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Steel Factor Induces Tyrosine Phosphorylation of CRKL and Binding of CRKL to a Complex Containing c-Kit, Phosphatidylinositol 3-Kinase, and p120CBL*

Open AccessPublished:April 11, 1997DOI:https://doi.org/10.1074/jbc.272.15.10248
      Steel factor (SF) is a growth and survival factor for hematopoietic cells. The receptor for SF, c-Kit, contains intrinsic tyrosine kinase activity, and binding of SF induces rapid tyrosine phosphorylation of several cellular proteins, including c-Kit itself. Activation of c-Kit is shown here to induce tyrosine phosphorylation of CRKL, and CRKL coprecipitated with c-Kit through an interaction that required the CRKL SH3 domains and not the SH2 domain. CRKL associated with c-Kit indirectly as part of a larger complex of proteins. Two proteins in this complex were identified as the p85 regulatory subunit of phosphatidylinositol 3-kinase (p85PI3K) and the proto-oncoprotein p120CBL. Because p85PI3K is known to bind to the activated c-Kit receptor, the possibility that CRKL interacted with c-Kit indirectly through p85PI3K was investigated. Far Western blotting with a CRKL-SH3 glutathione S-transferase fusion protein showed that CRKL binds directly to p85PI3K in vitro However, although a small amount of CRKL was preassociated with p85PI3K, the interaction was increased after SF stimulation, suggesting that the interactions of these three proteins are complex. We conclude that SF induces the formation of a signaling complex potentially containing CRKL and p120CBL, both of which bind to c-Kit through p85PI3K. These data suggest that one function of CRKL in normal cells might be to recruit signaling molecules such as CBL into a complex with PI3K. Such complexes could be important in propagating signals involving PI3K such as gene expression and adhesion.

      INTRODUCTION

      Steel factor (SF)
      The abbreviations used are: SF
      steel factor
      PI3K
      phosphatidylinositol 3-kinase
      GST
      glutathione S-transferase.
      supports growth and survival of immature hematopoietic cells of multiple lineages. The cytokine is synthesized as a transmembrane protein, and a soluble form is generated by a proteolytic cleavage. In vivo, the membrane form is required for normal hematopoiesis, but in vitro, both the membrane bound as well as the soluble form are biologically active. One interesting feature of SF is its ability to act synergistically with various other hematopoietic growth factors such as interleukin-3 or granulocyte macrophage-colony stimulating factor (
      • McNiece I.K.
      • Langley K.E.
      • Zsebo K.M.
      ,
      • Lowry P.A.
      • Zsebo K.M.
      • Deacon D.H.
      • Eichman C.E.
      • Quesenberry P.J.
      ,
      • Zsebo K.M.
      • Williams D.A.
      • Geissler E.N.
      • Broudy V.C.
      • Martin F.H.
      • Atkins H.L.
      • Hsu R.Y.
      • Birkett N.C.
      • Okino K.H.
      • Murdock D.C.
      • Jacobsen F.W.
      • Langley K.E.
      • Smith K.A.
      • Takeishi T.
      • Cattanach B.M.
      • Galli S.J.
      • Suggs S.V.
      ,
      • Martin F.H.
      • Suggs S.V.
      • Langley K.E.
      • Lu H.S.
      • Ting J.
      • Okino K.H.
      • Morris C.F.
      • McNiece I.K.
      • Jacobsen F.W.
      • Mendiaz E.A.
      • Birkett N.C.
      • Smith K.A.
      • Johnson M.J.
      • Parker V.P.
      • Flores J.C.
      • Patel A.C.
      • Fisher E.F.
      • Erjavec H.O.
      • Herrera C.J.
      • Wypych J.
      • Sachdev R.K.
      • Pope J.A.
      • Leslie I.
      • Wen D.
      • Lin C.-H.
      • Cupples R.L.
      • Zsebo K.M.
      ,
      • Zsebo K.M.
      • Wypych J.
      • McNiece I.K.
      • Lu H.S.
      • Smith K.A.
      • Karkare S.B.
      • Sachdev R.K.
      • Yuschenkoff V.N.
      • Birkett N.C.
      • Williams L.R.
      • Satyagal V.N.
      • Tung W.
      • Bosselman R.A.
      • Mendiaz E.A.
      • Langley K.E.
      ). The receptor for SF, the proto-oncoprotein c-Kit, belongs to a family of growth factor receptors with intrinsic tyrosine kinase activity, which also includes receptors such as c-Ret, the receptor for glia cell-derived neurotrophic factor, or the platelet-derived growth factor receptor (
      • Yarden Y.
      • Kuang W.J.
      • Yang-Feng T.
      • Coussens L.
      • Munemitsu S.
      • Dull T.J.
      • Chen E.
      • Schlessinger J.
      • Francke U.
      • Ullrich A.
      ,
      • Takahashi M.
      • Cooper G.M.
      ,
      • Yarden Y.
      • Escobedo J.A.
      • Kuang W.J.
      • Yang-Feng T.L.
      • Daniel T.O.
      • Tremble P.M.
      • Chen E.Y.
      • Ando M.E.
      • Harkins R.N.
      • Francke U.
      • Fried V.A.
      • Ullrich A.
      • Williams L.T.
      ). Mutations in the murine locus for SF, steel, and its receptor c-Kit, white spotting, have suggested pleiotropic functions for the SF/c-Kit pathway and include defects in melanogenesis, gametogenesis, or hematopoiesis (
      • Chabot B.
      • Stephenson D.A.
      • Chapman V.M.
      • Besmer P.
      • Bernstein A.
      ,
      • Geissler E.N.
      • Ryan M.A.
      • Housman D.E.
      ). The severity of the phenotype in mice depends on the mutation, with the most severe effects associated with absent kinase activity. Mice heterozygotic for Kit receptor mutations may also have a phenotype, possibly related to the fact that certain mutant receptors behave as dominant negatives (
      • Geissler E.N.
      • McFarland E.C.
      • Russell E.S.
      ,
      • Wiktor-Jedrzejczak W.
      • Szczylik C.
      • Gornas P.
      • Sharkis S.J.
      • Ahmed A.
      ). Most humans heterozygous for Kit mutations do not have a significant hematopoietic disorder but do have mild defects in hair or skin pigmentation (
      • Fleischman R.A.
      • Saltman D.L.
      • Stastny V.
      • Zneimer S.
      ,
      • Richmond A.
      • Balentien E.
      • Thomas H.G.
      • Flaggs G.
      • Barton D.E.
      • Spiess J.
      • Bordoni R.
      • Francke U.
      • Derynck R.
      ).
      SF and c-Kit have also been linked to several hematologic and nonhematologic neoplastic disorders. It has been suggested that one mechanism could include autocrine production of SF, leading to a clonal expansion of c-Kit expressing cells. For example, coexpression of SF and c-Kit has been reported in breast tumor cells, small cell lung cancer cells, and malignant glioma cell lines (
      • Hines S.J.
      • Organ C.
      • Kornstein M.J.
      • Krystal G.W.
      ,
      • Rygaard K.
      • Nakamura T.
      • Spang-Thomsen M.
      ,
      • Stanulla M.
      • Welte K.
      • Hadam M.R.
      • Pietsch T.
      ). Also, it has been shown that the tyrosine phosphorylation pattern of cellular proteins in chronic myelogenous leukemia resembles the pattern observed after SF stimulation in normal cells (
      • Wisniewski D.
      • Strife A.
      • Berman E.
      • Clarkson B.
      ).
      With the diverse effects of SF on cells of different lineages, there has been considerable interest in identifying the critical signal transduction pathways activated by c-Kit. The ligand is believed to induce dimerization or oligomerization of the receptor, activating the tyrosine kinase activity and inducing rapid but transient tyrosine phosphorylation of several cellular proteins, including c-Kit itself. Some substrates of c-Kit have been described to associate with c-Kit after activation, including phospholipase C-γ, p85PI3K, and SHP2 (
      • Rottapel R.
      • Reedijk M.
      • Williams D.E.
      • Lyman S.D.
      • Anderson D.M.
      • Pawson T.
      • Bernstein A.
      ,
      • Tauchi T.
      • Feng G.-S.
      • Marshall M.S.
      • Shen R.
      • Mantel C.
      • Pawson T.
      • Broxmeyer H.E.
      ,
      • Yi T.
      • Ihle J.N.
      ). These proteins are believed to link c-Kit directly to various enzymatic pathways. However, as is the case for most receptors, the intermediate signaling events are not well understood.
      In this study we have investigated the specific role of CRKL, an adapter protein containing one SH2 domain and two SH3 domains, in c-Kit signaling. CRKL is widely expressed and belongs to the family of CRK adapter proteins that includes v-Crk and c-CRK (
      • ten Hoeve J.
      • Morris C.
      • Heisterkamp N.
      • Groffen J.
      ). The CRKL SH2 domain can bind to the proto-oncoprotein p120CBL, p130CAS, and the focal adhesion protein paxillin (
      • de Jong R.
      • ten Hoeve J.
      • Heisterkamp N.
      • Groffen J.
      ,
      • Sattler M.
      • Salgia R.
      • Okuda K.
      • Uemura N.
      • Durstin M.A.
      • Pisick E.
      • Xu G.
      • Li J.L.
      • Prasad K.V.
      • Griffin J.D.
      ,
      • Petruzzelli L.
      • Takami M.
      • Herrera R.
      ,
      • Salgia R.
      • Uemura N.
      • Okuda K.
      • Li J.-L.
      • Pisick E.
      • Sattler M.
      • DeJong R.
      • Druker B.
      • Heisterkamp N.
      • Chen L.B.
      • Groffen J.
      • Griffin J.D.
      ,
      • Salgia R.
      • Pisick E.
      • Sattler M.
      • Li J.-L.
      • Uemura N.
      • Wong W.-K.
      • Burky S.A.
      • Hirai H.
      • Chen L.B.
      • Griffin J.D.
      ). The CRKL SH3 domain, like the c-CRK SH3 domain, binds in vitro and associates in vivo with c-ABL, SOS, and C3G (
      • Sattler M.
      • Salgia R.
      • Okuda K.
      • Uemura N.
      • Durstin M.A.
      • Pisick E.
      • Xu G.
      • Li J.L.
      • Prasad K.V.
      • Griffin J.D.
      ,
      • Feller S.M.
      • Knudsen B.
      • Hanafusa H.
      ,
      • Knudsen B.S.
      • Feller S.M.
      • Hanafusa H.
      ,
      • Matsuda M.
      • Hashimoto Y.
      • Muroya K.
      • Hasegawa H.
      • Kurata T.
      • Tanaka S.
      • Nakamura S.
      • Hattori S.
      ,
      • Feller S.M.
      • Knudsen B.
      • Hanafusa H.
      ,
      • Tanaka S.
      • Hattori S.
      • Kurata T.
      • Nagashima K.
      • Fukui Y.
      • Nakamura S.
      • Matsuda M.
      ). CRKL is therefore likely to be involved in the regulation of the enzymatic pathways associated with these proteins.
      Using a human megakaryoblastic cell line, MO7e, SF stimulation was found to result in the rapid tyrosine phosphorylation of CRKL and coprecipitation of CRKL with c-Kit. However, although this interaction was inducible, it was mediated through the CRKL SH3 domain. This suggested the possibility that CRKL was not binding to c-Kit directly, but associated with c-Kit as part of a larger complex of proteins. We found that the coprecipitation of CRKL with c-Kit is, at least in part, likely to be mediated through binding of p85PI3K to c-Kit as well as to CRKL. Other proteins are likely to be involved in this complex, with the most prominent identified as the proto-oncoprotein p120CBL. These data demonstrate that CRKL is involved in the formation of a complex of signaling proteins that bind to c-Kit through PI3K and further suggest that CRKL, and its SH2-binding partners such as p120CBL, may function downstream of PI3K.

      DISCUSSION

      Although there are a number of signaling molecules known to be tyrosine phosphorylated by c-Kit, most downstream signaling pathways are not well understood. After stimulation of c-Kit with SF, previous studies have demonstrated rapid tyrosine phosphorylation of c-Kit itself and cellular proteins including SHP2, phospholipase C-γ, SHC, and p120CBL (
      • Rottapel R.
      • Reedijk M.
      • Williams D.E.
      • Lyman S.D.
      • Anderson D.M.
      • Pawson T.
      • Bernstein A.
      ,
      • Tauchi T.
      • Feng G.-S.
      • Marshall M.S.
      • Shen R.
      • Mantel C.
      • Pawson T.
      • Broxmeyer H.E.
      ,
      • Yi T.
      • Ihle J.N.
      ,
      • Wisniewski D.
      • Strife A.
      • Clarkson B.
      ,
      • Cutler R.L.
      • Liu L.
      • Damen J.E.
      • Krystal G.
      ). These molecules in turn contribute signals to various pathways that influence growth, viability, adhesion, migration, or differentiation. PI3K has been the focus of several studies related to c-Kit signaling. For example, mutation of tyrosine 709 to phenylalanine in the Tyr-Xaa-Xaa-Met motif of the murine c-Kit receptor has been shown to reduce binding of the p85 subunit of PI3K (
      • Serve H.
      • Hsu Y.-C.
      • Besmer P.
      ). Further, this mutation also caused defects in SF-mediated adhesion and early gene expression, presumably by interfering with activation of PI3K (
      • Serve H.
      • Yee N.S.
      • Stella G.
      • Sepp-Lorenzino L.
      • Tan J.C.
      • Besmer P.
      ). However, despite the apparent importance of PI3K signaling in this and other receptors, downstream signaling events have been difficult to identify.
      In the studies reported here, we found that the adapter protein, CRKL, was tyrosine phosphorylated after c-Kit activation and coprecipitated with c-Kit. However, there was no evidence that CRKL bound to c-Kit directly, and the data presented here suggest rather that CRKL binds directly to p85PI3K and indirectly to c-Kit through p85PI3K. The interaction of CRKL with p85PI3K was found to utilize the CRKL SH3 domains and could be further increased in response to factor stimulation. Also, we found that after SF stimulation, p120CBL is tyrosine phosphorylated and coprecipitates with CRKL, suggesting the formation of a signaling complex that contains c-Kit, PI3K, CRKL, and p120CBL. Overall, the data suggest the possibility that CRKL and/or p120CBL play a role in sending or modulating signals from c-Kit that require PI3K.
      One of the most intriguing findings here is the specific, inducible association of CRKL with p85PI3K through the CRKL SH3 domains. Previous studies suggested that CRK is constitutively associated with c-ABL, C3G, or SOS through the CRK SH3 domains. These proteins were first described to bind to the CRKII SH3 domain, but we and others have shown that they also bind to the CRKL SH3 domain (
      • Sattler M.
      • Salgia R.
      • Okuda K.
      • Uemura N.
      • Durstin M.A.
      • Pisick E.
      • Xu G.
      • Li J.L.
      • Prasad K.V.
      • Griffin J.D.
      ,
      • Feller S.M.
      • Knudsen B.
      • Hanafusa H.
      ,
      • Knudsen B.S.
      • Feller S.M.
      • Hanafusa H.
      ,
      • Matsuda M.
      • Hashimoto Y.
      • Muroya K.
      • Hasegawa H.
      • Kurata T.
      • Tanaka S.
      • Nakamura S.
      • Hattori S.
      ,
      • Feller S.M.
      • Knudsen B.
      • Hanafusa H.
      ,
      • Tanaka S.
      • Hattori S.
      • Kurata T.
      • Nagashima K.
      • Fukui Y.
      • Nakamura S.
      • Matsuda M.
      ). SOS has known guanine exchange factor activity for p21Ras, whereas C3G appears to have specific guanine exchange activity for p21Rap1 (
      • Gotoh T.
      • Hattori S.
      • Nakamura S.
      • Kitayama H.
      • Noda M.
      • Takai Y.
      • Kaibuchi K.
      • Matsui H.
      • Hatase O.
      • Takahashi H.
      • Kurata T.
      • Matsuda M.
      ). C3G has unique binding affinities to the CRK family proteins, because it preferentially binds to the N-terminal SH3 domain (
      • Knudsen B.S.
      • Feller S.M.
      • Hanafusa H.
      ). The exact function of the tyrosine kinase c-ABL is unknown, although c-ABL has been shown to be involved in transcriptional activation and possibly is activated in response to certain types of DNA damage (
      • Kharbanda S.
      • Ren R.
      • Pandey P.
      • Shafman T.D.
      • Feller S.M.
      • Weichselbaum R.R.
      • Kufe D.W.
      ,
      • Welch P.J.
      • Wang J.Y.
      ). Recently a consensus sequence for binding to the CRK SH3 domain, Pro-Xaa-Leu-Pro-Xaa-Lys, has been described (
      • Matsuda M.
      • Ota S.
      • Tanimura R.
      • Nakamura H.
      • Matuoka K.
      • Takenawa T.
      • Nagashima K.
      • Kurata T.
      ,
      • Sparks A.B.
      • Rider J.E.
      • Hoffman N.G.
      • Fowlkes D.M.
      • Quilliam L.A.
      • Kay B.K.
      ). Consistent with our finding of CRKL SH3 binding to p85PI3K, a proline-rich motif with this consensus sequence, Pro-Ala-Leu-Pro-Pro-Lys (amino acids 305-310, human sequence), is present in p85PI3K (
      • Skolnik E.Y.
      • Margolis B.
      • Mohammadi M.
      • Lowenstein E.
      • Fischer R.
      • Drepps A.
      • Ullrich A.
      • Schlessinger J.
      ). The SH3 domain interaction of CRKL with p85PI3K may occur at this site, although this has not yet been directly tested. Inducible association of an adapter protein through SH3 domain interactions has been previously described for the binding of GRB2 to SOS in T lymphocytes, although most SH3 domain-mediated interactions are constitutive (
      • Ravichandran K.S.
      • Lorenz U.
      • Shoelson S.E.
      • Burakoff S.J.
      ). One explanation for this phenomenon would be the presence of other proteins binding to CRKL, p85PI3K, and c-Kit that are important for overall stability of the complex and that are brought into the complex in response to receptor activation. We believe that p120CBL may be important in this regard, and this will be discussed in more detail below. Overall, our data strongly suggest that PI3K should be added to the list of signaling proteins known to interact with the CRKL SH3 domains but in the specific situation of c-Kit activation.
      The fact that the CRKL SH3 domains can bind to several different, unrelated signaling proteins suggests that CRKL may play a role in several different signaling pathways. Thus, it is possible that the biological functions of CRKL may vary widely in different cells. Alternatively, it is possible that different stimuli may activate different signaling pathways involving CRKL in the same cell. CRKL has been shown to be tyrosine phosphorylated in cells transformed by onco-proteins including BCR/ABL, v-Abl, v-Src, or in normal signaling after EGF receptor stimulation (
      • ten Hoeve J.
      • Kaartinen V.
      • Fioretos T.
      • Haataja L.
      • Voncken J.W.
      • Heisterkamp N.
      • Groffen J.
      ,
      • Fukazawa T.
      • Miyake S.
      • Band V.
      • Band H.
      ,
      • Andoniou C.E.
      • Thien C.B.
      • Langdon W.Y.
      ). Binding of CRKL to p120CBL appears to be independent from CRKL tyrosine phosphorylation, because in other systems, including T cell signaling, CRKL is not tyrosine phosphorylated, although it binds to p120CBL (
      • Reedquist K.A.
      • Fukazawa T.
      • Panchamoorthy G.
      • Langdon W.Y.
      • Shoelson S.E.
      • Druker B.J.
      • Band H.
      ). Surprisingly, we did not find significant association of CRK with c-Kit in SF signaling in MO7e cells, despite abundant expression of CRK in this cell line (data not shown) and the reported similarity of target proteins selected by CRKL and CRK during in vitro binding studies. It may be worthwhile to look specifically for situations in which CRK and not CRKL is preferentially selected for tyrosine phosphorylation and activation in various cell lineages.
      Thus, the data presented here support the notion that CRKL is involved in a signaling pathway that also involves PI3K. Our data suggest that a substantial proportion of the PI3K activity associated with an activated c-Kit receptor is also associated with CRKL, because approximately the same amount of PI3K enzymatic activity is found in anti-CRKL as in anti-c-Kit immunoprecipitates. The biological effects of activated PI3K may vary widely from cell to cell, ranging from regulation of apoptosis, viability, or early gene expression to regulation of adhesion. CRKL may be involved in signaling to any or all of these different biological events. Specifically with regard to c-Kit, mutant c-Kit receptors that fail to bind PI3K, fail to induce c-fos or junB expression, and lack the ability to induce binding of cells to fibronectin and CRKL could be involved in one or all of these events (
      • Serve H.
      • Yee N.S.
      • Stella G.
      • Sepp-Lorenzino L.
      • Tan J.C.
      • Besmer P.
      ).
      As noted above, a protein known to bind to the CRKL SH2 domain in certain transformed cells, p120CBL, was found to coprecipitate with CRKL after c-Kit stimulation. In vitro binding studies suggested that the SH2 domains of both CRKL and p85PI3K can bind to p120CBL, and the SH3 domain of PI3K also interacted directly with p120CBL. Of course, other proteins that can interact with the CRKL SH2 domain may also be brought into any such complex, and in addition to p120CBL, the known possibilities include p130CAS, and paxillin (
      • Petruzzelli L.
      • Takami M.
      • Herrera R.
      ,
      • Salgia R.
      • Uemura N.
      • Okuda K.
      • Li J.-L.
      • Pisick E.
      • Sattler M.
      • DeJong R.
      • Druker B.
      • Heisterkamp N.
      • Chen L.B.
      • Groffen J.
      • Griffin J.D.
      ,
      • Salgia R.
      • Pisick E.
      • Sattler M.
      • Li J.-L.
      • Uemura N.
      • Wong W.-K.
      • Burky S.A.
      • Hirai H.
      • Chen L.B.
      • Griffin J.D.
      ). However, our data in MO7e cells suggest that p120CBL is the most abundant tyrosine phosphoprotein coprecipitating with CRKL after SF stimulation in these cells. The proto-oncoprotein p120CBL (for Casitas B-lineage lymphoma) is the cellular homolog of v-Cbl, the oncoprotein in the CAS NS-1 retrovirus (
      • Langdon W.Y.
      • Hyland C.D.
      • Grumont R.J.
      • Morse H.D.
      ,
      • Langdon W.Y.
      • Hartley J.W.
      • Klinken S.P.
      • Ruscetti S.K.
      • Morse H.D.
      ) that induces pre-B cell lymphomas and myelogenous leukemias in mice (
      • Fredrickson T.N.
      • Langdon W.Y.
      • Hoffman P.M.
      • Hartley J.W.
      • Morse H.D.
      ). p120CBL is also known to be a substrate of tyrosine kinases in response to T cell (
      • Donovan J.A.
      • Wange R.L.
      • Langdon W.Y.
      • Samelson L.E.
      ) and B cell (
      • Cory G.O.
      • Lovering R.C.
      • Hinshelwood S.
      • MacCarthy-Morrogh L.
      • Levinsky R.J.
      • Kinnon C.
      ) activation, FC-γ receptor cross-linking (
      • Marcilla A.
      • Rivero-Lezcano O.M.
      • Agarwal A.
      • Robbins K.C.
      ,
      • Tanaka S.
      • Neff L.
      • Baron R.
      • Levy J.B.
      ), and growth factors (
      • Sattler M.
      • Durstin M.A.
      • Frank D.A.
      • Okuda K.
      • Kaushansky K.
      • Salgia R.
      • Griffin J.D.
      ,
      • Galisteo M.L.
      • Dikic I.
      • Batzer A.G.
      • Langdon W.Y.
      • Schlessinger J.
      ,
      • Meisner H.
      • Czech M.P.
      ,
      • Odai H.
      • Sasaki K.
      • Iwamatsu A.
      • Hanazono Y.
      • Tanaka T.
      • Mitani K.
      • Yazaki Y.
      • Hirai H.
      ). In mammalian cells, the function of p120CBL is not known; however, it may act downstream of c-Src signaling for bone resorption by osteoclasts (
      • Tanaka S.
      • Amling M.
      • Neff L.
      • Peyman A.
      • Uhlmann E.
      • Levy J.B.
      • Baron R.
      ). Also, the p120CBL homolog Sli-1 in Caenorhabditis elegans is a negative regulator of the epidermal growth factor receptor tyrosine kinase homologue Let-23 (
      • Yoon C.H.
      • Lee J.
      • Jongeward G.D.
      • Sternberg P.W.
      ). The formation of a complex containing CRKL, p85PI3K, and p120CBL has been previously demonstrated by us in BCR/ABL-transformed cells (
      • Sattler M.
      • Salgia R.
      • Okuda K.
      • Uemura N.
      • Durstin M.A.
      • Pisick E.
      • Xu G.
      • Li J.L.
      • Prasad K.V.
      • Griffin J.D.
      ). Although the function of the CRK family proteins is not known, a model by Feller et al. shows a hypothetical role for the tyrosine phosphorylation of CRK (
      • Feller S.M.
      • Knudsen B.
      • Hanafusa H.
      ). In this model the N-terminal SH3 domain interacts with the proline-rich domain of c-ABL. Subsequent tyrosine phosphorylation of CRK leads to dissociation of c-ABL from CRK and binding of the CRK SH2 domain to the tyrosine phosphorylated site in CRK. However, the data presented here and our previous studies in BCR/ABL-transformed cells do not suggest that CRKL, unlike CRK, undergoes intramolecular binding to its own SH2 domain.
      Overall, one model consistent with our data would be that a tyrosine phosphorylated c-Kit receptor first attracts the p85PI3K subunit through its SH2 domain to Tyr709. Some CRKL and 120CBL may already be bound to PI3K and may become targets for the c-Kit tyrosine kinase or another kinase. The tyrosine phosphorylation of p120CBL is likely to provide binding sites for the SH2 domains of CRKL and p85PI3K, further potentially stabilizing the complex. Thereafter the complex may leave the receptor intact or disassembled, and it is not clear at this time if p120CBL and CRKL are regulators of PI3K enzymatic activity or downstream effectors that require PI3K activity for an as yet unknown signaling function. In any case, it is likely that further elucidation of the functions of this complex will be helpful in understanding the signaling of c-Kit and PI3K in particular.

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