Phosphatidylinositol 3-kinase P85 Research Articles

The K Protein Domain That Recruits the Interleukin 1-responsive K Protein Kinase Lies Adjacent to a Cluster of c-Src and Vav SH3-binding Sites: IMPLICATIONS THAT K PROTEIN ACTS AS A DOCKING PLATFORM

The heterogeneous ribonucleoprotein particle (hnRNP) K protein interacts with multiple molecular partners including DNA, RNA, serine/threonine, and tyrosine kinases and the product of the proto-oncogene, Vav. The K protein is phosphorylated in vivo and in vitro on serine/threonine residues by an interleukin 1 (IL-1)-responsive kinase with which it forms a complex. In this study we set out to map the K protein domains that bind kinases. We demonstrate that the K protein contains a cluster of at least three SH3-binding sites (P1, PPGRGGRPMPPSRR, amino acids 265-278; P2, PRRGPPPPPPGRG, 285-297; and P3, RARNLPLPPPPPPRGG, 303-318) and that each one of these sites is capable of selectively engaging c-Src and Vav SH3 domains but not SH3 domains of Abl, p85 phosphatidylinositol 3-kinase, Grb-2, and Csk. We demonstrate that the K protein domain that recruits and is phosphorylated in an RNA-dependent manner by the IL-1-responsive kinase, designated KPK for K protein kinase, is contained within the 338-425-amino acid stretch and thus is contiguous but does not include the cluster of the SH3-binding sites. K protein and KPK co-immunoprecipitate from cell extracts with either c-Src or Vav, suggesting that K protein-KPK-c-Src and K protein-KPK-Vav complexes exist in vivo. Furthermore, in the context of K protein, c-Src can reactivate KPK in vitro. The succession of kinase-binding sites contained within the K protein that allow it to form multienzyme complexes and facilitate kinase cross-talk suggest that K protein may serve as a docking platform that promotes molecular interactions occurring during signal transduction.

In vitro association of the phosphatidylinositol 3‐kinase regulatory subunit (p85) with the human insulin receptor

The insulin receptor, as a consequence of ligand binding, undergoes autophosphorylation of critical tyrosyl residues within the cytoplasmic portion of its beta-subunit. The 85 kDa regulatory subunit of phosphatidylinositol (PI) 3-kinase (p85), an SH2 domain protein, has been implicated as a regulatory molecule in the insulin signal transduction pathway. For the present study, glutathione S-transferase (GST) fusion proteins of p85 SH2 domains were used to determine if such motifs associate directly with the autophosphorylated human insulin receptor. The p85 N + C (amino plus carboxyl) SH2 domains were demonstrated to associate with the autophosphorylated beta-subunit, while neither the GTPase activator protein (GAP) N SH2 domain nor the phospholipase C-gamma 1 (PLC gamma 1) N + C SH2 domains exhibited measurable affinity for the activated receptor. The p85 N SH2 domain demonstrated weak association with the insulin receptor, while the p85 C SH2 domain alone formed no detectable complexes with the insulin receptor. The association of p85 N + C SH2 domains with the autophosphorylated receptor was competed efficiently by a 15-residue tyrosine-phosphorylated peptide corresponding to the carboxyl-terminal region of the insulin receptor, but not by phosphopeptides of similar length derived from the juxtamembrane or regulatory regions. The insulin receptor C domain phosphopeptide inhibited the p85 N + C SH2 domain-insulin receptor complex with an IC0.5 of 2.3 +/- 0.35 microM, whereas a 10-residue phosphopeptide derived from the insulin receptor substrate 1 (IRS-1) competed with an IC0.5 of 0.54 +/- 0.10 microM. These results demonstrate that, in vitro, there is an association between the p85 regulatory protein and the carboxyl-terminal region of the activated insulin receptor that requires the presence of both the N and C SH2 domains. Furthermore, formation of the p85/insulin receptor complex may lead to signaling pathways independent of IRS-1.

Compensatory alterations for insulin signal transduction and glucose transport in insulin-resistant diabetes.

Insulin binding activates the receptor tyrosine kinase toward the insulin receptor substrate-1 (IRS-1). Phosphorylated IRS-1 then interacts with the p85 alpha subunit of phosphatidylinositol 3-kinase (PI3K), Nck, growth factor receptor-bound protein 2 (GRB2), and Syp, thus branching insulin's signal for both mitogenic and metabolic responses. To determine whether the expression of these proteins is altered in insulin resistance, the levels of these proteins were compared in adipose and liver tissues of nondiabetic mice and obese insulin-resistant diabetic KKAy mice. IR and PI3K p85 alpha protein levels were significantly lower in KKAy mice than in control nondiabetic mice, whereas IRS-1 protein levels were not altered. In contrast, the protein levels of GRB2, Nck, Syp, and GLUT-1 were dramatically elevated in KKAy fat, with less striking changes in liver. Treatment of diabetic animals with pioglitazone, an insulin-sensitizing antihyperglycemic agent, partially corrected the expression of some of these proteins. Taken together, these findings suggest that the insulin-resistant diabetic condition is characterized by changes in expression of insulin signal transduction components that may be associated with altered glucose metabolism.

Heregulin-stimulated Signaling in Rat Pheochromocytoma Cells: EVIDENCE FOR ErbB3 INTERACTIONS WITH Neu/ErbB2 AND p85

We have reported that overexpression of Neu leads to heregulin-stimulated neurite outgrowth and the tyrosine-phosphorylation of Neu and other cellular proteins in PC12 cells. Considering that Neu/ErbB2 alone is not able to functionally couple to heregulin, we looked for the possible involvement of ErbB3 in these neurite outgrowth and tyrosine phosphorylation responses. We found that heregulin stimulates the tyrosine phosphorylation of endogenous ErbB3 protein in PC12 cells and that this phosphorylation, like that of Neu, is greatly enhanced in cells that overexpress Neu. Furthermore, overexpression of ErbB3 in PC12 cells led to heregulin-stimulated neurite extension. In addition to becoming tyrosine-phosphorylated, Neu/ErbB2 and ErbB3 associate with each other, and each associates with the 85-kDa regulatory subunit (p85) of phosphatidylinositol 3-kinase in a heregulin-dependent manner. Thus, Neu/ErbB2 and ErbB3 appear to cooperate to mediate the heregulin signal in PC12 cells. Like heregulin, epidermal growth factor (EGF) also stimulate the tyrosine phosphorylation of both Neu and ErbB3. However, there are clear differences between the EGF- and heregulin-stimulated phosphorylations of ErbB3. In the heregulin response, two tyrosine-phosphorylated forms of ErbB3 are detected. Of these, only the more quickly migrating form (on SDS-polyacrylamide gel electrophoresis) is found to be associated with Neu, whereas the other, more slowly migrating form is uniquely capable of forming stable complexes with p85. In the EGF response, at least two tyrosine-phosphorylated forms of ErbB3 are detected, but these phosphoproteins have distinctly lower apparent molecular weights compared with the heregulin-stimulated ErbB3 phosphoproteins and do not complex with p85. Thus the formation of a stable ErbB3-p85 complex in PC12 cells is a unique outcome of heregulin signaling that correlates with the differences in cell morphology induced by the activated EGF receptor and the Neu tyrosine kinase.

The MATK Tyrosine Kinase Interacts in a Specific and SH2-dependent Manner with c-Kit

We have cloned a protein tyrosine kinase, MATK, which is expressed abundantly in megakaryocytes and the brain. We investigated whether MATK participates in the c-Kit ligand/stem cell factor (KL/SCF) signaling pathway in the megakaryocytic cell line CMK. After KL/SCF stimulation, five major proteins of molecular masses of 145, 113, 92, 76, and 63 kDa were rapidly and transiently tyrosine-phosphorylated in a time-dependent manner, peaking within 5 min, and returning to basal levels within 60 min. To study the role of MATK in the KL/SCF signaling pathway, glutathione S-transferase (GST) fusion proteins containing SH2 and SH3 domains of MATK were cloned, expressed in Escherichia coli, and purified. MATK-SH2, but not MATK-SH3, precipitated the tyrosine-phosphorylated c-Kit (molecular mass of 145 kDa) in KL/SCF-stimulated CMK cells. Other GST fusion proteins containing the SH2 domain of p85 of phosphatidylinositol 3-kinase, phospholipase C gamma-1, and ras-GAP also precipitated c-Kit. The tyrosine-phosphorylated c-Kit was co-immunoprecipitated with anti-MATK and anti-p85 antibodies in KL/SCF-stimulated CMK cells, but not in granulocyte-macrophage colony stimulating factor or interleukin-6-stimulated cells, suggesting receptor specificity. These results indicate that MATK associates with the c-Kit receptor following specific stimulation by KL/SCF via its SH2 domain and likely participates in transduction of growth signals induced by this cytokine in megakaryocytes.

Phosphatidylinositol 3‐kinase activity is not essential for CD28 costimulatory activity in Jurkat T cells: studies with a selective inhibitor, wortmannin

The interaction of CD28 with its counter-receptor, B7-1 (CD 80), on antigen-presenting cells induces a co-signal in T cells required to promote antigen-dependent interleukin-2 (IL-2) production and to prevent clonal anergy. CD28 stimulation causes both protein-tyrosine kinase and phosphatidylinositol3-kinase (PI3-K) activation, suggesting a possible role for these enzyme activities in CD28 co-signal transduction. Here, we investigate the effect of wortmannin, a selective and irreversible PI3-K inhibitor on CD28 co-signaling events in the Jurkat T cell line. Wortmannin added to cell cultures partially inhibits CD28-induced tyrosine phosphorylation of the putative p110 catalytic subunit of PI3-K, but does not block CD28-induced association of the p85 PI3-K regulatory subunit with the CD28 receptor. Wortmannin inhibits in a dose-dependent manner both total cellular PI3-K activity and CD28-induced receptor-associated PI3-K activity. Wortmannin (1 microM) inhibits cellular PI3-K activity by 90% with complete inhibition achieved at 10 microM. The inhibitory effect of wortmannin on cellular PI3-K activity is prolonged ( > 18 h), suggesting that the drug is not readily metabolized by Jurkat T cells. Wortmannin, at concentrations that blocked PI3-K activity, fails to inhibit the synergistic effect of CD28 on IL-2 secretion in the presence of phorbol 12-myristate 13-acetate and ionomycin. These data demonstrate that CD28-induced signaling events other than the activation of PI3-K catalytic activity contribute to the control of IL-2 secretion.

Potentiation of insulin stimulation of phosphatidylinositol 3-kinase by thiazolidinedione-derived antidiabetic agents in Chinese hamster ovary cells expressing human insulin receptors and L6 myotubes.

Thiazolidinedione derivatives are insulin-sensitizing agents with proven antidiabetic activities in vivo. To explore the mechanism of action of this class of compounds, the effects of pioglitazone, CP-86,325, and AD-5075 on elements of the insulin signal transduction pathways were studied in Chinese hamster ovary cells overexpressing human insulin receptor (CHO.T) and L6 myotubes. In CHO.T cells, the binding of insulin to its receptor and the insulin-stimulated tyrosine kinase activity of the receptor were not altered by pioglitazone or CP-86,325. In contrast, treatment of CHO.T cells with the compounds resulted in significant increases in insulin-stimulated phosphatidylinositol (PI) 3-kinase activity. This insulin-enhancing effect was also observed in L6 myotubes treated with CP-86,325. The augmentations in kinase activity observed in CHO.T cells correlated with increases in the amount of PI 3-kinase (p85 subunit) in anti-phosphotyrosine immunoprecipitates of cell lysates. No gross changes in the tyrosine phosphorylation state of the insulin receptor substrate-1 were detected in insulin-stimulated CHO.T cells following treatment with the compounds. Furthermore, the compounds did not enhance insulin stimulation of mitogen-activated protein kinase or DNA synthesis in CHO.T cells. Thus, thiazolidinedione-derived antidiabetic agents may act as insulin sensitizers by augmenting insulin stimulation of PI 3-kinase activity in a rather specific manner.

Rapid and efficient purification of Src homology 2 domain-containing proteins: Fyn, Csk and phosphatidylinositol 3-kinase p85.

To analyse the regulation of Src family tyrosine kinases in vitro, we have purified Fyn and Csk, a kinase capable of regulating Fyn activity by phosphorylation, from baculovirus-infected insect cells. The proteins were purified by affinity purification over a phosphotyrosine column. Highly purified proteins were eluted from the resin by a salt gradient and further purified by ion-exchange chromatography. This purification scheme was successfully applied to a third, unrelated protein that also contains the Src homology 2 (SH2) domain, namely the 85 kDa subunit of phosphatidylinositol 3-kinase, indicating that this method is versatile and should prove applicable to any protein with an accessible SH2 domain. The binding of Csk to different phosphopeptides was tested, and specificity for the autophosphorylation site of Fyn was demonstrated. Pure Csk was used to phosphorylate Fyn and down-regulate its kinase activity, and the kinetic parameters of both the active and the repressed forms of Fyn were determined. Repression of Fyn activity by Csk reduced binding of Fyn to phosphopeptides to undetectable levels, supporting the model that predicts an intramolecular interaction of the Fyn SH2 domain with a C-terminal phosphotyrosine residue.

The activation of phosphatidylinositol 3-kinase by Ras.

Activation of the mammalian phosphatidylinositol 3-kinase complex can play a critical role in transducing growth factor responses. The lipid kinase complex, which is made up of p85 alpha and p110 alpha regulatory and catalytic subunits, becomes associated with a number of activated receptor protein tyrosine kinases, but the mechanism of its activation has not yet been defined. Recent evidence indicates that Ras can bind to the p85 alpha/p110 alpha complex. We describe here the functional regulation of the mammalian phosphatidylinositol 3-kinase complex by Ras. Expression of p110 alpha, the catalytic subunit of phosphatidylinositol 3-kinase, in the fission yeast, Schizosaccharomyces pombe, has been used to demonstrate an inhibitory effect of p85 alpha on p110 alpha activity in intact cells; inhibition did not result from a decrease in p110 alpha expression. In this cellular context, we have investigated the effect of a constitutively active mutant of Ras, v-Ras, either on p85 alpha or p110 alpha-alone, or on the p85 alpha/p110 alpha complex. In the presence of the p85 alpha/p110 alpha complex, v-Ras suppressed cell growth, but an effector-domain mutant of v-Ras did not. The growth-suppressive effect of v-Ras was not seen for any other combination of expressed proteins. The phenotype induced by v-Ras was consistent with activation of the p85 alpha/p110 alpha complex: it was sensitive to the phosphatidylinositol 3-kinase inhibitor, wortmannin, and the cells accumulated 3-phosphorylated polyphosphoinositides. Activation of purified p85 alpha/p110 alpha by purified recombinant Ras in vitro was also demonstrated. The phosphatidylinositol 3-kinase complex, p85 alpha/p110 alpha, shows a suppressed catalytic function in vivo when compared with free p110 alpha. This complex can, however, be activated by Ras. We suggest that the phosphatidylinositol 3-kinase p85 alpha/p110 alpha complex is a downstream effector of Ras.

Physical and functional interactions between SH2 and SH3 domains of the Src family protein tyrosine kinase p59fyn.

The Src family protein tyrosine kinases participate in signalling through cell surface receptors that lack intrinsic tyrosine kinase domains. All nine members of this family possess adjacent Src homology (SH2 and SH3) domains, both of which are essential for repression of the enzymatic activity. The repression is mediated by binding between the SH2 domain and a C-terminal phosphotyrosine, and the SH3 domain is required for this interaction. However, the biochemical basis of functional SH2-SH3 interaction is unclear. Here, we demonstrate that when the SH2 and SH3 domains of p59fyn (Fyn) were present as adjacent domains in a single protein, binding of phosphotyrosyl peptides and proteins to the SH2 domain was enhanced, whereas binding of a subset of cellular polypeptide ligands to the SH3 domain was decreased. An interdomain communication was further revealed by occupancy with domain-specific peptide ligands: occupancy of the SH3 domain with a proline-rich peptide enhanced phosphotyrosine binding to the linked SH2 domain, and occupancy of the SH2 domain with phosphotyrosyl peptides enhanced binding of certain SH3-specific cellular polypeptides. Second, we demonstrate a direct binding between purified SH2 and SH3 domains of Fyn and Lck Src family kinases. Heterologous binding between SH2 and SH3 domains of closely related members of the Src family, namely, Fyn, Lck, and Src, was also observed. In contrast, Grb2, Crk, Abl, p85 phosphatidylinositol 3-kinase, and GTPase-activating protein SH2 domains showed lower or no binding to Fyn or Lck SH3 domains. SH2-SH3 binding did not require an intact phosphotyrosine binding pocket on the SH2 domain; however, perturbations of the SH2 domain induced by specific high-affinity phosphotyrosyl peptide binding abrogated binding of the SH3 domain. SH3-SH2 binding was observed in the presence of proline-rich peptides or when a point mutation (W119K) was introduced in the putative ligand-binding pouch of the Fyn SH3 domain, although these treatments completely abolished the binding to p85 phosphatidylinositol 3-kinase and other SH3-specific polypeptides. These biochemical SH2-SH3 interactions suggest novel mechanisms of regulating the enzymatic activity of Src kinases and their interactions with other proteins.

Physical and functional interactions between SH2 and SH3 domains of the Src family protein tyrosine kinase p59fyn.

The Src family protein tyrosine kinases participate in signalling through cell surface receptors that lack intrinsic tyrosine kinase domains. All nine members of this family possess adjacent Src homology (SH2 and SH3) domains, both of which are essential for repression of the enzymatic activity. The repression is mediated by binding between the SH2 domain and a C-terminal phosphotyrosine, and the SH3 domain is required for this interaction. However, the biochemical basis of functional SH2-SH3 interaction is unclear. Here, we demonstrate that when the SH2 and SH3 domains of p59fyn (Fyn) were present as adjacent domains in a single protein, binding of phosphotyrosyl peptides and proteins to the SH2 domain was enhanced, whereas binding of a subset of cellular polypeptide ligands to the SH3 domain was decreased. An interdomain communication was further revealed by occupancy with domain-specific peptide ligands: occupancy of the SH3 domain with a proline-rich peptide enhanced phosphotyrosine binding to the linked SH2 domain, and occupancy of the SH2 domain with phosphotyrosyl peptides enhanced binding of certain SH3-specific cellular polypeptides. Second, we demonstrate a direct binding between purified SH2 and SH3 domains of Fyn and Lck Src family kinases. Heterologous binding between SH2 and SH3 domains of closely related members of the Src family, namely, Fyn, Lck, and Src, was also observed. In contrast, Grb2, Crk, Abl, p85 phosphatidylinositol 3-kinase, and GTPase-activating protein SH2 domains showed lower or no binding to Fyn or Lck SH3 domains. SH2-SH3 binding did not require an intact phosphotyrosine binding pocket on the SH2 domain; however, perturbations of the SH2 domain induced by specific high-affinity phosphotyrosyl peptide binding abrogated binding of the SH3 domain. SH3-SH2 binding was observed in the presence of proline-rich peptides or when a point mutation (W119K) was introduced in the putative ligand-binding pouch of the Fyn SH3 domain, although these treatments completely abolished the binding to p85 phosphatidylinositol 3-kinase and other SH3-specific polypeptides. These biochemical SH2-SH3 interactions suggest novel mechanisms of regulating the enzymatic activity of Src kinases and their interactions with other proteins.

Insulin-like growth factor-1-mediated association of p85 phosphatidylinositol 3-kinase with pp 185: requirement of SH2 domains for in vivo interaction.

Insulin-like growth factor-I (IGF-1) stimulates the production of 3-phosphoinositides and increases the phosphatidylinositol 3-kinase activity that is immunoprecipitated by antiphosphotyrosine antibodies, a small portion of which are also associated with the IGF-1 receptor. In vitro reconstitution experiments showed that p85 associates with high affinity to the IGF-1 receptor and this interaction is mediated through the p85 SH2 groups. Moreover, in vitro, p85 is a substrate for the IGF-1 receptor tyrosine kinase activity. In this study, we analyzed the in vivo association of p85 with tyrosyl- phosphorylated proteins and its tyrosyl phosphorylation state, in response to IGF-1. After stimulation with IGF-1, the major tyrosylphosphorylated protein that was associated with p85 was a 185-kilodalton protein, identified as IRS-1. Only a small fraction of p85 was associated with the IGF-1 receptor. In contrast, the PDGF receptor was the major protein associated with p85 upon stimulation. Neither ligand stimulated the tyrosyl phosphorylation of p85 in vivo. In order to determine whether the SH2 domains of p85 were involved in its association with p185 in vivo after IGF-1 stimulation, different SH2-constructs of p85 were expressed in COS-1 cells. After stimulation with IGF-1, the expressed SH2 proteins were immunoprecipitated with specific antibodies, and associated p185 was detected on Western blots. These results show that both the p85 N-SH2 and N+C-SH2 associate with IRS-1 after IGF-1 stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Insulin-like growth factor-1-mediated association of p85 phosphatidylinositol 3-kinase with pp 185: requirement of SH2 domains for in vivo interaction

Insulin-like growth factor-1-mediated association of p85 phosphatidylinositol 3-kinase with pp 185: requirement of SH2 domains for in vivo interaction

Activation of phosphoinositide 3-kinase activity by Cdc42Hs binding to p85.

The Ras-like GTPase Cdc42 is essential for cell polarity and bud site assembly in Saccharomyces cerevisiae by regulating cell cycle-dependent reorganization of cortical cytoskeletal elements. However, its role in mammalian cells is unknown. To identify potential effectors of Cdc42Hs, we incubated lysates from NIH 3T3 fibroblasts or PC12 cells with immobilized glutathione S-transferase (GST)-Cdc42Hs fusion proteins bound to different guanine nucleotides and observed a specific association between the 85-kDa subunit (p85) of phosphatidylinositol 3-kinase (PI 3-kinase) and GTP gamma S (guanosine 5'-3-O-(thio)triphosphate)-bound GST-Cdc42Hs. Recombinant p85 formed a complex with GTP gamma S-bound GST-Cdc42Hs and with a GTPase-defective GTP-bound GST-Cdc42Hs-Q61L mutant, but not with a GTP gamma S-bound, effector domain GST-Cdc42HsT35A mutant. Both the Rho-GAP homology domain of p85 and the Cdc42Hs-GAP competitively inhibited the binding of recombinant p85 to Cdc42Hs. In addition, PI 3-kinase activity immunoprecipitated from cell lysates with anti-p85 antibody was stimulated 2-4-fold by GST-Cdc42-GTP gamma S. Similar interactions were observed between p85 and GST-Rac1-GTP gamma S but not between p85 and GST-RhoA-GTP gamma S. These findings suggest that PI 3-kinase, through the Rho-GAP homology domain of p85, can couple to the effector domain of Cdc42Hs and that p85 may be a target for the GTP-bound forms of Cdc42Hs and Rac1.

Platelet tyrosine kinases and fibrinogen receptor activation.

Platelet adhesion and aggregation during hemostasis and thrombosis are usually limited to sites where the integrity of the vessel wall is disrupted. The high concentration of platelet agonists within these sites represents a putative control mechanism for targeting platelet activation. Although much has been learned about the intracellular signaling systems controlling platelet activation, our understanding of the connection between signaling molecules and platelet aggregation remains limited. Tyrosine kinases are important signaling enzymes in cells and are abundant in platelets. Previous reports indicate that binding of glycoprotein IIb-IIIa (GPIIb-IIIa) to fibrinogen can induce the tyrosine phosphorylation of specific substrates. We show that, in turn, protein tyrosine kinase activity is necessary for agonist-induced activation of GPIIb-IIIa. Genistein and the tyrphostin AG-18 are two specific tyrosine kinase inhibitors, and the former has been shown to inhibit platelet aggregation. We use genistein and AG-18 in the present study to demonstrate that aggregation inhibition is due to suppression of GPIIb-IIIa activation. In contrast, genistin, an isoflavone compound related to genistein, and acetylsalicylic acid do not affect the tyrosine kinase-signaling pathway, nor do they inhibit GPIIb-IIIa activation induced by strong agonists. On identifying prominent tyrosine kinase substrates in activated platelets, we confirm that several substrates correspond to proteins associated with the cytoskeleton: the 85-kD subunit of phosphatidylinositol 3-kinase, the SH3-containing and actin-associating p85, pp60Src, and pp125FAK.(ABSTRACT TRUNCATED AT 250 WORDS)

T cell receptor-associated alpha-phosphatidylinositol 3-kinase becomes activated by T cell receptor cross-linking and requires pp56lck.

The activation of a phosphatidylinositol (PI) 3-kinase is one of the earliest intracellular responses to T lymphocyte stimulation. We have used a human T cell line, Jurkat, and an antibody that recognizes the clonotype of its T cell receptor (TcR) to characterize the association of PI 3-kinase with the TcR and its activation in response to TcR cross-linking. We show that the TcR is constitutively associated with the alpha isoform of the PI 3-kinase p85 regulatory subunit. Stimulation of Jurkat cells through the TcR results in the rapid and transient activation of the associated PI 3-kinase. In addition, our results indicate that the tyrosine kinase pp56lck is essential for PI 3-kinase activation via the TcR.

SH3 domain-mediated dimerization of an n-terminal fragment of the phosphatidylinositol 3-kinase p85 subunit

A recombinant fragment of the phosphatidylinositol 3-kinase p85 subunit containing the SH3 domain and an adjacent proline-rich region was analyzed by fluorescence spectroscopy and size exclusion chromatography. The recombinant protein dimerizes in solution with dissociation constant of 0.9 ± 0.3 μM.

Mutagenic analysis of the roles of SH2 and SH3 domains in regulation of the Abl tyrosine kinase.

We have used in vitro mutagenesis to examine in detail the roles of two modular protein domains, SH2 and SH3, in the regulation of the Abl tyrosine kinase. As previously shown, the SH3 domain suppresses an intrinsic transforming activity of the normally nontransforming c-Abl product in vivo. We show here that this inhibitory activity is extremely position sensitive, because mutants in which the position of the SH3 domain within the protein is subtly altered are fully transforming. In contrast to the case in vivo, the SH3 domain has no effect on the in vitro kinase activity of the purified protein. These results are consistent with a model in which the SH3 domain binds a cellular inhibitory factor, which in turn must physically interact with other parts of the kinase. Unlike the SH3 domain, the SH2 domain is required for transforming activity of activated Abl alleles. We demonstrate that SH2 domains from other proteins (Ras-GTPase-activating protein, Src, p85 phosphatidylinositol 3-kinase subunit, and Crk) can complement the absence of the Abl SH2 domain and that mutants with heterologous SH2 domains induce altered patterns of tyrosine-phosphorylated proteins in vivo. The positive function of the SH2 domain is relatively position independent, and the effect of multiple SH2 domains appears to be additive. These results suggest a novel mechanism for regulation of tyrosine kinases in which the SH2 domain binds to, and thereby enhances the phosphorylation of, a subset of proteins phosphorylated by the catalytic domain. Our data also suggest that the roles of the SH2 and SH3 domains in the regulation of Abl are different in several respects from the roles proposed for these domains in the closely related Src family of tyrosine kinases.

Mutagenic Analysis of the Roles of SH2 and SH3 Domains in Regulation of the Abl Tyrosine Kinase

We have used in vitro mutagenesis to examine in detail the roles of two modular protein domains, SH2 and SH3, in the regulation of the Abl tyrosine kinase. As previously shown, the SH3 domain suppresses an intrinsic transforming activity of the normally nontransforming c-Abl product in vivo. We show here that this inhibitory activity is extremely position sensitive, because mutants in which the position of the SH3 domain within the protein is subtly altered are fully transforming. In contrast to the case in vivo, the SH3 domain has no effect on the in vitro kinase activity of the purified protein. These results are consistent with a model in which the SH3 domain binds a cellular inhibitory factor, which in turn must physically interact with other parts of the kinase. Unlike the SH3 domain, the SH2 domain is required for transforming activity of activated Abl alleles. We demonstrate that SH2 domains from other proteins (Ras-GTPase-activating protein, Src, p85 phosphatidylinositol 3-kinase subunit, and Crk) can complement the absence of the Abl SH2 domain and that mutants with heterologous SH2 domains induce altered patterns of tyrosine-phosphorylated proteins in vivo. The positive function of the SH2 domain is relatively position independent, and the effect of multiple SH2 domains appears to be additive. These results suggest a novel mechanism for regulation of tyrosine kinases in which the SH2 domain binds to, and thereby enhances the phosphorylation of, a subset of proteins phosphorylated by the catalytic domain. Our data also suggest that the roles of the SH2 and SH3 domains in the regulation of Abl are different in several respects from the roles proposed for these domains in the closely related Src family of tyrosine kinases.

Phosphatidylinositol 3-kinase activation is mediated by high-affinity interactions between distinct domains within the p110 and p85 subunits.

Domains of interaction between the p85 and p110 subunits of phosphatidylinositol 3-kinase (PI 3-kinase) were studied with the yeast two-hybrid expression system. A gene fusion between the GAL4 transactivation domain and p85 activated transcription from a GAL1-lacZ reporter gene when complemented with a gene fusion between the GAL4 DNA binding domain and p110. To define subdomains responsible for this interaction, a series of p85 deletion mutants were analyzed. A 192-amino-acid inter-SH2 (IS) fragment (residues 429 to 621) was the smallest determinant identified that specifically associated with p110. In analogous experiments, the subdomain within p110 responsible for interaction with p85 was localized to an EcoRI fragment encoding the amino-terminal 127 residues. Expression of these two subdomains [p85(IS) with p110RI] resulted in 100-fold greater reporter activity than that obtained with full-length p85 and p110. Although the p85(IS) domain conferred a strong interaction with the p110 catalytic subunit, this region was not sufficient to impart phosphotyrosine peptide stimulation of PI 3-kinase activity. In contrast, coexpression of the p110 subunit with full-length p85 or with constructs containing the IS sequences flanked by both SH2 domains of p85 [p85(n/cSH2)] or either of the individual SH2 domains [p85(nSH2+IS) or p85(IS+cSH2)] resulted in PI 3-kinase activity that was activated by a phosphotyrosine peptide. These data suggest that phosphotyrosine peptide binding to either SH2 domain generates an intramolecular signal propagated through the IS region to allosterically activate p110.