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SHP-1 Regulates Lck-induced Phosphatidylinositol 3-Kinase Phosphorylation and Activity*

Open AccessPublished:September 24, 1999DOI:https://doi.org/10.1074/jbc.274.39.27583
      Ligation of the T cell antigen receptor (TCR) activates the Src family tyrosine kinase p56 Lck, which, in turn, phosphorylates a variety of intracellular substrates. The phosphatidylinositol 3-kinase (PI3K) and the tyrosine phosphatase SHP-1 are two Lck substrates that have been implicated in TCR signaling. In this study, we demonstrate that SHP-1 co-immunoprecipitates with the p85 regulatory subunit of PI3K in Jurkat T cells, and that this association is increased by ligation of the TCR complex. Co-expression of SHP-1 and PI3K with a constitutively activated form of Lck in COS7 cells demonstrated the carboxyl-terminal SH2 domain of PI3K to inducibly associate with the full-length SHP-1 protein. By contrast, a truncated SHP-1 mutant lacking the Lck phosphorylation site (Tyr564) failed to bind p85. Wild-type but not catalytically inactive SHP-1 induced dephosphorylation of p85. Furthermore, expression of SHP-1 decreased PI3K enzyme activity in anti-phosphotyrosine immunoprecipitates and phosphorylation of serine 473 in Akt, a process dependent on PI3K activity. These results indicate the presence of a functional interaction between PI3K and SHP-1 and suggest that PI3K signaling, which has been implicated in cell proliferation, apoptosis, cytoskeletal reorganization, and many other biological activities, can be regulated by SHP-1 in T lymphocytes.
      TCR
      T cell antigen receptor
      PDGFR
      platelet-derived growth factor receptor
      PI3K
      phosphatidylinositol 3-kinase
      PH
      pleckstrin homology
      GST
      glutathione S-transferase
      PAGE
      polyacrylamide gel electrophoresis
      HAp85
      hemagglutinin epitope tag-labeled p85 construct
      SH2
      Src homology domain 2
      In the context of appropriate co-stimulatory signals, ligation of the T cell antigen receptor (TCR)1 by antigenic peptide bound to a major histocompatibility complex molecule leads to T cell activation and ultimately, a functional immune response. Activation of protein tyrosine kinases and consequent intracellular protein phosphorylation are among the first events elicited by TCR ligation and are crucial to the induction of biochemical pathways that regulate cell growth (
      • Cantrell D.
      ). This protein-tyrosine kinase activity, together with opposing protein-tyrosine phosphatase activity, plays a major role in regulating the magnitude of TCR-induced tyrosine phosphorylation, as well as the duration and termination of cell activation (
      • Cantrell D.
      ,
      • Qian D.
      • Weiss A.
      ). The counterbalance of tyrosine kinases by tyrosine phosphatases is integral to the maintenance of cellular homeostasis (
      • Neet K.
      • Hunter T.
      ,
      • Neel B.G.
      • Tonks N.K.
      ), and disruption of this balance has been shown to be a hallmark of cellular transformation (
      • Smith A.
      • Ashworth A.
      ).
      P56 Lck is a member of the Src family of non-receptor tyrosine kinases which is highly expressed in T lymphocytes (
      • Weil R.
      • Veillette A.
      ). Along with the Fyn Src family kinase and the ζ-associated protein 70 (ZAP-70), Lck has been implicated in the initial activation events resulting from TCR ligation (
      • Cantrell D.
      ,
      • Qian D.
      • Weiss A.
      ,
      • Weil R.
      • Veillette A.
      ). Lck has been shown to associate with the CD4 and CD8 T cell surface antigens (
      • Weil R.
      • Veillette A.
      ), and to play an integral role in the ligand-induced phosphorylation of the TCR intracellular components (
      • Cantrell D.
      ,
      • Qian D.
      • Weiss A.
      ,
      • Weil R.
      • Veillette A.
      ). Indeed, Lck-mediated phosphorylation of the ζ subunit of the TCR and ZAP-70 couples TCR ligation to a variety of downstream signaling molecules (
      • Qian D.
      • Weiss A.
      ), and the loss of Lck activity significantly reduces the capacity of the TCR to transduce activation signals (
      • Straus D.B.
      • Weiss A.
      ).
      SHP-1 is an SH2 domain-containing non-receptor tyrosine phosphatase implicated in the negative regulation of a number of growth factor receptors, including the B and T cell antigen, erythropoeitin, the platelet-derived growth factor (PDGFR), c-kit, and the granulocyte macrophage colony-stimulating factor receptors (
      • Siminovitch K.A.
      • Neel B.G.
      ,
      • Klingmuller U.
      • Lorenz U.
      • Cantley L.C.
      • Neel B.G.
      • Lodish H.F.
      ,
      • Yu Z.
      • Su L.
      • Hoglinger O.
      • Jaramillo M.L.
      • Banville D.
      • Shen S.H.
      ,
      • Paulson R.F.
      • Vesely S.
      • Siminovitch K.A.
      • Bernstein A.
      ,
      • Lorenz U.
      • Bergemann A.D.
      • Steinberg H.N.
      • Flanagan J.G.
      • Li X.
      • Galli S.J.
      • Neel B.G.
      ,
      • Jiao H.
      • Yang W.
      • Berrada K.
      • Tabrizi M.
      • Shultz L.
      • Yi T.
      ). SHP-1 is highly expressed in T cells (
      • Neel B.G.
      • Tonks N.K.
      ), and has also been linked to the negative regulation of TCR signaling (
      • Plas D.R.
      • Johnson R.
      • Pingel J.T.
      • Matthews R.J.
      • Dalton M.
      • Roy G.
      • Chan A.C.
      • Thomas M.L.
      ,
      • Plas D.R.
      • Thomas M.L.
      ,
      • Pani G.
      • Fischer K.D.
      • Mlinaric-Rascan I.
      • Siminovitch K.A.
      ). This effect of SHP-1 appears to reflect its capacity to down-regulate ZAP-70 (
      • Plas D.R.
      • Johnson R.
      • Pingel J.T.
      • Matthews R.J.
      • Dalton M.
      • Roy G.
      • Chan A.C.
      • Thomas M.L.
      ) and Lck (
      • Lorenz U.
      • Ravichandran K.S.
      • Burakoff S.J.
      • Neel B.G.
      ) activities and to also dephosphorylate TCR components and downstream signaling molecules (
      • Plas D.R.
      • Thomas M.L.
      ,
      • Pani G.
      • Fischer K.D.
      • Mlinaric-Rascan I.
      • Siminovitch K.A.
      ). SHP-1 has been shown to undergo tyrosine phosphorylation in response to CD4 or CD8 stimulation as well as Lck activation (
      • Lorenz U.
      • Ravichandran K.S.
      • Pei D.
      • Walsh C.T.
      • Burakoff S.J.
      • Neel B.G.
      ). As is consistent with an inhibitory effect of SHP-1 on TCR signaling, thymocytes from SHP-1-deficient viable motheaten exhibit a significantly increased proliferative response to stimulation by anti-CD3 antibodies as compared with normal mouse thymocytes (
      • Pani G.
      • Fischer K.D.
      • Mlinaric-Rascan I.
      • Siminovitch K.A.
      ,
      • Lorenz U.
      • Ravichandran K.S.
      • Burakoff S.J.
      • Neel B.G.
      ).
      Ligation of the TCR alters inositol lipid metabolism through induction of phosphatidylinositol 3′-kinase (PI3K) activity (
      • Cantrell D.
      ). PI3K consists of a p85 regulatory subunit with two SH2 domains and a SH3 domain, and a p110 catalytic subunit which phosphorylates the 3′-hydroxyl of the inositol ring of phosphatidylinositol (
      • Carpenter C.L.
      • Cantley L.C.
      ,
      • Vanhaesebroeck B.
      • Leevers S.J.
      • Panayotou G.
      • Waterfield M.D.
      ). The resulting PI3K products bind to pleckstrin homology (PH) domains of intracellular signaling molecules recruiting them to the cell membrane. Activation of the PH domain containing c-Akt (
      • Franke T.F.
      • Kaplan D.R.
      • Cantley L.C.
      • Toker A.
      ,
      • Andjelkovic M.
      • Alessi D.R.
      • Meier R.
      • Fernandez A.
      • Lamb N.J.
      • Frech M.
      • Cron P.
      • Cohen P.
      • Lucocq J.M.
      • Hemmings B.A.
      ) has been associated with cell cycle progression (
      • Klippel A.
      • Escobedo M.A.
      • Wachowicz M.S.
      • Apell G.
      • Brown T.W.
      • Giedlin M.A.
      • Kavanaugh W.M.
      • Williams L.T.
      ,
      • Pullen N.
      • Dennis P.B.
      • Andjelkovic M.
      • Dufner A.
      • Kozma S.C.
      • Hemmings B.A.
      • Thomas G.
      ) and the propagation of an anti-apoptotic signal (
      • Andjelkovic M.
      • Alessi D.R.
      • Meier R.
      • Fernandez A.
      • Lamb N.J.
      • Frech M.
      • Cron P.
      • Cohen P.
      • Lucocq J.M.
      • Hemmings B.A.
      ,
      • Dudek H.
      • Datta S.R.
      • Franke T.F.
      • Birnbaum M.J.
      • Yao R.
      • Cooper G.M.
      • Segal R.A.
      • Kaplan D.R.
      • Greenberg M.E.
      ,
      • Franke T.F.
      • Kaplan D.R.
      • Cantley L.C.
      ,
      • Kennedy S.G.
      • Wagner A.J.
      • Conzen S.D.
      • Jordan J.
      • Bellacosa A.
      • Tsichlis P.N.
      • Hay N.
      ). Jurkat T cell activation via anti-CD3 antibody binding to the TCR complex has been shown to result in the rapid phosphorylation of both PI3K subunits (
      • von Willebrand M.
      • Baier G.
      • Couture C.
      • Burn P.
      • Mustelin T.
      ), as well as an accumulation of PI3K products (
      • Ward S.G.
      • Ley S.C.
      • MacPhee C.
      • Cantrell D.A.
      ). TCR-induced tyrosine phosphorylation of Tyr688 in the p85 subunit of PI3K and the consequent activation of PI3K have been linked to the presence of Lck (
      • von Willebrand M.
      • Baier G.
      • Couture C.
      • Burn P.
      • Mustelin T.
      ,
      • von Willebrand M.
      • Williams S.
      • Saxena M.
      • Gilman J.
      • Tailor P.
      • Jascur T.
      • Amarante-Mendes G.P.
      • Green D.R.
      • Mustelin T.
      ), and other recent data provide additional evidence of a role for Lck in PI3K signaling (
      • Jascur T.
      • Gilman J.
      • Mustelin T.
      ). However, the phosphatase(s) that dephosphorylates PI3K has not been identified as of yet.
      In this study, we demonstrate that Lck activity is associated with an interaction of SHP-1 with the p85 subunit of PI3K, and also identify p85 as a target for SHP-1-mediated dephosphorylation. The association between p85 and SHP-1 requires tyrosine phosphorylation of SHP-1 and likely involves binding of SHP-1 phosphotyrosine 564 to the p85 carboxyl-terminal SH2 domain via a novel tyrosine recognition motif. This interaction is also associated with a reduction in the lipid kinase activity in total anti-phosphotyrosine immunoprecipitates and a reduction in PI3K-mediated phosphorylation of Akt. Together, these findings implicate the interaction of SHP-1 with PI3K in the modulation of the PI3K signaling cascade downstream of TCR engagement.

      DISCUSSION

      In the current study, the possibility that interaction between PI3K and SHP-1 contributes to the effects of these respective proteins on TCR signaling was investigated. The data reveal that SHP-1 interacts with the p85 subunit of PI3K in Jurkat T cells, and indicate this association to be enhanced by TCR stimulation. Furthermore, SHP-1 and PI3K are present in a complex including the TCR. Association of SHP-1 with p85 was also found to be inducible in COS7 cells by addition of activated Lck and to represent a phosphotyrosine-dependent interaction involving association of the p85 carboxyl-terminal SH2 domain likely with phosphorylated tyrosine 564 in the SHP-1 carboxyl-terminal tail. By further analysis of this interaction in COS7 cells, p85 was identified as a substrate for SHP-1, and the activity of tyrosine-phosphorylated PI3K shown to be markedly reduced in the presence of wild-type, but not catalytically inert SHP-1 (
      • Pani G.
      • Kozlowski M.
      • Cambier J.C.
      • Mills G.B.
      • Siminovitch K.A.
      ). SHP-1 expression did not, however, alter lipid kinase activity of total cellular PI3K. A role for SHP-1 in regulating PI3K signaling was also evidenced by the finding that SHP-1 expression in COS7 cells engenders a decrease in phosphorylation of Akt Ser473. Phosphorylation of Akt at this site involves association of the Akt PH domain with phosphorylated PI3K lipid substrates in the cell membrane and is known to be completely dependent on PI3K activation (
      • Andjelkovic M.
      • Alessi D.R.
      • Meier R.
      • Fernandez A.
      • Lamb N.J.
      • Frech M.
      • Cron P.
      • Cohen P.
      • Lucocq J.M.
      • Hemmings B.A.
      ). Taken together, these observations provide evidence that SHP-1 not only interacts with PI3K, but also impacts upon PI3K activation and downstream signaling.
      The current data indicate the SHP-1/PI3K interaction to be mediated by binding of the PI3K p85 subunit carboxyl-terminal SH2 domain to phosphorylated SHP-1 and to require that the most carboxyl-terminal located 35-amino acid segment of SHP-1 be intact. As Tyr564, which has been identified as the primary target for Lck effects on SHP-1, maps within this region (
      • Lorenz U.
      • Ravichandran K.S.
      • Pei D.
      • Walsh C.T.
      • Burakoff S.J.
      • Neel B.G.
      ), it appears likely that Tyr564 represents the site on SHP-1 which interacts with the p85 SH2 domain. Interestingly, the results of these studies also revealed the truncated SHP-1 Δ35 protein to exhibit decreased phosphatase activity (data not shown), a result which contrasts with previous data suggesting catalytic activity of this mutant form of SHP-1 to be enhanced (
      • Pei D.
      • Lorenz U.
      • Klingmuller U.
      • Neel B.G.
      • Walsh C.T.
      ). This discrepancy may reflect the differences in the conditions used for the respective phosphatase assays, the previous study involving analysis of PTP activity at pH 5.5. In the current study, the assay was performed at pH 7.3, which would presumably more closely approximate physiologic conditions. In any case, in view of the potential for this truncation mutation to alter SHP-1 activity, the SHP-1 Δ35 protein was used here only in binding studies, and its effects on p85 phosphorylation and PI3K activity were not examined.
      Although p85 SH2 domains have been previously shown to specifically target YMXM phosphotyrosine motifs, the current data suggest that the carboxyl-terminal SH2 domain of p85 binds a SHP-1 phosphotyrosine residue (Tyr564) embedded within a YENV motif. This divergence in the SH2 domain specificity is, however, not without precedent (
      • von Willebrand M.
      • Williams S.
      • Saxena M.
      • Gilman J.
      • Tailor P.
      • Jascur T.
      • Amarante-Mendes G.P.
      • Green D.R.
      • Mustelin T.
      ,
      • Rameh L.E.
      • Chen C.S.
      • Cantley L.C.
      ). The SHP-1 SH2 domains, for example, have been demonstrated to interact with several distinct phosphotyrosine motifs (
      • Kozlowski M.
      • Larose L.
      • Lee F.
      • Le D.M.
      • Rottapel R.
      • Siminovitch K.A.
      ). Furthermore, in vitro phosphorylation of the p85 carboxyl-terminal SH2 domain has been shown to alter its capacity to bind certain targets in activated Jurkat cells (
      • von Willebrand M.
      • Williams S.
      • Saxena M.
      • Gilman J.
      • Tailor P.
      • Jascur T.
      • Amarante-Mendes G.P.
      • Green D.R.
      • Mustelin T.
      ), a finding which again raises the possibility that the SH2 domain may interact with phosphotyrosines in more than one structural context.
      Interestingly, p85 association with SHP-1 in PDGFR-stimulated MCF-7 cells has been shown to be mediated by binding of the SHP-1 amino-terminal SH2 domain to phosphorylated p85 (
      • Yu Z.
      • Su L.
      • Hoglinger O.
      • Jaramillo M.L.
      • Banville D.
      • Shen S.H.
      ). By contrast, interaction of the SHP-1 SH2 domains with phosphorylated p85 was not detected in the current study, a discrepancy which may reflect differences in the PI3K sites targeted by Lck and PDGFR, respectively (
      • Lorenz U.
      • Ravichandran K.S.
      • Pei D.
      • Walsh C.T.
      • Burakoff S.J.
      • Neel B.G.
      ,
      • Bouchard P.
      • Zhao Z.
      • Banville D.
      • Dumas F.
      • Fischer E.H.
      • Shen S.H.
      ). It is also not clear whether p85 is a direct PDGFR targetin vivo. However, taken together, these findings raise the possibility that association of SHP-1 with PI3K and the consequent modulation of PI3K signaling occurs in a variety of cell stimulatory contexts.
      The data reported here concur with other data in the literature revealing the phosphorylation of p85 and the in vitro lipid kinase activity of immunoprecipitated PI3K to be poorly correlated (
      • von Willebrand M.
      • Williams S.
      • Saxena M.
      • Gilman J.
      • Tailor P.
      • Jascur T.
      • Amarante-Mendes G.P.
      • Green D.R.
      • Mustelin T.
      ). However, wild-type SHP-1 decreases PI3K activity in anti-phosphotyrosine immunoprecipitates and PI3K-dependent phosphorylation of Akt in intact cells. Interestingly, both p85 phosphorylation and PI3K activity, as revealed by Akt S473 phosphorylation, were found to be up-regulated in the presence of catalytically inactive SHP-1 C453S protein. As SHP-1 C453S does not enhance activity of Lck Y505F (Fig. 3 C), these data suggest that SHP-1 C453S acts in this context as a “substrate trap,” binding phosphorylated targets, but failing to dephosphorylate or release these phosphoproteins, thus protecting them from dephosphorylation by other cellular phosphatases. The increased level of phospho-Akt in the SHP-1 C453S-transfected cells may also reflect the capacity of mutant SHP-1 C453S protein bound to PI3K to impede PI3K interaction with a negative regulator of PI3K, or, alternatively, the capacity of PI3K bound SHP-1 C453S to induce conformational changes in PI3K which favor its activation, possibly by mimicking the effects of a positive modulator of PI3K. Both of these latter hypotheses suggest the involvement of a third molecule in the PI3K/SHP-1 interaction, a possibility also suggested by our finding that SHP-1 and PI3K can be co-immunoprecipitated from the membrane fraction of resting, serum-starved Jurkat cells in which protein phosphorylation would be expected to be minimal. Therefore, SHP-1 may also associate with PI3K by a phosphotyrosine-independent mechanism, such as interactions with an SH3 domain containing protein (
      • Fusaki N.
      • Iwamatsu A.
      • Iwashima M.
      • Fujisawa J.
      ). This possibility however, remains purely speculative at present.
      In summary, the data shown here reveal a functional relationship between Lck, SHP-1, and PI3K signaling proteins, which have each been identified as key elements in the induction of T cell activation. While Lck acts primarily to promote TCR signaling (
      • Weil R.
      • Veillette A.
      ), SHP-1 effects on TCR signal relay are largely inhibitory (
      • Pani G.
      • Fischer K.D.
      • Mlinaric-Rascan I.
      • Siminovitch K.A.
      ,
      • Lorenz U.
      • Ravichandran K.S.
      • Burakoff S.J.
      • Neel B.G.
      ). The current data suggest that this inhibitory effect of SHP-1 is realized at least in part through the down-regulation of PI3K activity. However, in view of the limited understanding of the role for PI3K activity in TCR signaling, further studies are required to address the physiological significance of SHP-1 effects on PI3K. It also remains to be determined whether SHP-1 effects on PI3K signaling in vivo reflect direct modulation of PI3K activity by SHP-1 and/or the capacity of SHP-1 to influence other PI3K modulatory signaling effectors by virtue of its interaction with PI3K. Investigation of these various possibilities represents a promising avenue to further elucidating the mechanisms whereby both SHP-1 and PI3K impact upon the signaling cascades linking TCR stimulation to cell response.

      Acknowledgments

      We thank Dr. A. Veillette for his gift of the Lck Y505F cDNA construct, Dr. P. Beverly for the UCHT1 hybridoma, Dr. B. Su for the 12CA5 hybridoma, Dr. T. Pawson for the GST fusion constructs, and Dr. Julian Downward for HAAkt the cDNA construct.

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