dependent on tyrosine phosphorylation since it persists while the amount of phosphotyrosine residues in pp97 decreases. The modifications of pp97 migration profile may be due to additional post-translational modifications such as ubiquitination or serine phosphorylation that require further investigation.
We also provide evidence, by peptide competition assays and immunodepletion experiments, that a large proportion of pp97 is simultaneously associated with CrkL, p85, and SHP-2. Furthermore, p85 and SHP-2 were found to associate directly with pp97 through their SH2 domains, whereas the interaction between p85 and SHP-2 is likely to depend upon an intermediary molecule. The observation that this p85-SHP-2 interaction is nevertheless disrupted by a pYXXM peptide, and thus depends upon the p85 SH2, and is only observed in the presence of pp97, led us to propose a hypothetical model (Fig. 10) in which pp97 mediates the interaction between p85 and SHP-2. The tyrosine phosphorylation of SHP-2 has been shown recently, but it is the first time that SHP-2 is reported to interact with other signaling molecules in response to IL-2. The function of SHP-2 tyrosine phosphorylation is not yet understood and may lead to SHP-2 activation or may be a required event to mediate protein-protein interactions. Regarding IL-2 signaling, it is not yet known if SHP-2 phosphatase activity is required or if it functions solely as an adaptor molecule. We provide evidence that p85 interacts with both a phosphorylated and a non-phosphorylated form of SHP-2 through a YXXM motif-dependent mechanism. YXXM motifs are present in SHP-2, but there are no indications as to whether they are phosphorylated or not in response to IL-2, and we have found no evidence that such motifs, if phosphorylated, are involved in p85/SHP-2 association. Rather the model would be consistent with our data showing that p85 can interact with SHP-2 independently of its tyrosine phosphorylation. This model also depicts the direct association of pp97 with CrkL and accounts for its presumed role in the association of CrkL with SHP-2. Since we have observed in far Western experiments that CrkL-SH2 did not bind pp97 very efficiently, CrkL may primarily associate with SHP-2 via a very indirect mechanism involving p85 as well as pp97, and the size of this complex probably explains at least in part the low amount of SHP-2 detectable in CrkL immunoprecipitates. On the other hand pp97 interacts with Grb2, and the scheme shown in Fig. 10 only illustrates the Grb2-SH3-pp97 interaction, as supported by our pull-down experimental data. However, Grb2 may also participate in this complex in several other ways, recognizing p85 proline motifs or phosphorylated SHP-2 (48, 49). 2 Grb2/SHP-2 association has already been reported in response to PDGF and was shown to mediate the recruitment of Grb2 to the PDGF receptor leading to the activation of Ras and the mitogen-activated protein kinase pathway (32,33). The scheme shown in Fig. 10 is based upon pull-down and immunodepletion experiments performed at 5 min following IL-2 stimulation. It is possible, although this has not been tested, that the complex behaves in a dynamic way, with a stoichiometry that could vary with time. Such an hypothesis would be consistent with our observation that at a later stimulation time pp97 appears to be present in larger amounts on p85 and SHP-2 than on CrkL and Grb2 immunoprecipitates.
The multiple protein interaction domains present in this 2 F. Gesbert, C. Guenzi, and J. Bertoglio, personal observations FIG. 10. Hypothetical model of pp97 interactions induced by IL-2. This model is a scheme showing protein interactions involving specific SH2 and SH3 domains based upon data described in this report. For SH2-mediated associations, the environment of phosphotyrosine is indicated referring to consensus binding sites reported in the literature although no sequence information on pp97 is yet available. See the "Discussion" for details. large complex provide several possible means by which it could be recruited to the cell membrane and the IL-2R. Although pp97 is largely a cytosolic protein, a significant proportion can be found in membrane fractions after IL-2 stimulation (not shown). It is of interest that PI3K activity has been reported to associate with IL-2 receptor in response to IL-2 despite the absence of YXXM motifs in either chains of the IL-2R (14). IRS-1 and IRS-2 proteins are large, heavily tyrosine-phosphorylated docking proteins primarily described in insulin receptor signaling (50). IRS-2, also called 4PS, was initially reported to be tyrosine-phosphorylated in response to IL-4 in hematopoietic cells. The tyrosine phosphorylation of these proteins has recently been described in response to stimulation of the cytokine receptors that share the common ␥ chain (i.e. IL-2, -4, -7, and -15) (51). Indeed, we observed a pp180 associated to p85 in response to IL-2 that may represent IRS-1 or IRS-2 (Fig. 1, lane  4 and Fig. 8A, lane 8). Additionally it has been demonstrated that IRS-2 may serve as an alternate pathway to activate PI3K in response to erythropoietin (52). Not withstanding the possible role of IRS-1 or IRS-2, it is hypothesized that pp97 may play a central role in the recruitment of PI3K to the IL-2 receptor or to the membrane. In in vitro experiments, fusion proteins containing the p85-SH2 domains precipitated a large amount of IL-2R␤. However, IL-2R␤ was not evidenced in p85 immunoprecipitates and pp97 was not detectable in IL-2R␤ immunoprecipitates (not shown). This may be related to relatively labile interactions due to indirect interactions or to a low stoichiometry of these complexes in vivo. Alternatively, PI3K may be recruited to the membrane, where its preferred substrate phosphatidylinositol 4,5-bisphosphate is located, via CrkL interactions with cytoskeletal proteins, which would be consistent with the rapid phosphorylation of the CrkL-associated pp97. Consistent with this is the recent observation that CrkL plays a central role in ␤ 1 integrin-mediated adhesion mechanisms and has been shown to tightly interact with focal adhesion-associated proteins (53).
Our data thus suggest that pp97 may represent a novel example of large docking proteins such as IRS-1, IRS-2, or Gab-1 and may play important functions in several of the signal transduction pathways activated by IL-2. The formal identification of pp97 or the generation of specific antibodies should help in understanding these functions. Furthermore, the identification of pp97 should help in providing new insights in the respective functions of CrkL and SHP-2 in IL-2 signaling.


s2fieldsofstudy: Biology Chemistry

text:
Evidence for the formation of dinor isoprostanes E1 from alpha-linolenic acid in plants.

The free radical oxidation of arachidonic acid is known to generate complex metabolites, termed isoprostanes, that share structural features of prostaglandins and exert potent receptor-mediated biological activities. In the present study, we show that alpha-linolenic acid can undergo a similar oxidation process, resulting in a series of isomeric dinor isoprostanes E1. E-ring dinor isoprostane formation from linolenate was found to be catalyzed by soybean lipoxygenase. The main enzymatic products were 13- and 9-hydroperoxylinolenate but in addition, two dinor isoprostane E1 regioisomers were formed with a yield of 0.31%. Identification and quantification of two dinor isoprostane E1 regioisomers in plant cell cultures was achieved by a negative chemical ionization gas chromatography-mass spectrometry method using [18O]dinor isoprostanes E1 as internal standards. Endogenous levels of these compounds were determined in four taxonomically distant plant species and found to be in the range of 4.5 to 60.9 ng/g of dry weight. Thus analogous pathways in animals and plants exist, each leading to a family of prostaglandin-like compounds derived from polyunsaturated fatty acids. It remains to be shown whether the dinor isoprostanes exert biological activities in plants as has been demonstrated for their C20 congeners in mammals.

In 1990, a series of prostaglandin (PG) 1 F2-like compounds termed isoprostanes F 2␣ (iPF 2␣ ) was discovered to be produced in abundance in humans by a free radical mechanism independent of the cyclooxygenase enzyme from arachidonic acid (1). Formation of these prostanoids proceeds through bicyclic endoperoxide intermediates that are reduced in vivo to isoprostanes F 2␣ . In addition, the unstable bicyclic endoperoxide intermediates (2) may also readily rearrange in an aqueous environment to isothromboxane (3) and isoprostanes of the D 2 and E 2 type (4). Four regioisomers for each of the D 2 , E 2 , and F 2 isoprostanes are formed from arachidonic acid, each of which is comprised of 16 stereoisomers (5,6). Interest in these molecules stems not only from the fact that they have been shown to provide a valuable index of free radical-induced lipid peroxidation in vitro and in vivo but also from the finding that isoprostanes E 2 and F 2 exert potent biological activities in mammals (6).
As early as the 1960s, it was shown by Nutgeren et al. (7) that prostaglandin-like compounds could be formed by autoxidation of 8,11,14-eicosatrienoic acid. Some years later Pryor et al. (8) autoxidized methyl linolenate and obtained mass spectra evidence for the formation of PGF-type products after reduction of the autoxidation mixture. Porter and co-workers prepared a single hydroperoxy fatty acid from ␥-linolenic acid and allowed it to react in the presence of oxygen and a free radical source. After reduction, GC-MS analysis indicated the presence of a number of PGF-like compounds, one of which was identical with an authentic sample of dinor-PGF 1␣ (9).
The mechanism of formation of dinor isoprostanes involves initial hydrogen abstraction from one of the two bisallylic methylene groups of ␣-linolenic acid at carbons 11 and 14. Two bicyclic endoperoxide regioisomeric families, each comprising 16 members, result from the subsequent reaction of the initial linolenate free radical with two oxygen molecules, where each of the isomers has a cyclopentane ring containing a cis-1,3endoperoxide moiety. Two alkyl chains extend from the cyclopentane ring, one containing the original carboxylic acid moiety, and the second chain containing the methyl terminus (see Fig. 1).
The stereochemical aspects of the formation of bicyclic endoperoxides from hydroperoxy methyl ␣and ␥-linolenate have been studied, and it was found that the side chains of the bicyclic endoperoxides are almost exclusively oriented cis similar to the C20 isoprostanes (10,11). This is in contrast to prostaglandins formed by cyclooxygenase in which the side chains are exclusively oriented trans.
The bicyclic endoperoxide moiety of the molecules easily rearranges in the presence of water to E-and D-ring compounds having a hydroperoxy group in the side chain, which in turn may be reduced in vitro or in vivo to dinor isoprostanes E 1 and D 1 (see Fig. 1; see Ref. 12 for a discussion of the nomenclature).
In the present study, we demonstrate that ␣-linolenic acid autoxidizes readily via the 12-and 13-hydroperoxyradical intermediate to dinor isoprostanes of the E-type in aqueous buffers, a reaction that can be catalyzed by soybean lipoxygenase. Dinor isoprostanes were prepared in the mg scale and characterized by mass spectroscopy and NMR. Analytical methods were developed that allowed us to quantitate the formation of the two dinor isoprostane E 1 (dinor-iPE 1 ) regioisomers in vitro by HPLC and GC-MS and to demonstrate that plants form dinor-iPE 1 in vivo. cherey and Nagel, Dü ren, Germany. Silica gel 60 (particle size 63-100 m) for column chromatography was from Merck. Oxygen-18 O 2 gas (99.1 atom % 18 O) was obtained from Isotec, Miamisburg, OH. Plant suspension cultures were obtained from the departmental culture collection and grown as described (13).
Preparation of 18 O-Labeled and Unlabeled Dinor Isoprostanes E 1 -Linolenic acid (6 g) was dissolved in chloroform, loaded on a silica column (10 g), and eluted with hexane/diethyl ether (9:1, v/v) to remove any contaminating oxygenated fatty acids. The eluate was taken to dryness in a 500-ml round bottom flask and dissolved in 12 ml of methanol/water (1:1, v/v). In the case of the labeling experiment, the flask was evaporated in vacuo and filled with 500 ml of oxygen-18 O 2 gas (99.1 atom % 18 O). After stirring the mixture for 7 days at room temperature, the autoxidation reaction was stopped by the addition of 6 g of triphenylphosphine in 50 ml of chloroform. After 30 min of incubation time, 12 ml of saturated NaCl solution was added. The organic phase was evaporated in vacuo, reconstituted in 60 ml of chloroform, and loaded on a silica column (30 g of silica gel 60). The remaining linolenic acid, monohydroperoxides, and triphenylphosphine were eluted with 600 ml of diethyl ether/acetic acid (99:1, v/v) and discarded. A crude mixture of [ 18 O]dinor-iPE 1 s labeled with three 18 O atoms was obtained by eluting the column with 500 ml of diethyl ether/acetone/acetic acid (7:3:0.1, v/v). The eluate containing an isomeric mixture of [ 18 O]dinor-iPE 1 s was quantitated by HPLC (see below) and used without further purification as the internal standard for GC-MS analysis. Unlabeled dinor-iPE 1 compounds were produced analogously by using 16 O 2 gas.
Analysis of Dinor Isoprostanes E 1 by HPLC-Aqueous samples containing dinor-iPE 1 were obtained from autoxidation of linolenic acid (5 mg) in methanol/water or LOX-catalyzed oxidation of linolenic acid (5 mg) in borate buffer (see below). For HPLC analysis, samples were spiked with 5 g of PGE 1 as internal standard. After the addition of 0.05% butylated hydroxytoluene (BHT) (w/v), the incubation mixture was acidified with citric acid to pH 3 and extracted with diethyl ether. The ether phase was taken to dryness under a stream of nitrogen and reconstituted in chloroform. The sample was incubated with 20 mg of triphenylphosphine to reduce peroxides to their corresponding hydroxy derivatives for 5 min and applied to a silica solid phase extraction column (500 mg). The column was washed with 9 ml of diethyl ether/ acetic acid (98:2, v/v) and 3 ml of chloroform/acetone/acetic acid (8:2:0.1, v/v), thereby removing remaining fatty acids and monohydroperoxides thereof. Any isoprostane-like compounds of the B-type, if present, will also be lost during this procedure. Dinor-iPE 1 compounds were eluted with 6 ml of diethyl ether/acetone/acetic acid (6:4:0.1, v/v). The eluate was taken to dryness, reconstituted in 100 l of a solution of 0.25% BHT (w/v) in methanol, and treated with 400 l of 1 M KOH for 10 min at 40°C. Under these conditions, dinor-iPE 1 and PGE 1 undergo basecatalyzed dehydration (14) to form the corresponding B 1 -ring compounds (see Fig. 2). The incubation mixture was acidified with citric acid to pH 3 and extracted with diethyl ether. The diethyl ether phase was taken to dryness and analyzed by HPLC. Dinor isoprostanes B 1 and PGB 1 were separated on a LiChrospher 100 reversed phase18 endcapped column (5-m particle size, 250 ϫ 4 mm, Merck) eluted with a mixture of acetonitrile/methanol/water/acetic acid with the ratio of 24: 28:48:0.01 (v/v) for 15 min and then with 30:35:35:0.01 (v/v) at a flow rate of 1 ml/min. UV spectra were recorded on a Hewlett Packard 1040A diode array detector. The chromatogram showed the presence of three strongly UV-absorbing compounds ( max at 278 nm) representing dinor-iPB 1 type I and type II and the derivative of the internal standard, PGB 1 (see Fig. 3). Because the dinor isoprostanes B 1 contain the same chromophore as PGB 1 , we used the molar absorption coefficient of PGB 1 (⑀ ϭ 30,000 cm -1 M -1 ) to quantitate the dinor-iPB 1 s.
In a series of preparative HPLC runs, the two peaks corresponding to dinor-iPB 1 type I and type II were collected and analyzed by twodimensional NMR, which revealed unambiguously their structures (data not shown).
Trace Analysis of Dinor Isoprostanes E 1 by Negative Ion Chemical Ionization (NCI) GC-MS-Cells (10 g) were harvested from plant cell suspension cultures 3 days after transfer into fresh medium by suction filtration, shock frozen in liquid nitrogen and suspended in 10 ml of a saturated NaCl solution in water containing 0.05% BHT (w/v), 20 mg of triphenylphosphine, and 0.2 ml of 1 M citric acid. As internal standard, a mixture of [ 18 O]dinor isoprostanes E 1 containing 100 ng of type I regioisomers and 92 ng of type II regioisomers was added. The frozen cells were broken up and extracted with 20 ml of diethyl ether using a high speed disperser. The ether phase was taken to dryness under a stream of nitrogen, reconstituted in 2 ml of chloroform, and further purified as described above. The sample obtained after the base-catalyzed dehydration of E-ring compounds containing the corresponding B-ring derivatives was reconstituted in chloroform and loaded on a silica solid phase extraction column. The column was washed with 3 ml of hexane/diethyl ether/acetic acid (2:1:0.03, v/v), and B-ring compounds were eluted from the column with 6 ml of diethyl ether/acetic acid (98:2, v/v). Because all contaminants that can be eluted from the silica column with diethyl ether/acetic acid (98:2, v/v) have been removed in the first step of purification, the diethyl ether/acetic acid (98:2, v/v) eluate of the second silica column represents a highly purified dinor-iPB 1 extract.
For NCI GC-MS analysis, dinor-iPB 1 s were converted to pentafluorobenzyl (PFB) ester and trimethylsilyl (TMS) ethers as described (5). NCI GC-MS analysis was carried out on a Finnigan SSQ 700 mass spectrometer. GC was performed using a 25-m, 0.25-m film thickness Optima 5-fused silica capillary column (Macherey and Nagel, Dü ren, Germany). The column temperature was programmed from 175°C to 285°C at 30°C/min and from 285°C to 300°C at 2.5°C/min and kept at 300°C for 5 min. The major ion generated in the NCI mass spectra of the PFB ester, TMS ether derivatives of unlabeled and 18 O-labeled dinor-iPB 1 is the m/z 379 and m/z 381 carboxylate anion (M-181), respectively. Quantitation was performed by comparison of the peak areas of unlabeled and labeled dinor-iPB 1 after correction for the fraction (1.5%) of unlabeled dinor-iPB 1 derived from the internal standard.
For electron impact ionization (EI) GC-MS, dinor isoprostanes B 1 were methylated in ethanol with an excess of ethereal diazomethane and silylated with N,O-bis(trimethylsilyl)trifluoroacetamide as described (5). High resolution EI mass spectra from the dinor-iPB 1 derivatives were obtained on a Finnigan MAT 95Q mass spectrometer.
Measurement of Conjugated Diene and Hydroperoxide Formation-Fatty acid peroxidation was assessed by two different approaches. Autoxidation of ␣-linolenic acid is accompanied by an increase in UV absorbance at 234 nm because of the formation of diene-conjugated peroxides. An aliquot of the reaction mixture was diluted into 100 mM borate buffer, pH 7.5, and from the absorption at 234 nm, the concentration of conjugated dienes was calculated by using the molar absorption coefficient of hydroperoxylinolenate of 23,000 cm -1 M -1 . To identify and quantitate different positional isomers of the monohydroperoxylinolenates formed by the action of LOX or during autoxidation, samples were treated with Na ϩ to reduce the hydroperoxides to their corresponding hydroxy derivatives. The mixture was then extracted with diethyl ether, taken to dryness, and analyzed by straight phase HPLC on a LiChrospher Si60 column (5-m particle size, 250 ϫ 4 mm, Merck) operated isocratically with hexane/isopropanol/acetic acid (98:2:0.05, v/v) at a flow rate of 1 ml/min. Hydroxylinolenates were quantitated at 234 nm and collected for GC-MS analysis. Purified isomers were hydrogenated, methylated, and silylated essentially as described (15,16). By EI GC-MS it was possible to assign the position of oxygenation unambiguously.
Hydroperoxides were determined by the ferrous oxidation-xylenol orange assay as described (17).  18 O atoms originally present in dinor-iPE 1 were lost during the base-catalyzed dehydration process.

Synthesis and Analysis of [ 18 O]Dinor
Lipoxygenase-catalyzed Formation of Two Series of Dinor Isoprostane E 1 Regioisomers from ␣-Linolenic Acid-We first analyzed linolenic acid for the presence of any autoxidized material. Interestingly, on a molar basis linolenic acid (puriss. p.a., stored under argon at Ϫ20°C) was found to be contaminated with 0.72% hydroperoxy compounds and 0.022% dinor isoprostanes E 1 type I and type II in a ratio of 1:0.8. A ratio of dinor-iPE 1 type I/type II of 1:0.75 Ϯ 0.2 is typically found when linolenic acid is autoxidized in water/methanol or without any solvent (see Fig. 3). Therefore, the dinor isoprostane E 1 concentration was checked in every batch of linolenic acid before use, and the results were corrected accordingly. Alternatively, linolenic acid was purified by silica column chromatography (see above) before use.
To assess the time course of the formation of linolenic acid oxidation products generated by the action of LOX, linolenic acid (60 mg, 216 mol) was dissolved in 1.2 ml of n-octane and incubated with 48 mg of soybean lipoxygenase in 600 ml of 100 mM sodium borate buffer, pH 7.5. The incubation mixture was sonicated for 1 min and stirred at room temperature. The linolenic acid hydroperoxide (HPOTE) concentration increased linearly over the first 2 h of incubation and then reached a plateau (see Fig. 4). Two h after onset of the reaction, an aliquot of the mixture was analyzed for hydroperoxy fatty acids by straight phase HPLC and GC-MS as described under "Experimental Procedures." The main compounds formed were 13-and 9-HPOTE in a ratio of 9:1. Only minor amounts of the 12-and 16-hydroperoxy isomers (Ͻ2%, see "Experimental Procedures" for details) were found, whereas 10-and 15-hydroperoxides were not detectable with HPLC.
In addition, dinor isoprostanes E 1 were quantitated by HPLC as their B 1 -type derivatives. The time course of product formation is shown in Fig. 4. Synthesis of linolenic acid hydroperoxides and dinor isoprostanes E 1 occurred almost in parallel. Both theoretically possible dinor-iPE 1 regioisomers were formed: type II compounds, which are derived from 13-HPOTE radicals, and type I compounds, which must have been formed via the 12-HPOTE radical (see Fig. 1). The ratio of type I to type II regioisomers detected at different time points after the addition of LOX linearly increased from 1:0.80 at zero time (ϭE 1 -dinor isoprostane contaminants of the starting material) to 1:1.5 after 5 h.
To explore the effect of LOX on dinor isoprostane E 1 formation in more detail, linolenic acid (5 mg, 18 mol) dissolved in 100 l of n-octane was incubated in 50 ml of borate buffer, pH 7.5, for 5 h in the presence and absence of LOX (4 mg) and analyzed for conjugated diene, HPOTE, and dinor-iPE 1 formation. Without LOX or with heat-inactivated LOX, only minute amounts of these products were formed (Fig. 5). Inclusion of the radical scavenger, BHT, resulted in a significant increase of conjugated diene compounds and hydroperoxides formed within 5 h (ϩ61%) but efficiently suppressed the formation of dinor-iPE 1 s (Ϫ78%), indicating that dinor-iPE 1 synthesis involves free radicals. To further ascertain that LOX catalytic activity rather than a LOX product is responsible for the generation of dinor-iPE 1 s, we incubated linolenic acid (18 mol) with 13-hydroperoxylinolenate (9 mol) and t-butylhydroperoxide (18 mol) in two separate experiments. The 13-HPOTE used for the assay was produced by LOX-catalyzed oxidation of linolenic acid, purified by reversed phase-HPLC, and quantitated before incubation with linolenic acid. However, both types of hydroperoxides had no significant effect on the autoxidation of ␣-linolenic acid as indicated by only minor formation of conjugated dienes, hydroperoxides, and dinor-iPE 1 s, suggesting that LOX activity is required to form significant amounts of dinor isoprostanes (Fig. 5).
In all these experiments, n-octane has been added to facilitate the dispersion of ␣-linolenic acid and to increase the yield of prostaglandin-like cyclization products. However, omission of octane in the standard incubation mixture still resulted in the formation of dinor-iPE 1 s but reduced the yield to 39%. Thus, a hydrophobic environment, i.e. n-octane droplets, increased the formation of dinor isoprostanes but was not strictly required. Increasing the pH of the borate buffer in the standard incubation to pH 9.6 also decreased the yield of dinor-iPE 1 s (49%), which may be because of the base-catalyzed conversion of E 1 into B 1 -type compounds, which will not be detected in our assay system.
Presence of Dinor Isoprostanes E 1 in Plants-Although the HPLC analysis method described above is rapid and allows the detection of 100 ng of dinor-iPE 1 in linolenate autoxidation mixtures, the sensitivity and specificity is not high enough to analyze these compounds in plants. For high sensitivity detection of these compounds, a NCI GC-MS method was employed. To identify unesterified dinor isoprostanes E 1 in plant cell cultures, cells of four taxonomically distant plant species were collected and shock frozen with liquid N 2 . Dinor-iPE 1 s were extracted, purified, converted into the corresponding dinor- Free radical-catalyzed autoxidation of ␣-linolenic acid leads to the formation of two bicyclic endoperoxide regioisomers. These intermediates are prone to undergo rearrangement to form E-and D-ring compounds, which are reduced to two regioisomers (type I and II), each of which theoretically comprises a mixture of eight racemic diastereomers. For simplicity, formation of D-ring compounds and stereochemical orientation is not indicated. Red., deduced. iPB 1 s, and quantified by NCI GC-MS as outlined under "Experimental Procedures." Nicotiana tabacum (Solanaceae), Glycine max (Fabaceae), Rauvolfia serpentina (Apocynaceae), and Agrostis tenuis (Poaceae) contained dinor isoprostanes E 1 of the type I and type II in the range of 4.5 -61 ng/g of dry weight (Table I). A representative NCI GC-MS chromatogram (R. serpentina) is shown in Fig. 6. Because of the low concentrations of unesterified dinor isoprostanes E 1 in plants, it was not possible to obtain EI mass spectra. To further ascertain the identity of these compounds, we prepared the dinor isoprostane B 1 t-butyldimethylsilyl ether and PFB ester derivatives from the plant dinor-iPE 1 s. These derivatives showed the same GC-MS retention time as authentic reference compounds and yielded identical quantitative data as obtained with the TMS ether derivatives (Fig. 6).

DISCUSSION
Analysis of isoprostanes in vitro and in vivo generally requires a multistep purification procedure. In addition, quantification is difficult because of the presence of up to 16 theoretically possible stereoisomers for each regioisomer, which cannot all be separated by GC or HPLC procedures. The conversion of E-ring into B-ring compounds has been shown to facilitate the work-up procedure for GC-MS analysis leading to highly purified dinor-iPB 1 samples (see "Experimental Procedures" for details). Furthermore, by this procedure the 32 stereoisomers of dinor-iPE 1 are converted into 2 racemic dinor-iPB 1 compounds (Fig. 2). On GC and HPLC, the two dinor-iPB 1 peaks are well separated ( Fig. 3 and 6) and represent an integrative measure for the type I and type II dinor-iPE 1 regioisomers, thereby enhancing the limit of detection considerably. In contrast to E-, D-and F-ring (dinor) isoprostanes, it is possible to detect B-ring compounds at a convenient wavelength (278 nm) that allows the straightforward quantification of these compounds by HPLC. In vitro synthesis of 18 O-labeled and unlabeled dinor isoprostanes yielded mg quantities of these compounds, which were ideally suited as internal standards and reference compounds for GC-MS analysis.
We speculated that bicycloendoperoxides might be generated more easily and in a regioisomeric pure form from autoxidation of 13(S)-HPOTE, which is the main product produced from linolenic acid by many plant lipoxygenases. Therefore, we incubated linolenic acid with soybean lipoxygenase, which catalyses the formation of predominately 13(S)-and 9(S)-HPOTE in a ratio of 9:1. Because 13-HPOTE but not 9-HPOTE can autoxidize to bicycloendoperoxides, we expected the formation of regioisomer II almost exclusively. Furthermore, the initially formed bicycloendoperoxides are prone to rapid rearrangement of the endoperoxide group to yield dinor isoprostanes E 1 in an aqueous environment with a t1 ⁄2 of less than 10 min as has been shown for the corresponding C20 endoperoxide PGH 2 (2). Surprisingly, we observed not only the formation of type II-but also of type I-dinor isoprostanes E 1 in lipoxygenase/linolenic acid incubations (Fig. 4 and 5). This result together with the finding that dinor-iPE 1 formation was strongly inhibited by BHT suggests that both regioisomers are formed predominately from linolenic acid by a free radical-catalyzed mecha-nism. However, some of the dinor isoprostane E 1 type II compounds were apparently formed from LOX-dependent free radical-catalyzed oxidation of 13-HPOTE, because the ratio of type II to type I dinor-iPE 1 s increased from 0.75 (typical ratio observed after linolenate autoxidation) to 1.5 during the first   It is well known that (especially under low oxygen tension) many lipoxygenase isozymes can generate singlet oxygen and free alkoxyl radicals that may leak out from the active site during the catalytic cycle, i.e. formation of 1-hydroperoxy-2(E),4(Z)-pentadiene systems from 1(Z),4(Z)-pentadiene structures, reviewed in Ref. 18. These radicals may attack the lipoxygenase leading to time-dependent suicide inhibition of the enzyme but are also responsible for so-called secondary reactions such as carotenoid and chlorophyll bleaching in vivo (19). Thus, BHT may have a dual effect by scavenging free radicals generated by LOX anaerobic cycling, namely preventing suicide inactivation of LOX resulting in higher HPOTE formation and suppression of dinor-iPE 1 formation. Hydroperoxides such as 13-HPOTE and t-butyl hydroperoxide have been shown to be inefficient inducers of linolenate autoxidation (Fig. 5). Therefore, a free radical source such as LOX appears to be required to catalyze dinor isoprostane formation. In plants, the respiratory chain, the photosynthetic apparatus, and cytochrome P450 enzymes may be important additional sources of reactive oxygen species.
Interestingly, synthesis of PGF 2 from arachidonic acid by soybean LOX-2 was reported by Axelrod and co-workers in 1978 (20). However, the reaction mechanism leading to the formation of apparently several prostaglandin-like compounds has not been clarified. Because the stereochemistry of the PGF 2 -like product(s) has not been established, it is possible if not likely that not PGF 2 but rather isoprostanes F 2 were formed, some of which exhibit similar EI GC-MS properties as PGF 2␣ .
To investigate if dinor isoprostanes can be generated in vivo, we analyzed different plant cell cultures for the presence of dinor isoprostanes E 1 . Linolenic acid is a major constituent of plant membranes where it is predominately esterified in glycolipids, phospholipids and neutral lipids. However, in contrast to mammalian cells, up to 0.25% of the total fatty acids are found unesterified as free acids in comparable high concentra-tions (about 8 g/g of dry weight (21)). Analysis of four different plant species revealed that both theoretically possible dinor isoprostane E 1 regioisomers (Fig. 6, Table I) are present as free acids at low concentrations. In analogy to the pathway in animals, it is likely that even higher levels of dinor isoprostanes are found esterified in membranes rather than unesterified in the cytosol and that dinor isoprostanes F 1␣ and D 1 may be also present in plants.
Isoprostanes, however, cannot generally be formed in plants because arachidonate is rarely found in significant quantities in higher plants (22). In contrast, dinor isoprostane formation from ␣-linolenate may not be significant in animals because of the relatively low concentration of this fatty acid in mammalian membranes.
Thus, parallel pathways in plants and animals are operative, each leading to a series of characteristic prostaglandin-like compounds. Dinor isoprostanes may be suitable markers of lipid peroxidation in plants or plant products similar to the isoprostanes, which have been demonstrated to represent specific and reliable markers of oxidant stress in the animal kingdom. In the future, it will be exciting to study if there is also a functional analogy between the mammalian isoprostane and the plant dinor isoprostane pathway. These are fundamental issues to be addressed in the future.