Adaptor ShcA protein binds tyrosine kinase Tie2 receptor and regulates migration and sprouting but not survival of endothelial cells

Angiopoietin-1 can promote migration, sprouting, and survival of endothelial cells through activation of different signaling pathways triggered by the Tie2 tyrosine kinase receptor. ShcA adapter proteins are targets of activated tyrosine kinases and are implicated in the transmission of activation signals to the Ras/mitogen-activated protein kinase pathway. Here we report the identification of an interaction between the adapter protein ShcA and the cytoplasmic domain of Tie2 through in vitro co-immunoprecipitation analysis. Stimulation of endogenous Tie2 in endothelial cells with its ligand angiopoietin-1 increased its association with ShcA and phosphorylation of the adapter protein. The interaction requires the SH2 domain of ShcA and the tyrosine phosphorylation of Tie2 as shown by pull-down experiments. Furthermore, Tyr-1101 of Tie2 was identified as the primary binding site for the SH2 domain of ShcA. Overexpression of a dominant-negative form of ShcA affects angiopoietin-1-induced chemotaxis and sprouting, although it has no effect on survival of endothelial cells. Furthermore, this mutant partially reduces the tyrosine phosphorylation of the regulatory p85 subunit of phosphatidylinositol 3-kinase. Together, our results identified a novel interaction between Tie2 with the adapter molecule ShcA and suggested that this interaction may play a role in the regulation of migration and three-dimensional organization of endothelial cells induced by angiopoietin-1.     

The SH2 domain is required for stable phosphorylation of p56lck at tyrosine 505, the negative regulatory site.

The catalytic function of Src-related tyrosine protein kinases is repressed by phosphorylation of a conserved carboxy-terminal tyrosine residue. Recent studies suggest that this inhibitory event is not the result of autophosphorylation but that it is mediated by another cytoplasmic tyrosine protein kinase, termed p50csk. In this report, we have evaluated the processes regulating the extent of phosphorylation of the inhibitory carboxy-terminal tyrosine residue of p56lck, a lymphocyte-specific member of the Src family. By analyzing kinase-defective variants of p56lck expressed in mouse NIH 3T3 cells, we have found that the noncatalytic Src homology 2 (SH2) domain, but not the SH3 sequence or the sites of Lck myristylation and autophosphorylation, is necessary for stable phosphorylation at the carboxy-terminal tyrosine 505. Further studies in which Lck and Csk were coexpressed in S. cerevisiae indicated that the absence of the SH2 domain did not affect the ability of Csk to phosphorylate p56lck at tyrosine 505. However, we observed that incubation of cells with the tyrosine phosphatase inhibitor pervanadate restored the tyrosine 505 phosphorylation of Lck polypeptides devoid of the SH2 motif. Additionally, the presence of the SH2 sequence protected tyrosine 505 from in vitro dephosphorylation by the hemopoietic tyrosine protein phosphatase CD45. Taken together, these findings raised the possibility that the SH2 motif contributes to the physiological suppression of the catalytic function of p56lck at least in part through its ability to stabilize phosphorylation at the inhibitory site.     

Phosphotyrosine binding domain-dependent upregulation of the platelet-derived growth factor receptor alpha signaling cascade by transforming mutants of Cbl: implications for Cbl's function and oncogenicity.

Recent studies have demonstrated that Cbl, the 120-kDa protein product of the c-cbl proto-oncogene, serves as a substrate of a number of receptor-coupled tyrosine kinases and forms complexes with SH3 and SH2 domain-containing proteins, pointing to its role in signal transduction. Based on genetic evidence that the Caenorhabditis elegans Cbl homolog, SLI-1, functions as a negative regulator of the LET-23 receptor tyrosine kinase and our demonstration that Cbl's evolutionarily conserved N-terminal transforming region (Cbl-N; residues 1 to 357) harbors a phosphotyrosine binding (PTB) domain that binds to activated ZAP-70 tyrosine kinase, we examined the possibility that oncogenic Cbl mutants may activate mitogenic signaling by deregulating cellular tyrosine kinase machinery. Here, we show that expression of Cbl-N and two other transforming Cbl mutants (CblY368 delta and Cbl366-382 delta or Cb170Z), but not wild-type Cbl, in NIH 3T3 fibroblasts leads to enhancement of endogenous tyrosine kinase signaling. We identified platelet-derived growth factor receptor alpha (PDGFR alpha) as one target of mutant Cbl-induced deregulation. In mutant Cbl transfectants, PDGFR alpha was hyperphosphorylated and constitutively complexed with a number of SH2 domain-containing proteins. PDGFR alpha hyperphosphorylation and enhanced proliferation of mutant Cbl-transfected NIH 3T3 cells were drastically reduced upon serum starvation, and PDGF-AA substituted for the maintenance of these traits. PDGF-AA stimulation of serum-starved Cbl transfectants induced the in vivo association of transfected Cbl proteins with PDGFR alpha. In vitro, Cbl-N directly bound to PDGFR alpha derived from PDGF-AA-stimulated cells but not to that from unstimulated cells, and this binding was abrogated by a point mutation (G306E) corresponding to a loss-of-function mutation in SLI-1. The Cbl-N/G306E mutant protein, which failed to induce enhanced growth and transformation of NIH 3T3 cells, also failed to induce hyperphosphorylation of PDGFR alpha. Altogether, these findings identify a novel mechanism of Cbl's physiological function and oncogenesis, involving its PTB domain-dependent direct interaction with cellular tyrosine kinases.     

The association of Sam68 with Vav1 contributes to tumorigenesis

Vav1 functions in the hematopoietic system as a specific GDP/GTP nucleotide exchange factor regulated by tyrosine phosphorylation. An intact C-terminal SH3 domain of Vav1 (Vav1SH3C) was shown to be necessary for Vav1-induced transformation, yet the associating protein(s) necessary for this activity have not yet been identified. Using a proteomics approach, we identified Sam68 as a Vav1SH3C-associating protein. Sam68 (Src-associated in mitosis of 68 kD) belongs to the heteronuclear ribonucleoprotein particle K (hnRNP-K) homology (KH) domain family of RNA-binding proteins. The Vav1/Sam68 interaction was observed in vitro and in vivo. Mutants of Vav1SH3C previously shown to lose their transforming potential did not associate with Sam68. Co-expression of Vav1 and Sam68 in Jurkat T cells led to increased localization of Vav1 in the nucleus and changes in cell morphology. We then tested the contribution of Sam68 to known functions of Vav1, such as focus-forming in NIH3T3 fibroblasts and NFAT stimulation in T cells. Co-expression of oncogenic Vav1 with Sam68 in NIH3T3 fibroblasts resulted in a dose-dependent increase in foci, yet no further enhancement of NFAT activity was observed in Jurkat T cells, as compared to cells overexpressing only Vav1 or Sam68. Our results strongly suggest that Sam68 contributes to transformation by oncogenic Vav1.     

A novel Src homology 2 domain-containing molecule, Src-like adapter protein-2 (SLAP-2), which negatively regulates T cell receptor signaling

We have cloned a novel adapter protein containing Src homology 2 and Src homology 3 domains similar to the Src family of tyrosine kinases. This molecule lacks a catalytic tyrosine kinase domain and is related to a previously identified protein, Src-like adapter protein (SLAP), and is therefore designated SLAP-2. Northern blot analysis indicates that SLAP-2 is predominantly expressed in the immune system. Jurkat T cells express SLAP-2 protein and overexpression of SLAP-2 in these cells negatively regulates T cell receptor signaling as assessed by interleukin-2 promoter or NF-AT promoter reporter constructs. Mutational analysis revealed that an intact SH2 domain of SLAP-2 is essential for this inhibitory effect, whereas mutation of the SH3 domain alone has no effect. This inhibitory effect is upstream of the activation of Ras and increase of intracellular calcium levels, as no inhibition was observed when the cells were activated by phorbol ester plus ionomycin. SLAP-2 interacts with Cbl in vivo in a phosphorylation independent manner and with ZAP-70 and T cell receptor zeta chain upon T cell receptor activation. Finally, we show that the mutation of a predicted myristoylation site within the NH(2)-terminal of SLAP-2 is essential for its inhibitory effect. This report therefore implicates SLAP and SLAP-2 as a family of adapter proteins that negatively regulate T cell receptor signaling.     

Tyrosine-phosphorylated SOCS3 interacts with the Nck and Crk-L adapter proteins and regulates Nck activation

Suppressors of cytokine signaling (SOCS) are negative feedback inhibitors of cytokine and growth factor signal transduction. Although the affect of SOCS proteins on the Jak-STAT pathway has been well characterized, their role in the regulation of other signaling modules is not well understood. In the present study, we demonstrate that SOCS3 physically interacts with the SH2/SH3-containing adapter proteins Nck and Crk-L, which are known to couple activated receptors to multiple downstream signaling pathways and the actin cytoskeleton. Our data show that the SOCS3/Nck and SOCS3/Crk-L interactions depend on tyrosine phosphorylation of SOCS3 Tyr(221) within the conserved SOCS box motif and intact SH2 domains of Nck and Crk-L. Furthermore, SOCS3 Tyr(221) forms a YXXP motif, which is a consensus binding site for the Nck and Crk-L SH2 domains. Expression of SOCS3 in NIH3T3 cells induces constitutive recruitment of a Nck-GFP fusion protein to the plasma membrane and constitutive tyrosine phosphorylation of endogenous Nck. Our findings suggest that SOCS3 regulates multiple cytokine and growth factor-activated signaling pathways by acting as a recruitment factor for adapter proteins.     

GRID: a novel Grb-2-related adapter protein that interacts with the activated T cell costimulatory receptor CD28

Adapter proteins such as Grb2 play a central role in the formation of signaling complexes through their association with multiple protein binding partners. These interactions are mediated by specialized domains such as the well-characterized Src homology SH2 and SH3 motifs. Using yeast three-hybrid technology, we have identified a novel adapter protein, expressed predominantly in T lymphocytes, that associates with the activated form of the costimulatory receptor, CD28. The protein is a member of the Grb2 family of adapter proteins and contains an SH3-SH2-SH3 domain structure. A unique glutamine/proline-rich domain (insert domain) of unknown function is situated between the SH2 and N-terminal SH3 domains. We term this protein GRID for Grb2-related protein with insert domain. GRID coimmunoprecipitates with CD28 from Jurkat cell lysates following activation of CD28. Using mutants of CD28 and GRID, we demonstrate that interaction between the proteins is dependent on phosphorylation of CD28 at tyrosine 173 and integrity of the GRID SH2 domain, although there are also subsidiary stabilizing contacts between the PXXP motifs of CD28 and the GRID C-terminal SH3 domain. In addition to CD28, GRID interacts with a number of other T cell signaling proteins, including SLP-76 (SH2 domain-containing leukocyte protein of 76 kDa), p62dok, and RACK-1 (receptor for activated protein kinase C-1). These findings suggest that GRID functions as an adapter protein in the CD28-mediated costimulatory pathway in T cells.     

Association of p62, a multifunctional SH2- and SH3-domain-binding protein, with src family tyrosine kinases, Grb2, and phospholipase C gamma-1.

src family tyrosine kinases contain two noncatalytic domains termed src homology 3 (SH3) and SH2 domains. Although several other signal transduction molecules also contain tandemly occurring SH3 and SH2 domains, the function of these closely spaced domains is not well understood. To identify the role of the SH3 domains of src family tyrosine kinases, we sought to identify proteins that interacted with this domain. By using the yeast two-hybrid system, we identified p62, a tyrosine-phosphorylated protein that associates with p21ras GTPase-activating protein, as a src family kinase SH3-domain-binding protein. Reconstitution of complexes containing p62 and the src family kinase p59fyn in HeLa cells demonstrated that complex formation resulted in tyrosine phosphorylation of p62 and was mediated by both the SH3 and SH2 domains of p59fyn. The phosphorylation of p62 by p59fyn required an intact SH3 domain, demonstrating that one function of the src family kinase SH3 domains is to bind and present certain substrates to the kinase. As p62 contains at least five SH3-domain-binding motifs and multiple tyrosine phosphorylation sites, p62 may interact with other signalling molecules via SH3 and SH2 domain interactions. Here we show that the SH3 and/or SH2 domains of the signalling proteins Grb2 and phospholipase C gamma-1 can interact with p62 both in vitro and in vivo. Thus, we propose that one function of the tandemly occurring SH3 and SH2 domains of src family kinases is to bind p62, a multifunctional SH3 and SH2 domain adapter protein, linking src family kinases to downstream effector and regulatory molecules.     

SKAP55 recruits to lipid rafts and positively mediates the MAPK pathway upon T cell receptor activation

T cell receptor (TCR) engagement triggers a series of events including protein tyrosine kinase activation, tyrosine phosphorylation of adapter proteins, and multiple protein-protein interactions. We observed that adapter protein SKAP55, the Src kinase-associated phosphoprotein, formed homodimers through its SH3 domain and SK region. SKAP55 as a substrate interacted with Fyn kinase in vivo. In Jurkat cells, interaction between SKAP55 and Fyn kinase depended on TCR activation. Stable overexpression of SKAP55 in Jurkat cells caused mitogen-activated protein kinase activation following TCR engagement. Anti-CD3 stimulation also promoted the interaction of SKAP55 with Grb-2 in T cells. Mutational analysis revealed that tyrosine 271 in SKAP55 played a pivotal role for interaction with both Fyn kinase and adapter protein Grb-2, indicating that the Fyn-phosphorylated SKAP55 transiently associates with adapter Grb-2 to mediate mitogen-activated protein kinase activation. Intriguingly, T cell receptor engagement dramatically induced the translocation of endogenous SKAP55 to lipid rafts where SKAP55 was found to interact with Fyn kinase, suggesting that the positive function of SKAP55 via its association with Fyn and other signaling components may have been involved in raft-mediated T cell activation.     

Epidermal Growth Factor Stimulates the Tyrosine Phosphorylation of SHPS-1 and Association of SHPS-1 with SHP-2, a SH2 Domain-Containing Protein Tyrosine Phosphatase

SHPS-1 is a 120 kDa glycosylated receptor-like protein that contains immunoglobulin-like domains in its extracellular region and four potential tyrosine phosphorylation for SH2 domain binding sites in its cytoplasmic region. Epidermal growth factor (EGF) stimulated the rapid tyrosine phosphorylation of SHPS-1 and subsequent association of SHPS-1 with SHP-2, a protein tyrosine phosphatase containing SH2 domains, in Chinese hamster ovary cells overexpressing human EGF receptors. In the cells overexpressing SHPS-1, the tyrosine phosphorylation of SHPS-1 was more evident than that observed in parent cells. However, overexpression of SHPS-1 alone did not affect the activation of MAP kinase in response to EGF. These results suggest that SHPS-1 may be involved in the recruitment of SHP-2 from the cytosol to the plasma membrane in response to EGF.     

The v-Crk oncogene enhances cell survival and induces activation of protein kinase B/Akt

The v-Crk oncogene encodes an adaptor protein containing an SH2 domain and an SH3 domain. v-Crk-transformed fibroblast cells display enhanced tyrosine phosphorylation levels, and the v-Crk protein localizes in focal adhesions, suggesting that transformation may be due to enhanced focal complex signaling. Here we investigated the mechanism of transformation and found that v-Crk-transformed NIH 3T3 cells display growth rates and serum requirements similar to control cells. However, v-Crk enhanced survival in conditions of serum starvation. Both an intact SH2 and SH3 domain are required; moreover, SH2 mutants displayed dominant interfering properties, enhancing cell death. Using other cell death-inducing stimuli, it appeared that v-Crk in general inhibits apoptosis and enhances cell survival. In search of the signaling pathways involved, we found that v-Crk-transformed cells show constitutively higher levels of phospho-protein kinase B (PKB)/Akt and PKB/Akt activity, especially in conditions of serum starvation. These data strongly suggest involvement of the phosphatidylinositol 3-kinase/PKB survival pathway in the v-Crk-induced protection against apoptosis. In accordance, inhibition of this pathway by wortmannin or LY924002 reduced protection against starvation-induced apoptosis. In addition to the phosphatidylinositol 3-kinase/PKB pathway, a MEK-dependent pathway and an unknown additional pathway are also implicated in resistance against apoptosis. Activation of survival pathways may be the most important function of v-Crk in its oncogenic properties.     

CrkL is recruited through its SH2 domain to the erythropoietin receptor and plays a role in Lyn-mediated receptor signaling.

The erythropoietin (Epo) receptor transduces its signals by activating physically associated tyrosine kinases, mainly Jak2 and Lyn, and thereby inducing tyrosine phosphorylation of various substrates including the Epo receptor (EpoR) itself. We previously demonstrated that, in Epo-stimulated cells, an adapter protein, CrkL, becomes tyrosine-phosphorylated, physically associates with Shc, SHP-2, and Cbl, and plays a role in activation of the Ras/Erk signaling pathway. Here, we demonstrate that Epo induces binding of CrkL to the tyrosine-phosphorylated EpoR and SHIP1 in 32D/EpoR-Wt cells overexpressing CrkL. In vitro binding studies showed that the CrkL SH2 domain directly mediates the EpoR binding, which was specifically inhibited by a synthetic phosphopeptide corresponding to the amino acid sequences at Tyr(460) in the cytoplasmic domain of EpoR. The CrkL SH2 domain was also required for tyrosine phosphorylation of CrkL in Epo-stimulated cells. Overexpression of Lyn induced constitutive phosphorylation of CrkL and activation of Erk, whereas that of a Lyn mutant lacking the tyrosine kinase domain attenuated the Epo-induced phosphorylation of CrkL and activation of Erk. Furthermore, Lyn, but not Jak2, phosphorylated CrkL on tyrosine in in vitro kinase assays. Together, the present study suggests that, upon Epo stimulation, CrkL is recruited to the EpoR through interaction between the CrkL SH2 domain and phosphorylated Tyr(460) in the EpoR cytoplasmic domain and undergoes tyrosine phosphorylation by receptor-associated Lyn to activate the downstream signaling pathway leading to the activation of Erk and Elk-1.     

Lad, an adapter protein interacting with the SH2 domain of p56lck, is required for T cell activation.

T cell-specific Src family tyrosine kinase, p56lck, plays crucial roles in T cell differentiation, activation, and proliferation. These multiple functions of p56lck are believed to be conducted through the protein-protein interactions with various cellular signaling proteins. To clarify the mechanisms through which p56lck contributes to T cell signaling, we identified the proteins binding to the Src homology 2 (SH2) domain of p56lck through a tyrosine phosphorylation-dependent yeast two-hybrid screening. Subsequent characterization of positive clones revealed the presence of a protein of 366 aa named Lad (Lck-associated adapter protein), which is a potential murine homologue of previously reported TSAd, a T cell-specific adapter protein. Lad contains several protein-protein interaction domains including a zinc-finger motif, an SH2 domain, a proline-rich SH3 binding motif, and several phosphotyrosine sites. Furthermore, Lad was tyrosine phosphorylated and associated with p56lck in vivo and redistributed from cytoplasm to the plasma membrane in a T cell activation-dependent manner. Moreover in T cells, IL-2 promoter activity was enhanced upon coexpression of Lad but was inhibited by the coexpression of antisense Lad RNA. These characteristics of Lad suggest that Lad play an essential role as an adapter protein in p56lck-mediated T cell signaling.     

Modulation of Src Homology 3 Proteins by the Proline-Rich Adaptor Protein Shb

In the present study we have investigated a possible role for the proline-rich SH2 domain protein Shb as a regulator of expression or activity of certain SH3 domain proteins and MAP kinase. The expression of the Shb binding proteins Eps8, Src, and p85 PI3-kinase, PI3-kinase activity, and MAP kinase activation were assessed in wild-type NIH3T3 cells and in NIH3T3 cells overexpressing the Shb cDNA. In addition, the expression of the SH3 domain STAT1 proteins was assessed in wild-type and Shb overexpressing cells. The Eps8 protein content and Eps8 mRNA steady-state levels were downregulated, whereas the protein contents of Src and p85 PI3-kinase were unaffected by Shb overexpression. There was, however, an increased basal PI3-kinase activity in Shb transfected cells after a 3-h serum starvation. Increased steady-state levels of STAT1 mRNA were accompanied by an increased STAT1 protein content in Shb overexpressing cells. Shb overexpression was not associated with an altered activation of p44 or p42 MAP kinases in response to PDGF stimulation. The data presented in this study suggest novel functions for the adaptor protein Shb regulating the expression of certain signal-transducing SH3 domain proteins and modulating PI3-kinase activity.     

RACK1, a Receptor for Activated C Kinase and a Homolog of the β Subunit of G Proteins, Inhibits Activity of Src Tyrosine Kinases and Growth of NIH 3T3 Cells

To isolate and characterize proteins that interact with the unique domain and SH3 and SH2 domains of Src and potentially regulate Src activity, we used the yeast two-hybrid assay to screen a human lung fibroblast cDNA library. We identified RACK1, a receptor for activated C kinase and a homolog of the β subunit of G proteins, as a Src-binding protein. Using GST-Src fusion proteins, we determined that RACK1 binds to the SH2 domain of Src. Coimmunoprecipitation of Src and RACK1 was demonstrated with NIH 3T3 cells. Purified GST-RACK1 inhibited the in vitro kinase activity of Src in a concentration-dependent manner. GST-RACK1 (2 μM) inhibited the activities of purified Src and Lck tyrosine kinases by 40 to 50% but did not inhibit the activities of three serine/threonine kinases that we tested. Tyrosine phosphorylation on many cellular proteins decreased in 293T cells that transiently overexpressed RACK1. Src activity and cell growth rates decreased by 40 to 50% in NIH 3T3 cells that stably overexpressed RACK1. Flow cytometric analyses revealed that RACK1-overexpressing cells do not show an increased rate of necrosis or apoptosis but do spend significantly more time in G₀/G₁ than do wild-type cells. Prolongation of G₀/G₁ could account for the increased doubling time of RACK1-overexpressing cells. We suggest that RACK1 exerts its effect on the NIH 3T3 cell cycle in part by inhibiting Src activity.     

Genetic analysis of SH2D4A, a novel adapter protein related to T cell-specific adapter and adapter protein in lymphocytes of unknown function, reveals a redundant function in T cells

T cell-specific adapter (TSAd) protein and adapter protein in lymphocytes of unknown function (ALX) are two related Src homology 2 (SH2) domain-containing signaling adapter molecules that have both been shown to regulate TCR signal transduction in T cells. TSAd is required for normal TCR-induced synthesis of IL-2 and other cytokines in T cells and acts at least in part by promoting activation of the LCK protein tyrosine kinase at the outset of the TCR signaling cascade. By contrast, ALX functions as a negative-regulator of TCR-induced IL-2 synthesis through as yet undetermined mechanisms. In this study, we report a novel T cell-expressed adapter protein named SH2D4A that contains an SH2 domain that is highly homologous to the TSAd protein and ALX SH2 domains and that shares other structural features with these adapters. To examine the function of SH2D4A in T cells we produced SH2D4A-deficient mice by homologous recombination in embryonic stem cells. T cell development, homeostasis, proliferation, and function were all found to be normal in these mice. Furthermore, knockdown of SH2D4A expression in human T cells did not impact upon their function. We conclude that in contrast to TSAd and ALX proteins, SH2D4A is dispensable for TCR signal transduction in T cells.     

Interaction of fibroblast growth factor receptor 3 and the adapter protein SH2-B. A role in STAT5 activation

Fibroblast growth factor receptor 3 (FGFR3) influences a diverse array of biological processes, including cell growth, differentiation, and migration. Activating mutations in FGFR3 are associated with multiple myeloma, cervical carcinoma, and bladder cancer. To identify proteins that interact with FGFR3 and which may mediate FGFR3-dependent signaling, a yeast two-hybrid screen was employed using the cytoplasmic kinase domain of FGFR3 as bait. We identified the adapter protein SH2-B as an FGFR3-interacting protein. Coimmunoprecipitation experiments demonstrate binding of the SH2-B beta isoform to FGFR3 in 293T cells. Tyrosine phosphorylation of SH2-B beta was observed when coexpressed with activated FGFR3 mutants such as the weakly activated mutant N540K or the strongly activated mutant K650E, both associated with human developmental syndromes. The extent of tyrosine phosphorylation of SH2-B beta correlates with receptor activation, suggesting that FGFR3 activation mediates tyrosine phosphorylation of SH2-B beta. Furthermore, two tyrosine phosphorylation sites of FGFR3, Tyr-724 and Tyr-760, are required for optimal binding of the Src homology-2 (SH2) domain of SH2-B beta. We also demonstrate the phosphorylation and nuclear translocation of Stat5 by activated FGFR3, which increases in response to overexpression of SH2-B beta. Taken together, our results identify SH2-B beta as a novel FGFR3 binding partner that mediates signal transduction.     

Activation of phosphatidylinositol-3 kinase by nerve growth factor involves indirect coupling of the trk proto-oncogene with src homology 2 domains.

Growth factor receptor tyrosine kinases can form stable associations with intracellular proteins that contain src homology (SH) 2 domains, including the p85 regulatory subunit of phosphatidylinositol (PI)-3 kinase. The activation of this enzyme by growth factors is evaluated in PC12 pheochromocytoma cells and NIH 3T3 fibroblasts expressing the pp140c-trk nerve growth factor (NGF) receptor (3T3-c-trk). NGF causes the rapid stimulation of PI-3 kinase activity detected in anti-phosphotyrosine, but not in anti-trk, immunoprecipitates. This effect coincides with the tyrosine phosphorylation of two proteins, with molecular masses of of 100 kd and 110 kd, that coimmunoprecipitate with p85. Similar phosphorylation patterns are induced when an immobilized fusion protein containing the amino-terminal SH2 domain of p85 is used to precipitate tyrosine-phosphorylated proteins. Thus, although NGF produces the rapid activation of PI-3 kinase through a mechanism that involves tyrosine phosphorylation, there is no evidence for tyrosine phosphorylation of p85, or for its ligand-dependent association with the NGF receptor. Perhaps another phosphoprotein may link the NGF receptor to this enzyme.