1 Oncogene 2003 Vol: 22(16):2493-2503. DOI: 10.1038/sj.onc.1206318

70Z/3 Cbl induces PLCγ1 activation in T lymphocytes via an alternate Lat- and Slp-76-independent signaling mechanism

The oncoprotein 70Z/3 Cbl signals in an autonomous fashion or through blockade of endogenous c-Cbl, a negative regulator of signaling. The mechanism of 70Z/3 Cbl-induced signaling was investigated by comparing the molecular requirements for 70Z/3 Cbl- and TCR-induced phospholipase C1 (PLC1) activation. 70Z/3 Cbl-induced PLC1 tyrosine phosphorylation required, in addition to the PLC1 N-terminal SH2 domain, the C-terminal SH2 and SH3 domains that were dispensable for TCR-induced phosphorylation. Deletion of the leucine zipper of 70Z/3 Cbl did not eliminate 70Z/3 Cbl-induced PLC1 phosphorylation, suggesting that blockage of c-Cbl via dimerization with 70Z/3 Cbl cannot fully explain 70Z/3 Cbl activating characteristics. The complete elimination of PLC1 phosphorylation required deleting the SH3 domain-binding region of 70Z/3 Cbl, consistent with 70Z/3 Cbl binding the PLC1 SH3 domain. 70Z/3 Cbl-induced PLC1 phosphorylation required Zap-70, as for the TCR, and the tyrosine kinase binding domain of 70Z/3 Cbl, which binds Zap-70, but did not require PLC1 binding to Lat, a crucial interaction in TCR-induced PLC1 phosphorylation. Furthermore, 70Z/3 Cbl-induced activation of NFAT, a PLC1/Ca2+-dependent transcriptional event, required Zap-70, but was independent of Slp-76, an adapter required for TCR-induced NFAT activation. These results suggest that 70Z/3 Cbl and PLC1 form a TCR-, Lat- and Slp-76-independent complex that leads to PLC1 phosphorylation and activation.

Mentions
Figures
Figure 1: Schematic representation of the main structural domains of c-Cbl and variants used in this study. The TKB domain is connected to the RING finger by a short linker domain (-linker). The proline-rich region (Pro) and tyrosine phosphorylation sites (pY) are potential binding sites for SH3 and SH2 domains. Ub/LZ refers to a region with homology to ubiquitin-associated domains and leucine zippers. An oncogenic form of Cbl contains a 17-amino acid deletion in the -linker and RING domains (70Z/3 del) Figure 2: Z/3 Cbl expression does not affect the kinetics of TCR-induced PLC1 phosphorylation. (a) Jurkat cells were cotransfected with HA-tagged WT PLC1 and pCI-neo or untagged 70Z/3 Cbl. Cells were left unstimulated or were stimulated at 37°C for the indicated time points with C305. The amount of phosphorylated PLC1 (pY PLC1(HA)) was determined by immunoblotting anti-HA immunoprecipitates with the antiphosphotyrosine antibody 4G10. Stripped blots were probed with anti-HA to determine the amount of immunoprecipitated PLC1 (PLC1(HA)). Expression of 70Z/3 Cbl was determined by immunoblotting whole-cell lysates with anti-Cbl. (b) The amount of phosphorylated PLC1 in (a) was normalized to the amount of PLC1 immunoprecipitated and the data expressed as the percentage of maximum response (4-min time point) Figure 3: The enhancement of basal and TCR-stimulated PLC1 phosphorylation by 70Z/3 Cbl requires the carboxyl-terminal region of 70Z/3 Cbl and does not occur with c/v-Cbl. (a) Jurkat cells were cotransfected with HA-tagged WT PLC1 and the indicated Cbl constructs or pCI-neo. Cells were left unstimulated or were stimulated with C305 for 2 min at 37°C. Anti-HA immunoprecipitates were immunoblotted with 4G10. The membrane was then stripped and reprobed with anti-HA. (b) Jurkat cells were cotransfected with HA-tagged WT PLC1 along with pCI-neo, full length HA-tagged 70Z/3 Cbl (70Z/3(FL)), or the indicated HA-tagged truncation mutants of 70Z/3 Cbl. Cells and anti-HA immunoprecipitates were treated as in (a) Figure 4: PLC1 phosphorylation in 70Z/3 Cbl-expressing Jurkat cells can occur independently of Lat and the SH2N domain of PLC1. Jurkat cells were cotransfected with the indicated HA-tagged PLC1 constructs along with either untagged 70Z/3 Cbl or pCI-neo. Cells and anti-HA immunoprecipitates were treated as in a Figure 5: The enhancement of basal and TCR-induced PLC1 phosphorylation by 70Z/3 Cbl requires the SH2C and SH3 domains of PLC1. Jurkat T cells were cotransfected with the indicated HA-tagged PLC1 constructs along with either untagged 70Z/3 Cbl or pCI-neo. Cells and anti-HA immunoprecipitates were treated as in a Figure 6: Z/3 Cbl-induced PLC1 phosphorylation requires the TKB domain but not the tyrosine phosphorylation sites of 70Z/3 Cbl. (a) Jurkat T cells were cotransfected with HA-tagged WT PLC1 and the indicated untagged 70Z/3 Cbl constructs or pCI-neo. Cells and anti-HA immunoprecipitates were treated as in a. Whole-cell lysates were immunoblotted for anti-Cbl to confirm 70Z/3 Cbl expression. (b) The amount of phosphorylated PLC1 in (a) was normalized by the amount of anti-HA reactivity Figure 7: Z/3 Cbl-induced NFAT activation in Jurkat cells is independent of PTEN and occurs in SupT1 cells. (a) Jurkat T cells were cotransfected with pCI-neo or 70Z/3 Cbl, and PTEN or PTEN C/S. At 24 h after transfection, cell lysates were immunoblotted with anti-phospho-AKT (pAKT, Ser473). (b) Jurkat cells were transfected with an NFAT-luciferase reporter construct, PTEN or PTEN C/S, and pCI-neo or 70Z/3 Cbl-HA, as indicated. At 24 h after transfection, the cells were immunblotted for PTEN expression (left panel) or were incubated with medium alone or 10 ng/ml PMA plus 1 M ionomycin for an additional 5 h (right panel). These cells were then lysed and assayed for luciferase activity. Data are expressed as the percentage of maximum activation observed with PMA plus ionomycin. Shown are the means.e.m. of triplicates. (c) Left panel: SupT1 cells were cotransfected with HA-tagged WT PLC1 and pCI-neo or untagged 70Z/3 Cbl. The amount of phosphorylated PLC1 in unstimulated cells was determined by immunoprecipitating with anti-HA, followed by immunoblotting with an anti-phosphotyrosine antibody. The membrane was then stripped and probed with anti-HA. Whole cell lysates were immunoblotted with an anti-Cbl antibody to confirm 70Z/3 Cbl expression. Right panel: Jurkat and SupT1 cells were transfected with an NFAT-luciferase construct reporter construct, and pCI-neo or 70Z/3 Cbl-HA as indicated, and assayed for basal NFAT activity as in (b) Figure 8: Zap-70 is required for 70Z/3 Cbl-induced PLC1 phosphorylation. The Zap-70-deficient Jurkat T cell line P116 (Zap-70- Jurkat) or P116 cells stably reconstituted with Zap-70 (Zap-70+ Jurkat) were cotransfected with HA-tagged WT PLC1 and untagged 70Z/3 Cbl or pCI-neo. Cells and anti-HA immunoprecipitates were treated as in a. Expression of HA-tagged PLC1 in different cell lines was confirmed by stripping the membrane and probing with anti-HA. 70Z/3 Cbl expression was determined by immunoblotting whole-cell lysates with anti-Cbl Figure 9: Z/3 Cbl-induced NFAT activation requires Zap-70 but is independent of Slp-76. (a) The Zap-70-deficient Jurkat T cell line P116 and a P116-derived clone stably reconstituted with Zap-70, or (b) the Slp-76 deficient Jurkat line J14 and J14 cells reconstituted with Slp-76 were transfected with 70Z/3 Cbl or pCI-neo along with an NFAT-luciferase reporter. Cells were then treated and analysed as in a and b. Also shown in (c) is an anti-phosphotyrosine Western blot analysis of anti-HA immunoprecipitates from unstimulated or C305-stimulated, Slp-76-deficient or reconstituted cells transfected with HA-tagged PLC1 and untagged 70Z/3 Cbl (left panel). The amount of immunoprecipitated PLC1 was determined by stripping and reprobing with anti-HA. The expression of 70Z/3 Cbl was determined by immunoblotting the whole cell lysates with an anti-Cbl antibody
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References
  1. Andoniou CE, Thien CBF and Langdon WY.. EMBO J., 13, 4515-4523 , (1994) .
    • . . . It should also be noted that 70Z/3 Cbl-induced in vitro transformation is quantitatively and qualitatively more pronounced than that observed with c/v-Cbl (Andoniou et al., 1994) . . .
  2. Bartkiewicz M, Houghton A and Baron R.. J. Biol. Chem., 274, 300887-330895 , (1999) .
    • . . . It is also possible that the signaling and oncogenic activity of 70Z/3 Cbl requires its ability to dimerize with endogenous c-Cbl via the C-terminal leucine zipper, resulting in blockade of the physiologic inhibitory function of c-Cbl (Bartkiewicz et al., 1999; van Leeuwen et al., 1999a,1999b). . . .
    • . . . It has been suggested that cell activation by 70Z/3 Cbl can be explained by a direct blockade of endogenous c-Cbl through heterodimerization (Bartkiewicz et al., 1999; van Leeuwen et al., 1999a,1999b) . . .
  3. Boerth NJ, Sadler JJ, Bauer DE, Clements JL, Gheith SM and Koretzky G.. J. Exp. Med., 192, 1047-1058 , (2000) .
    • . . . During TCR signaling, Lat, once phosphorylated, acts as a scaffold that recruits PLC1 via a direct interaction with the SH2N domain of PLC1 and indirectly via a ternary complex involving phospho-Lat binding to Gads, an interaction of Gads with Slp-76 (Zhang et al., 1998; Boerth et al., 2000), and a constitutive interaction of Slp-76 with the SH3 domain of PLC1 (Yablonski et al., 2001) . . .
  4. Bunnell SC, Henry PA, Kolluri R, Kirchhausen T, Rickles RJ and Berg LJ.. J. Biol. Chem., 271, 25646-25656 , (1996) .
    • . . . Interestingly, in addition to Slp-76, Itk has been shown to bind c-Cbl (Bunnell et al., 1996), a property that is likely to be shared by 70Z/3 Cbl. . . .
  5. Chang JS, Noh DY, Park IA, Kim MJ, Song H, Ryu SH and Suh PG.. Cancer Res., 57, 5465-5468 , (1997) .
    • . . . Does 70Z/3 Cbl-induced PLC activation play a role in the transformation process? PLC1 overexpression has been associated with transformation in NIH 3T3 and 3YL fibroblasts (Chang et al., 1997; Smith et al., 1998) and PLC1 expression and activity are elevated in several human tumors (Johnson et al., 1994; Noh, 1994; Lee et al., 1995) . . .
  6. DeBell KE, Stoica BA, Veri MC, Di Baldassarre A, Miscia S, Graham LJ, Rellahan BL, Ishia M, Kurosaki T and Bonvini E.. Mol. Cell. Biol., 19, 7388-7398 , (1999) .
    • . . . The assembly of HA-tagged bovine PLC1 in pCI-neo (WT PLC1(HA)) and the site-directed mutagenesis of the SH2N, SH2C, and SH3 domains have also been previously described (Stoica et al., 1998; DeBell et al., 1999) . . .
  7. Fournel M, Davidson D, Weil R and Veillette A.. J. Exp. Med., 183, 301-306 , (1996) .
    • . . . Since Zap-70 interacts with the TKB domain of Cbl (Fournel et al., 1996; Lupher et al., 1996,1997), we utilized a 70Z/3 Cbl TKB domain mutant (G306E 70Z/3 Cbl, Figure 1) . . .
  8. Graham LJ, DeBell KE, Veri M, Stoica B, Mostowski H, Bonvini E and Rellahan B.. FEBS Lett., 470, 273-280 , (2000) .
    • . . . Accumulating evidence indicates that c-Cbl negatively regulates signaling by growth factor receptors by mediating the multiubiquitination and degradation of the receptors (Sawasdikosol et al., 2000). c-Cbl has also been demonstrated to negatively regulate the activation of the nonreceptor protein tyrosine kinase, Syk, and that of phospholipase C1 (PLC1) (Ota and Samelson, 1997; Graham et al., 2000). . . .
    • . . . We have previously reported that, contrary to wild-type c-Cbl that inhibits antigen receptor-induced PLC1 activation and PLC1/Ca2+-dependent transcriptional events (Graham et al., 2000; Yasuda et al., 2000), ectopic expression of an oncogenic form of c-Cbl, the 70Z/3 Cbl mutant, leads to augmented basal and TCR-induced PLC1 signaling . . .
    • . . . The oncogenic c-Cbl variant 70Z/3 Cbl has been shown to induce the phosphorylation and activation of PLC1 (Graham et al., 2000) . . .
    • . . . We and others have previously established that 70Z/3 Cbl can induce activation of both the Ras and PLC1 pathways in T cells (van Leeuwen et al., 1999a,1999b; Graham et al., 2000) . . .
    • . . . Consistent with this hypothesis, we have found that c-Cbl overexpression in T cells negatively regulates TCR-induced PLC1 activation, albeit it did not affect PLC1 phosphorylation (Graham et al., 2000) . . .
    • . . . The assembly of Cbl and 70Z/3 Cbl in pCI-neo (pCI-Cbl, pCI-70Z/3) has been previously described (Rellahan et al., 1997; Graham et al., 2000) . . .
  9. Graham LJ, Stoica BA, Shapiro M, DeBell KE, Rellahan B, Laborda J and Bonvini E.. Biochem. Biophys. Res. Commun., 249, 537-541 , (1998) .
    • . . . Furthermore, PLC1 and 70Z/3 Cbl interact in T cells in a constitutive manner (data not shown), consistent with our previous finding that c-Cbl is constitutively bound to the SH3 domain of PLC1 (Graham et al., 1998) . . .
  10. Irvin BJ, Williams BL, Nilson AE, Maynor HO and Abraham RT.. Mol. Cell. Biol., 20, 9149-9161 , (2000) .
    • . . . Interestingly, reconstitution of PLC1-deficient Jurkat cells with an SH3 domain-negative mutant of PLC1 was shown to induce increased transcriptional activity in T cells (Irvin et al., 2000), suggesting that this domain interacts with a negative regulatory protein, such as c-Cbl . . .
  11. Joazeiro CAP, Wing SS, Huang H, Leverson JD, Hunter T and Liu Y-C.. Science, 286, 309-312 , (1999) .
    • . . . 70Z/3 Cbl differs from c-Cbl only in a 17-amino acid deletion that disrupts the linker -helix and the RING finger domain, structures that contribute to the E3 ubiquitin-ligase activity of c-Cbl (Joazeiro et al., 1999; Sawasdikosol et al., 2000; Zheng et al., 2000) . . .
  12. Johnson MD, Horiba M, Winnier AR and Arteaga CL.. Hum. Pathol., 25, 146-153 , (1994) .
    • . . . Does 70Z/3 Cbl-induced PLC activation play a role in the transformation process? PLC1 overexpression has been associated with transformation in NIH 3T3 and 3YL fibroblasts (Chang et al., 1997; Smith et al., 1998) and PLC1 expression and activity are elevated in several human tumors (Johnson et al., 1994; Noh, 1994; Lee et al., 1995) . . .
  13. Kane LP, Lin J and Weiss A.. Curr. Opin. Immunol., 12, 242-249 , (2000) .
    • . . . T-cell receptor (TCR)-induced PLC1 activation is controlled by nonreceptor protein tyrosine kinases, including Lck and Zap-70 (Williams et al., 1998,1999; Kane et al., 2000), the phosphorylation of the adapters Lat and Slp-76 (Yablonski et al., 1998; Zhang et al., 1999a,1999b), and the tyrosine phosphorylation of PLC1 itself (Todderud et al., 1990; Kim et al., 1991). . . .
  14. Kim HK, Kim JW, Zilberstein A, Margolis B, Kim JG, Schlessinger J and Rhee SG.. Cell, 65, 435-441 , (1991) .
    • . . . T-cell receptor (TCR)-induced PLC1 activation is controlled by nonreceptor protein tyrosine kinases, including Lck and Zap-70 (Williams et al., 1998,1999; Kane et al., 2000), the phosphorylation of the adapters Lat and Slp-76 (Yablonski et al., 1998; Zhang et al., 1999a,1999b), and the tyrosine phosphorylation of PLC1 itself (Todderud et al., 1990; Kim et al., 1991). . . .
  15. Lee SJ, Lee SD, Park JG, Kim CM, Ryu SH and Suh PG.. J. Biol. Chem., 270, 16378-16384 , (1995) .
    • . . . Does 70Z/3 Cbl-induced PLC activation play a role in the transformation process? PLC1 overexpression has been associated with transformation in NIH 3T3 and 3YL fibroblasts (Chang et al., 1997; Smith et al., 1998) and PLC1 expression and activity are elevated in several human tumors (Johnson et al., 1994; Noh, 1994; Lee et al., 1995) . . .
  16. Liu Y-C and Altman A.. Cell. Signal., 10, 377-385 , (1998) .
    • . . . The proto-oncoprotein c-Cbl is involved in the regulation of signaling initiated by several different receptor types and acts principally as a negative regulator of signal transduction (Liu and Altman, 1998; Thien and Langdon, 2001). c-Cbl is a complex adapter with multiple, diverse, protein interaction domains (Figure 1) . . .
  17. Lupher ML, Rao N, Eck MJ and Band H.. Immunol. Today, 20, 375-382 , (1999) .
    • . . . The structure of c-Cbl includes a tyrosine kinase binding (TKB) domain, a RING finger domain, which confers ubiquitin E3 ligase activity and recruits E2 ubiquitin-conjugating enzymes, numerous potential SH3 and SH2 domain-binding motifs, as well as an ubiquitin-associated domain and a leucine zipper (Lupher et al., 1999; Thien and Langdon, 2001) . . .
  18. Lupher ML, Reedquist KA, Miyake S, Langdon WY and Band H.. J. Biol. Chem., 271, 24063-24068 , (1996) .
    • . . . Since Zap-70 interacts with the TKB domain of Cbl (Fournel et al., 1996; Lupher et al., 1996,1997), we utilized a 70Z/3 Cbl TKB domain mutant (G306E 70Z/3 Cbl, Figure 1) . . .
  19. Lupher Jr ML, Songyang Z, Shoelson SE, Cantley LC and Band H.. J. Biol. Chem., 272, 33140-33144 , (1997) .
    • . . . Since Zap-70 interacts with the TKB domain of Cbl (Fournel et al., 1996; Lupher et al., 1996,1997), we utilized a 70Z/3 Cbl TKB domain mutant (G306E 70Z/3 Cbl, Figure 1) . . .
  20. Neumeister EN, Zhu Y, Richard S, Terhorst C, Chan AC and Shaw AS.. Mol. Cell. Biol., 15, 3171-3178 , (1995) .
    • . . . This includes the possibility that Zap-70 may function upstream of other kinases required for PLC1 activation, including Itk (Shan and Wange, 1999), or as an adapter that mediates the translocation of 70Z/3 Cbl to the proximity of the TCR (Neumeister et al., 1995), favoring the association of 70Z/3 Cbl with other regulatory proteins. . . .
  21. Noh DY, Lee YH, Kim YI, Ryu SH, Suh PG and Park JG.. Cancer, 73, 36-41 , (1994) .
    • . . . Does 70Z/3 Cbl-induced PLC activation play a role in the transformation process? PLC1 overexpression has been associated with transformation in NIH 3T3 and 3YL fibroblasts (Chang et al., 1997; Smith et al., 1998) and PLC1 expression and activity are elevated in several human tumors (Johnson et al., 1994; Noh, 1994; Lee et al., 1995) . . .
  22. Ota Y and Samelson LE.. Science, 276, 418-420 , (1997) .
    • . . . Accumulating evidence indicates that c-Cbl negatively regulates signaling by growth factor receptors by mediating the multiubiquitination and degradation of the receptors (Sawasdikosol et al., 2000). c-Cbl has also been demonstrated to negatively regulate the activation of the nonreceptor protein tyrosine kinase, Syk, and that of phospholipase C1 (PLC1) (Ota and Samelson, 1997; Graham et al., 2000). . . .
  23. Rellahan BL, Graham LJ, Stoica B, DeBell KE and Bonvini E.. J. Biol. Chem., 272, 30806-30811 , (1997) .
    • . . . A CD3-high Jurkat T-cell line was as previously described (Rellahan et al., 1997) . . .
    • . . . The assembly of Cbl and 70Z/3 Cbl in pCI-neo (pCI-Cbl, pCI-70Z/3) has been previously described (Rellahan et al., 1997; Graham et al., 2000) . . .
  24. Rellahan BL, Jensen JP and Weissman AM.. J. Exp. Med., 180, 1529-1534 , (1994) .
    • . . . All transfections were performed by electroporation with the indicated amounts of DNA as previously described (Rellahan et al., 1994). . . .
  25. Rhee SG and Bae YS.. J. Biol. Chem., 272, 15045-15048 , (1997) .
    • . . . PLC1 catalyzes the hydrolysis of phosphatidylinositol (4,5)-bisphosphate to inositol (1,4,5)-trisphosphate and diacylglycerol, which control calcium mobilization and protein kinase C activation, respectively (Rhee and Bae, 1997) . . .
  26. Sawasdikosol S, Pratt JC, Meng W, Eck MJ and Burakoff SJ.. Biochem. Biophys. Acta, 1471, M1-M12 , (2000) .
    • . . . Accumulating evidence indicates that c-Cbl negatively regulates signaling by growth factor receptors by mediating the multiubiquitination and degradation of the receptors (Sawasdikosol et al., 2000). c-Cbl has also been demonstrated to negatively regulate the activation of the nonreceptor protein tyrosine kinase, Syk, and that of phospholipase C1 (PLC1) (Ota and Samelson, 1997; Graham et al., 2000). . . .
    • . . . We have previously reported that, contrary to wild-type c-Cbl that inhibits antigen receptor-induced PLC1 activation and PLC1/Ca2+-dependent transcriptional events (Graham et al., 2000; Yasuda et al., 2000), ectopic expression of an oncogenic form of c-Cbl, the 70Z/3 Cbl mutant, leads to augmented basal and TCR-induced PLC1 signaling (Graham et al., 2000). 70Z/3 Cbl and WT Cbl differ in a 17-amino acid deletion in 70Z/3 Cbl (aa 366–382) that abrogates E3 ubiquitin-ligase activity (Figure 1) (Sawasdikosol et al., 2000), suggesting that the E3 function is required for c-Cbl to negatively regulate PLC1 . . .
    • . . . 70Z/3 Cbl differs from c-Cbl only in a 17-amino acid deletion that disrupts the linker -helix and the RING finger domain, structures that contribute to the E3 ubiquitin-ligase activity of c-Cbl (Joazeiro et al., 1999; Sawasdikosol et al., 2000; Zheng et al., 2000) . . .
  27. Schaeffer EM and Schwartzber PL.. Curr. Opin. Immunol., 12, 282-288 , (2000) .
    • . . . Previous studies have established a role for the Tec kinases in the phosphorylation and activation of PLC1 (Schaeffer and Schwartzber, 2000) . . .
  28. Shan X, Czar MJ, Bunnel SC, Liu P, Liu Y, Schwartzberg PI and Wange RL.. Mol. Cell. Biol., 20, 6945-6957 , (2000) .
    • . . . Since Jurkat T cells lack PTEN, there is a constitutive high amount of D3-phosphoinositides, which results in a constitutive membrane localization of the Tec kinase Itk (Shan et al., 2000) . . .
    • . . . The pSRa-Flag-PTEN and pSRa-Flag-PTEN C124S (PTEN C/S) were as described previously (Shan et al., 2000) . . .
  29. Shan X and Wange RL.. J. Biol. Chem., 274, 29323-29330 , (1999) .
    • . . . This includes the possibility that Zap-70 may function upstream of other kinases required for PLC1 activation, including Itk (Shan and Wange, 1999), or as an adapter that mediates the translocation of 70Z/3 Cbl to the proximity of the TCR (Neumeister et al., 1995), favoring the association of 70Z/3 Cbl with other regulatory proteins. . . .
  30. Smith MR, Court DW, Kim H, Park JB, Rhee SG and Kung H.. Carcinogenesis, 19, 177-185 , (1998) .
    • . . . Does 70Z/3 Cbl-induced PLC activation play a role in the transformation process? PLC1 overexpression has been associated with transformation in NIH 3T3 and 3YL fibroblasts (Chang et al., 1997; Smith et al., 1998) and PLC1 expression and activity are elevated in several human tumors (Johnson et al., 1994; Noh, 1994; Lee et al., 1995) . . .
  31. Stoica B, DeBell KE, Graham L, Rellahan BL, Alava MA, Laborda J and Bonvini E.. J. Immunol., 160, 1059-1066 , (1998) .
    • . . . We have previously established that TCR-induced PLC1 tyrosine phosphorylation is predominantly dependent upon an intact N-terminal SH2 (SH2N) domain and independent of the C-terminal SH2 (SH2C) domain (Stoica et al., 1998) or the SH3 domain (data not shown) . . .
    • . . . In the absence of 70Z/3 Cbl expression, TCR-induced PLC1 phosphorylation was, as previously reported (Stoica et al., 1998), dramatically decreased by the lack of a functional SH2N domain (Figure 4, compare lanes 2 and 6) . . .
    • . . . The assembly of HA-tagged bovine PLC1 in pCI-neo (WT PLC1(HA)) and the site-directed mutagenesis of the SH2N, SH2C, and SH3 domains have also been previously described (Stoica et al., 1998; DeBell et al., 1999) . . .
    • . . . Cells were lysed as previously described (Stoica et al., 1998) . . .
  32. Su YW, Zhang Y, Schweikert J, Koretzky GA, Reth M and Wienands J.. Eur. J. Immunol., 29, 3702-3711 , (1999) .
    • . . . Slp-76 plays a role in TCR-induced PLC1 activation (Yablonski et al., 1998), possibly by recruiting the Tec kinase Itk (Su et al., 1999) . . .
  33. Thien CB and Langdon WY.. Nat. Rev., 2, 294-305 , (2001) .
    • . . . The proto-oncoprotein c-Cbl is involved in the regulation of signaling initiated by several different receptor types and acts principally as a negative regulator of signal transduction (Liu and Altman, 1998; Thien and Langdon, 2001). c-Cbl is a complex adapter with multiple, diverse, protein interaction domains (Figure 1) . . .
  34. Thien CBF, Walker F and Langdon WY.. Mol. Cell, 7, 355-365 , (2001) .
    • . . . In support of multiple mechanisms, recent evidence suggests that the inability of 70Z/3 Cbl to mediate the multiubiquitination and degradation of growth-factor receptors is not the sole basis for its transforming abilities (Thien et al., 2001). . . .
  35. Todderud G, Wahl MI, Rhee SG and Carpenter G.. Science, 249, 296-298 , (1990) .
    • . . . T-cell receptor (TCR)-induced PLC1 activation is controlled by nonreceptor protein tyrosine kinases, including Lck and Zap-70 (Williams et al., 1998,1999; Kane et al., 2000), the phosphorylation of the adapters Lat and Slp-76 (Yablonski et al., 1998; Zhang et al., 1999a,1999b), and the tyrosine phosphorylation of PLC1 itself (Todderud et al., 1990; Kim et al., 1991). . . .
  36. Tsygankov AY, Teckchandani AM, Feshchenko EA and Swaminathan G.. Oncogene, 20, 6382-6402 , (2001) .
    • . . . There are also examples, such as the c-Met receptor and the interleukin 4 receptor signaling pathways, where c-Cbl positively regulates signal transduction (for a complete review see Tsygankov et al., 2001), suggesting that c-Cbl can, under specific conditions, enucleate a productive activation complex. . . .
  37. van Leeuwen JEM, Paik PK and Samelson LE. (1999a). Mol. Cell. Biol., 19, 6652-6664 , .
    • . . . It is also possible that the signaling and oncogenic activity of 70Z/3 Cbl requires its ability to dimerize with endogenous c-Cbl via the C-terminal leucine zipper, resulting in blockade of the physiologic inhibitory function of c-Cbl (Bartkiewicz et al., 1999; van Leeuwen et al., 1999a,1999b). . . .
    • . . . Although the 7X 70Z/3 Cbl mutant shows no detectable tyrosine phosphorylation either constitutively or in response to TCR cross-linking (van Leeuwen et al., 1999a,1999b), it did induce similar levels of PLC1 phosphorylation compared to WT 70Z/3 Cbl (Figure 6a and b) . . .
    • . . . As previously reported (van Leeuwen et al., 1999a,1999b), Zap-70 was required for 70Z/3 Cbl-induced NFAT activity (Figure 9a) . . .
    • . . . We and others have previously established that 70Z/3 Cbl can induce activation of both the Ras and PLC1 pathways in T cells (van Leeuwen et al., 1999a,1999b; Graham et al., 2000) . . .
    • . . . It has been suggested that cell activation by 70Z/3 Cbl can be explained by a direct blockade of endogenous c-Cbl through heterodimerization (Bartkiewicz et al., 1999; van Leeuwen et al., 1999a,1999b) . . .
    • . . . PLC1, however, does not appear to be a direct substrate of Zap-70 (Veri et al., 2001) and no increase in Zap-70 phosphorylation or kinase activity has been observed in Jurkat cells expressing 70Z/3 Cbl (van Leeuwen et al., 1999a,1999b; Zhang et al., 1999a,1999b), making it unlikely that 70Z/3 Cbl alters the kinase activity of Zap-70 . . .
    • . . . The G306E, 7X (Y674F, Y700F, Y731F, Y735F, Y774F, Y869F, Y871F), and 1–840 mutants of 70Z/3 Cbl in pSX were a gift from Dr L Samelson (Laboratory of Cellular and Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA) (van Leeuwen et al., 1999a,1999b) . . .
  38. van Leeuwen JEM, Peik PK and Samelson LE. (1999b). J. Biol. Chem., 274, 5153-5162 , .
    • . . . It is also possible that the signaling and oncogenic activity of 70Z/3 Cbl requires its ability to dimerize with endogenous c-Cbl via the C-terminal leucine zipper, resulting in blockade of the physiologic inhibitory function of c-Cbl (Bartkiewicz et al., 1999; van Leeuwen et al., 1999a,1999b). . . .
    • . . . Although the 7X 70Z/3 Cbl mutant shows no detectable tyrosine phosphorylation either constitutively or in response to TCR cross-linking (van Leeuwen et al., 1999a,1999b), it did induce similar levels of PLC1 phosphorylation compared to WT 70Z/3 Cbl (Figure 6a and b) . . .
    • . . . As previously reported (van Leeuwen et al., 1999a,1999b), Zap-70 was required for 70Z/3 Cbl-induced NFAT activity (Figure 9a) . . .
    • . . . We and others have previously established that 70Z/3 Cbl can induce activation of both the Ras and PLC1 pathways in T cells (van Leeuwen et al., 1999a,1999b; Graham et al., 2000) . . .
    • . . . It has been suggested that cell activation by 70Z/3 Cbl can be explained by a direct blockade of endogenous c-Cbl through heterodimerization (Bartkiewicz et al., 1999; van Leeuwen et al., 1999a,1999b) . . .
    • . . . PLC1, however, does not appear to be a direct substrate of Zap-70 (Veri et al., 2001) and no increase in Zap-70 phosphorylation or kinase activity has been observed in Jurkat cells expressing 70Z/3 Cbl (van Leeuwen et al., 1999a,1999b; Zhang et al., 1999a . . .
    • . . . The G306E, 7X (Y674F, Y700F, Y731F, Y735F, Y774F, Y869F, Y871F), and 1–840 mutants of 70Z/3 Cbl in pSX were a gift from Dr L Samelson (Laboratory of Cellular and Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA) (van Leeuwen et al., 1999a,1999b) . . .
  39. Veri M-C, DeBell KE, Seminario M-C, DiBaldassarre A, Reischl I, Rawat R, Graham L, Noviello C, Rellahan BL, Miscia S, Wange RL and Bonvini E.. Mol. Cell. Biol., 21, 6939-6950 , (2001) .
    • . . . PLC1, however, does not appear to be a direct substrate of Zap-70 (Veri et al., 2001) and no increase in Zap-70 phosphorylation or kinase activity has been observed in Jurkat cells expressing 70Z/3 Cbl (van Leeuwen et al., 1999a,1999b; Zhang et al., 1999a,1999b), making it unlikely that 70Z/3 Cbl alters the kinase activity of Zap-70 . . .
  40. Wange RL and Samelson LE.. Immunity, 5, 197-205 , (1996) .
    • . . . In T cells, the PLC1-regulated Ca2+ mobilization controls Ca2+-dependent transcriptional events that govern cellular responses, such as cytokine production (Wange and Samelson, 1996) . . .
  41. Williams BL, Irvin BJ, Sutor SL, Chini CCS, Yacyshyn E, Wardenburg JB, Dalton M, Chan AC and Abraham RT.. EMBO J., 18, 1832-1844 , (1999) .
    • . . . T-cell receptor (TCR)-induced PLC1 activation is controlled by nonreceptor protein tyrosine kinases, including Lck and Zap-70 (Williams et al., 1998,1999; Kane et al., 2000), the phosphorylation of the adapters Lat and Slp-76 (Yablonski et al., 1998; Zhang et al., 1999a . . .
    • . . . Since Zap-70 is also required for TCR-induced PLC1 phosphorylation (Williams et al., 1998,1999), one can speculate that Zap-70 itself is responsible for the phosphorylation of PLC1 . . .
  42. Williams BL, Schreiber KL, Zhang W, Wange RL, Samelson LE, Leibson PJ and Abraham RT.. Mol. Cell. Biol., 18, 1388-1399 , (1998) .
    • . . . T-cell receptor (TCR)-induced PLC1 activation is controlled by nonreceptor protein tyrosine kinases, including Lck and Zap-70 (Williams et al., 1998,1999; Kane et al., 2000), the phosphorylation of the adapters Lat and Slp-76 (Yablonski et al., 1998; Zhang et al., 1999a,1999b), and the tyrosine phosphorylation of PLC1 itself (Todderud et al., 1990; Kim et al., 1991). . . .
    • . . . Additional evidence for a Zap-70 requirement in the effect of 70Z/3Cbl on PLC1 phosphorylation was obtained by expressing WT PLC1-HA together with either 70Z/3 Cbl or pCI-neo in the Zap-70-deficient Jurkat cell line P116 (Williams et al., 1998), and comparing the effect to that observed in P116 cells stably reconstituted with Zap-70 . . .
    • . . . Since Zap-70 is also required for TCR-induced PLC1 phosphorylation (Williams et al., 1998,1999), one can speculate that Zap-70 itself is responsible for the phosphorylation of PLC1 . . .
    • . . . The Zap-70 negative (P116) and reconstituted cell lines were from Dr RT Abraham (Duke University, Durham, NC, USA) (Williams et al., 1998) . . .
  43. Yablonski D, Kadlecek T and Weiss A.. Mol. Cell. Biol., 21, 4208-4218 , (2001) .
    • . . . During TCR signaling, Lat, once phosphorylated, acts as a scaffold that recruits PLC1 via a direct interaction with the SH2N domain of PLC1 and indirectly via a ternary complex involving phospho-Lat binding to Gads, an interaction of Gads with Slp-76 (Zhang et al., 1998; Boerth et al., 2000), and a constitutive interaction of Slp-76 with the SH3 domain of PLC1 (Yablonski et al., 2001) . . .
    • . . . If Slp-76 also interacts with PLC1 SH3 domain (Yablonski et al., 2001), however, this observation stands in conflict with the positive role of Slp-76 in PLC1 regulation (Yablonski et al., 1998), in that an SH3 domain-negative PLC1 mutant would be expected to be deficient in activation potential . . .
  44. Yablonski D, Kuhne MR, Kadlecek T and Weiss A.. Science, 281, 413-416 , (1998) .
    • . . . T-cell receptor (TCR)-induced PLC1 activation is controlled by nonreceptor protein tyrosine kinases, including Lck and Zap-70 (Williams et al., 1998,1999; Kane et al., 2000), the phosphorylation of the adapters Lat and Slp-76 (Yablonski et al., 1998; Zhang et al., 1999a,1999b), and the tyrosine phosphorylation of PLC1 itself (Todderud et al., 1990; Kim et al., 1991). . . .
    • . . . Slp-76, a Zap-70 substrate, is an adapter protein that is required for PLC1 phosphorylation and activation in T cells (Yablonski et al., 1998) . . .
    • . . . Slp-76 plays a role in TCR-induced PLC1 activation (Yablonski et al., 1998), possibly by recruiting the Tec kinase Itk (Su et al., 1999) . . .
    • . . . If Slp-76 also interacts with PLC1 SH3 domain (Yablonski et al., 2001), however, this observation stands in conflict with the positive role of Slp-76 in PLC1 regulation (Yablonski et al., 1998), in that an SH3 domain-negative PLC1 mutant would be expected to be deficient in activation potential . . .
    • . . . The Slp-76 deficient (J14-v-29) and reconstituted (J14-76-11) clones as well as the anti-TCR antibody C305 were from Dr A Weiss (UCSF, San Francisco, CA, USA) (Yablonski et al., 1998) . . .
  45. Yasuda T, Maeda A, Kurosaki M, Tezuka T, Hironaka K, Yamamoto T and Kurosaki T.. J. Exp. Med., 191, 641-650 , (2000) .
    • . . . We have previously reported that, contrary to wild-type c-Cbl that inhibits antigen receptor-induced PLC1 activation and PLC1/Ca2+-dependent transcriptional events (Graham et al., 2000; Yasuda et al., 2000), ectopic expression of an oncogenic form of c-Cbl, the 70Z/3 Cbl mutant, leads to augmented basal and TCR-induced PLC1 signaling (Graham et al., 2000). 70Z/3 Cbl and WT Cbl differ in a 17-amino acid deletion in 70Z/3 Cbl (aa 366–382) that abrogates E3 ubiquitin-ligase activity (Figure 1) (Sawasdikosol et al., 2000), suggesting that the E3 function is required for c-Cbl to negatively regulate PLC1 . . .
  46. Zhang W, Irvin BJ, Trible RP, Abraham RT and Samelson LE. (1999a). Int. Immunol., 11, 943-950 , .
    • . . . T-cell receptor (TCR)-induced PLC1 activation is controlled by nonreceptor protein tyrosine kinases, including Lck and Zap-70 (Williams et al., 1998,1999; Kane et al., 2000), the phosphorylation of the adapters Lat and Slp-76 (Yablonski et al., 1998; Zhang et al., 1999a,1999b), and the tyrosine phosphorylation of PLC1 itself (Todderud et al., 1990; Kim et al., 1991). . . .
    • . . . The fact that 70Z/3 Cbl expression can lead to a decrease in NFAT activation in TCR-stimulated cells has been previously reported, but the reason for the decrease remains unclear (Zhang et al., 1999a,1999b) . . .
    • . . . PLC1, however, does not appear to be a direct substrate of Zap-70 (Veri et al., 2001) and no increase in Zap-70 phosphorylation or kinase activity has been observed in Jurkat cells expressing 70Z/3 Cbl (van Leeuwen et al., 1999a,1999b; Zhang et al., 1999a,1999b), making it unlikely that 70Z/3 Cbl alters the kinase activity of Zap-70 . . .
  47. Zhang W, Sloan-Lancaster J, Kitchen J, Trible RP and Samelson LE.. Cell, 92, 83-92 , (1998) .
    • . . . Since it has previously been established that TCR-induced PLC1 phosphorylation requires the interaction of the PLC1 SH2N domain with phospho-Lat (Stoica et al., 1998; Zhang et al., 1998,2000), we examined whether pp36-38/Lat was required for 70Z/3 Cbl-induced PLC1 phosphorylation . . .
    • . . . During TCR signaling, Lat, once phosphorylated, acts as a scaffold that recruits PLC1 via a direct interaction with the SH2N domain of PLC1 and indirectly via a ternary complex involving phospho-Lat binding to Gads, an interaction of Gads with Slp-76 (Zhang et al., 1998; Boerth et al., 2000), and a constitutive interaction of Slp-76 with the SH3 domain of PLC1 (Yablonski et al., 2001) . . .
  48. Zhang W, Trible RP, Zhu M, Liu SK, McGlade CJ and Samelson LE.. J. Biol. Chem., 275, 23355-23361 , (2000) .
    • . . . Since it has previously been established that TCR-induced PLC1 phosphorylation requires the interaction of the PLC1 SH2N domain with phospho-Lat (Stoica et al., 1998; Zhang et al., 1998,2000), we examined whether pp36-38/Lat was required for 70Z/3 Cbl-induced PLC1 phosphorylation . . .
  49. Zhang Z, Elly C, Altman A and Liu Y-C. (1999b). J. Biol. Chem., 274, 4883-4889 , .
    • . . . T-cell receptor (TCR)-induced PLC1 activation is controlled by nonreceptor protein tyrosine kinases, including Lck and Zap-70 (Williams et al., 1998,1999; Kane et al., 2000), the phosphorylation of the adapters Lat and Slp-76 (Yablonski et al., 1998; Zhang et al., 1999a,1999b), and the tyrosine phosphorylation of PLC1 itself (Todderud et al., 1990; Kim et al., 1991). . . .
    • . . . As previously reported (van Leeuwen et al., 1999a,1999b), Zap-70 was required for 70Z/3 Cbl-induced NFAT activity (Figure 9a) . . .
    • . . . PLC1, however, does not appear to be a direct substrate of Zap-70 (Veri et al., 2001) and no increase in Zap-70 phosphorylation or kinase activity has been observed in Jurkat cells expressing 70Z/3 Cbl (van Leeuwen et al., 1999a,1999b; Zhang et al., 1999a . . .
  50. Zheng N, Wang P, Jeffrey PD and Pavletich NP.. Cell, 102, 533-539 , (2000) .
    • . . . 70Z/3 Cbl differs from c-Cbl only in a 17-amino acid deletion that disrupts the linker -helix and the RING finger domain, structures that contribute to the E3 ubiquitin-ligase activity of c-Cbl (Joazeiro et al., 1999; Sawasdikosol et al., 2000; Zheng et al., 2000) . . .
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