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The c-Myc and E2F transcription factors are among the most potent regulators of cell cycle progression in higher eukaryotes. This report describes the isolation of a novel, highly conserved 434 kDa protein, designated TRRAP, which interacts specifically with the c-Myc N terminus and has homology to the ATM/PI3-kinase family. TRRAP also interacts specifically with the E2F-1 transactivation domain. Expression of transdominant mutants of the TRRAP protein or antisense RNA blocks c-Myc- and E1A-mediated oncogenic transformation. These data suggest that TRRAP is an essential cofactor for both the c-Myc and E1A/E2F oncogenic transcription factor pathways.

ErbB3 (HER3), a unique member of the ErbB receptor family, lacks intrinsic protein tyrosine kinase activity and contains six Tyr-Xaa-Xaa-Met (YXXM) consensus binding sites for the SH2 domains of the p85 regulatory subunit of phosphoinositide 3-kinase. ErbB3 also has a proline-rich sequence that forms a consensus binding site for the SH3 domain of p85. Here we have investigated the interacting domains of ErbB3 and p85 by a unique application of the yeast two-hybrid system. A chimaeric ErbB3 molecule containing the epidermal growth factor receptor protein tyrosine kinase domain was developed so that the C-terminal domain of ErbB3 could become phosphorylated in the yeast system. We also generated several ErbB3 deletion and Tyr-->Phe site-specific mutants, and observed that a single ErbB3 YXXM motif was necessary and sufficient for the association of ErbB3 with p85. The incorporation of multiple YXXM motifs into the ErbB3 C-terminus enabled a stronger ErbB3/p85 interaction. The proline-rich region of ErbB3 was not necessary for interaction with p85. However, either deletion or mutation of the p85 SH3 domain decreased the observed ErbB3/p85 association. Additionally an ErbB3/p85 SH3 domain interaction was detected by an assay in vitro. These results were consistent with a model in which pairs of phosphorylated ErbB3 YXXM motifs co-operate in binding to the tandem SH2 domains of p85. Although a contributing role for the p85 SH3 domain was suggested, the N- and C-terminal SH2 domains seemed to be primarily responsible for the high-affinity association of p85 and ErbB3.

Background: Leukocyte activation results in a rapid increase in adhesion to the extracellular matrix due to the activation of beta 1 integrin receptors. A role for phosphatidylinositol (PI) 3-kinase in integrin activation has been proposed, as activation of integrins by many receptors can be blocked by PI 3-kinase inhibitors. One receptor that regulates integrins is the CD28 surface antigen; here, we investigated the mechanisms responsible for CD28-mediated integrin activation., Results: CD28-mediated integrin activation was blocked by mutation of the binding site for the p85 catalytic subunit of PI 3-kinase in the CD28 cytoplasmic domain, and by expression of a dominant-negative form of the p85 subunit. Substitution of the Src homology 2 (SH2)-binding motif in the CD28 cytoplasmic domain for the corresponding motif in the CD28-related CTLA-4 surface antigen also blocked integrin activation but did not affect the recruitment and activation of PI 3-kinase. Mutations of the CD28 cytoplasmic domain that blocked integrin activation also impaired the tyrosine phosphorylation of the Cbl adaptor protein and the activation of the PI 3-kinase that was associated with Cbl. This Cbl-associated PI 3-kinase was distinct from the PI 3-kinase that coprecipitated with the CD28 cytoplasmic domain. CD28-mediated activation of beta 1 integrins was inhibited by expression of a mutant Cbl protein that shows reduced association with PI 3-kinase., Conclusions: Cbl is required for PI-3-kinase-dependent regulation of integrin receptors by CD28. Furthermore, CD28 is coupled to two distinct pools of PI 3-kinase, one directly associated with the CD28 cytoplasmic tail and the other associated with Cbl.

Phosphatidylinositol 3-kinase (PI3-kinase) is a heterodimeric enzyme composed of a catalytic subunit of 110 kDa and an adaptor regulatory subunit. We investigated the presence and localization of five isoforms of the regulatory subunits, p55 alpha, p55 gamma, p85 alpha, p85 beta, and p50 alpha, in the rat brain. In situ hybridization histochemistry using isoform-specific cRNA probes revealed that all five isoforms were expressed in the neurons of the brain. Interestingly, most neuronal cells including Purkinje cells in the cerebellum and pyramidal cells in the cerebrum expressed all five isoforms. Immunohistochemical staining also showed the localization of p55 alpha, p55 gamma, p85 alpha, and p50 alpha in the neuronal cells in the brain. Expression of multiple isoforms in neurons suggests that they may play important roles in signal transduction in the brain.

Because insulin-like growth factor-I (IGF-I), insulin, and interleukin-4 (IL-4) have known biological effects in breast cancer cells and signal through insulin-receptor substrate (IRS) adaptor proteins, we examined the expression and function of IRS-1 and IRS-2 in breast tumors and cell lines. IRS-1 and IRS-2 were expressed by cell lines and primary breast tumor specimens. IGF-I, insulin, and IL-4 treatment of MCF-7 and ZR-75, and IGF-I treatment of T47-D breast cancer cells, resulted in much greater tyrosine phosphorylation of IRS-1 compared with IRS-2. Furthermore, IGF-I stimulated greater tyrosine phosphorylation of IRS-1 than either insulin or IL-4. IGF-I treatment also enhanced association of the p85 regulatory subunit of phosphatidylinositol 3-kinase with IRS-1 and stimulated increased enzymatic activity compared with IL-4 and insulin in all three cell lines. Similarly, mitogen-activated protein kinase activity was greater in IGF-I-stimulated cells. To determine the functional significance of the activation of these pathways, we inhibited activation of phosphatidylinositol 3-kinase with wortmannin and mitogen-activated protein kinase with PD098059. Both compounds inhibited IGF-stimulated growth, suggesting that both pathways contributed to the mitogenic response to IGF-I. We conclude that IRS-1, and not IRS-2, is the predominant signaling molecule activated by IGF-I, insulin, and IL-4. Furthermore, enhanced tyrosine phosphorylation of IRS-1 by IGF-I, compared with either insulin or IL-4, is associated with greater activation of mitogenic downstream signaling pathways resulting in enhanced cell growth.

The SH3 domain is a well characterized small protein module with a simple fold found in many proteins. At acid pH, the SH3 domain (PI3-SH3) of the p85alpha subunit of bovine phosphatidylinositol 3-kinase slowly forms a gel that consists of typical amyloid fibrils as assessed by electron microscopy, a Congo red binding assay, and x-ray fiber diffraction. The soluble form of PI3-SH3 at acid pH (the A state by a variety of techniques) from which fibrils are generated has been characterized. Circular dichroism in the far- and near-UV regions and 1H NMR indicate that the A state is substantially unfolded relative to the native protein at neutral pH. NMR diffusion measurements indicate, however, that the effective hydrodynamic radius of the A state is only 23% higher than that of the native protein and is 20% lower than that of the protein denatured in 3.5 M guanidinium chloride. In addition, the A state binds the hydrophobic dye 1-anilinonaphthalene-8-sulfonic acid, which suggests that SH3 in this state has a partially formed hydrophobic core. These results indicate that the A state is partially folded and support the hypothesis that partially folded states formed in solution are precursors of amyloid deposition. Moreover, that this domain aggregates into amyloid fibrils suggests that the potential for amyloid deposition may be a common property of proteins, and not only of a few proteins associated with disease.

Phosphoinositide 3-kinases (PI3Ks) have been shown to play critical roles in cell growth, differentiation, survival, and vesicular transport. Class II PI3Ks have been recently identified in mouse and human (PI3K-C2 alpha/m-p170/m-cpk and HsC2-PI3K) and in Drosophila (PI3K 68D/cpk) which contain C2 domain at the C-terminus. However, their physiological function is largely unknown. We report here cloning and characterization of murine PI3K-C2 gamma, a novel class II PI3K. The catalytic domain as well as C2 domain are highly conserved in the Class II PI3K family, while the N-terminal regions of these proteins share little similarity. Unlike other Class II PI3Ks, PI3K-C2 gamma exclusively expressed in the liver, and a N-terminal truncated form was found in lung and a certain hematopoietic cell line. Specific antiserum against PI3K-C2 gamma precipitated PI3K activity from the membrane fraction of mouse liver but not from heart. Recombinant PI3K-C2 gamma exhibited a restricted lipid substrate specificity; it phosphorylated phosphatidylinositol (PtdIns) and PtdIns4P but not PtdIns(4,5)P2. Deletion mutations revealed that both the N-terminal region and the C2 domain were critical for enzymatic activity. The murine PI3K-C2 gamma gene locus was mapped to the distal region of mouse chromosome 6 in a region of homology with human chromosome 12p, which is distinct from the position of HsC2-PI3K. Cloning and biochemical characterization of the third member of class II PI3Ks provide a new insight into the function of this subfamily of PI3Ks.

We have used the yeast two-hybrid system to identify proteins that interact with the intracellular domain of the insulin-like growth factor-I receptor (IGFIR). In a search of a human fetal brain library we identified a cDNA encoding a protein that is the human homologue of mouse p55PIK, a regulatory subunit of phosphatidylinositol 3-kinase (hp55 gamma). The hp55 gamma protein interacts strongly with the activated IGFIR but not with the kinase-negative mutant receptor. hp55 gamma also interacts with the insulin receptor (IR) in the yeast two-hybrid system. The putative hp55 gamma protein is composed of a unique amino terminal region followed by a proline-rich motif and two Src homology 2 (SH2) domains, which are highly homologous to those in mouse p55PIK, rat p55 gamma, human p85 alpha and bovine p85 beta; it contains no SH3 domain. hp55 gamma mRNAs are expressed in most human fetal and adult tissues with particularly high abundance in adult testis. Splice variant(s) of hp55 gamma, one of which has a deletion of 36 amino acids at the amino terminus and another which has an insertion of 59 amino acids at position 256 between the SH2 domains, were also identified. A GST-hp55 gamma fusion protein interacts in vitro with both the activated IGFIR and IR derived from mammalian cells. Our findings suggest that hp55 gamma interacts with the IGFIR and IR and may be involved in PI 3-kinase activation by these receptors.

The refolding kinetics of the chemically denatured SH3 domain of phosphatidylinositol 3'-kinase (PI3-SH3) have been monitored by real-time one-dimensional 1H NMR coupled with a variety of other biophysical techniques. These experiments indicate that the refolding kinetics of PI3-SH3 are biphasic. The slow phase (27 (+/- 8)% amplitude) is due to a population of substantially unfolded molecules with an incorrectly configured cis proline residue. The fast phase (73 (+/- 8)% amplitude) corresponds to the folding of protein molecules with proline residues in a trans configuration in the unfolded state. NMR experiments indicate that the first species populated after the initiation of folding exhibit poor chemical shift dispersion and have spectra very similar to that of the denatured protein in 8 M guanidine hydrochloride. Linear combinations of the first spectrum and of the spectrum of the native protein accurately reconstruct all of the spectra acquired during refolding. Consistent with this, native side-chain and backbone H alpha atom packing (NMR), secondary structure (far-UV circular dichroism), burial of aromatic residues (near-UV circular dichroism), intrinsic fluorescence and peptide binding activity are all recovered with effectively identical kinetics. Equilibrium unfolding and folding/unfolding kinetics yield, within experimental error, identical values for the free energy of unfolding (delta Gu-H2O = 3.38 kcal mol-1) and for the slope of the free energy of unfolding versus denaturant concentration (meq = 2.33 kcal mol-1 M-1). Together, these data provide strong evidence that PI3-SH3 folds without significant population of kinetic well-structured intermediates. That PI3-SH3 folds slowly (time constant 2.8 seconds in H2O at 20 degrees C) indicates that slow refolding is not always a consequence of kinetic traps but may be observed even when a protein appears to fold via a simple, two-state mechanism.

Heterodimeric class IA phosphoinositide 3-kinase (PI 3-kinase) plays a crucial role in a variety of cellular signalling events downstream of a number of cell-surface receptor tyrosine kinases. Activation of the enzyme is effected in part by the binding of two Src homology-2 domains (SH2) of the 85 kDa regulatory subunit to specific phosphotyrosine-containing peptide motifs within activated cytoplasmic receptor domains. The solution structure of the uncomplexed C-terminal SH2 (C-SH2) domain of the p85 alpha subunit of PI 3-kinase has been determined by means of multinuclear, double and triple-resonance NMR experiments and restrained molecular-dynamics simulated-annealing calculations. The solution structure clearly indicates that the uncomplexed C-SH2 domain conforms to the consensus polypeptide fold exhibited by other SH2 domains, with an additional short helical element at the N terminus. In particular, the C-SH2 structure is very similar to both the p85 alpha N-terminal SH2 domain (N-SH2) and the Src SH2 domain with a root mean square difference (rmsd) for 44 C alpha atoms of 1.09 and 0.89 A, respectively. The canonical BC, EF and BG loops are less well-defined by the experimental restraints and show greater variability in the ensemble of C-SH2 conformers. The lower level of definition in these regions may reflect the presence of conformational disorder, an interpretation supported by the absence or broadening of backbone and side-chain NMR resonances for some of these residues. NMR experiments were performed, where C-SH2 was titrated with phosphotyrosine-containing peptides corresponding to p85 alpha recognition sites in the cytoplasmic domain of the platelet-derived growth-factor receptor. The ligand-induced chemical-shift perturbations indicate the amino-acid residues in C-SH2 involved in peptide recognition follow the pattern predicted from homologous complexes. A series of C-SH2 mutants was generated and tested for phosphotyrosine peptide binding by surface plasmon resonance. Mutation of the invariant Arg36 (beta B5) to Met completely abolishes phosphopeptide binding. Mutation of each of Ser38, Ser39 or Lys40 in the BC loop to Ala reduces the affinity of C-SH2 for a cognate phosphopeptide, as does mutation of His93 (BG5) to Asn. These effects are consistent with the involvement of the BC loop and BG loops regions in ligation of phosphopeptide ligands. Mutation of Cys57 (beta D5) in C-SH2 to Ile, the corresponding residue type in the p85 alpha N-SH2 domain, results in a change in peptide binding selectivity of C-SH2 towards that demonstrated by p85 alpha N-SH2. This pattern of p85 alpha phosphopeptide binding specificity is interpreted in terms of a model of the p85 alpha/PDGF-receptor interaction.

The Src homology 2 (SH2)-containing protein tyrosine phosphatase 1, SHP-1, is highly expressed in all hematopoietic cells as well as in many non-hematopoietic cells, particularly in some malignant epithelial cell lines. In hematopoietic cells, SHP-1 negatively regulates multiple cytokine receptor pathways. The precise function and the targets of SHP-1 in non-hematopoietic cells, however, are largely unknown. Here we demonstrate that SHP-1 associates with both the tyrosine-phosphorylated platelet-derived growth factor (PDGF) receptor and the p85 subunit of phosphatidylinositol 3-kinase in MCF-7 and TRMP cells. Through the use of mutant PDGF receptors and performing peptide competition for immunoprecipitation, it was determined that SHP-1 independently associates with the PDGF receptor and p85 and that its N-terminal SH2 domain is directly responsible for the interactions. Overexpression of SHP-1 in TRMP cells transfected with the PDGF receptor markedly inhibited PDGF-induced c-fos promoter activation, whereas the expression of three catalytically inactive SHP-1 mutants increased the c-fos promoter activation in response to PDGF stimulation. These results indicate that SHP-1 might negatively regulate PDGF receptor-mediated signaling in these cells. Identification of the association of SHP-1 with the PDGF receptor and p85 in MCF-7 and TRMP cells furthers our understanding of the function of SHP-1 in non-hematopoietic cells.

p85/p110 phosphoinositide 3-kinase (PI3K) is a heterodimer composed of a p85-regulatory and a p110-catalytic subunit, which is involved in a variety of cellular responses including cytoskeletal organization, cell survival and proliferation. We describe here the cloning and characterization of p65-PI3K, a mutant of the regulatory subunit of PI3K, which includes the initial 571 residues of the wild type p85alpha-protein linked to a region conserved in the eph tyrosine kinase receptor family. We demonstrate that this mutation, obtained from a transformed cell, unlike previously engineered mutations of the regulatory subunit, induces the constitutive activation of PI3K and contributes to cellular transformation. This report links the PI3K enzyme to mammalian tumor development for the first time.

Phosphatidylinositol 3-kinase (PI3K) is a heterodimer lipid kinase consisting of an 85-kD subunit bound to a 110-kD catalytic subunit that also possesses intrinsic, Mn(2+)-dependent protein serine kinase activity capable of phosphorylating the 85-kD subunit. Here, we examine the Mn(2+)-dependent protein kinase activity of PI3K alpha immunoprecipitated from normal resting or thrombin-stimulated platelets, and characterize p85/p110 phosphorylation, in vitro. Phosphoamino acid analysis of phosphorylated PI3K alpha showed p85 and p110 were phosphorylated on serine, but in contrast to previous results, were also phosphorylated on threonine and tyrosine. Wortmannin and LY294002 inhibited p85 phosphorylation; however, p110 phosphorylation was also inhibited suggesting p110 autophosphorylation on serine/threonine. The protein tyrosine kinase inhibitor, erbstatin analog, partially inhibited p85 and p110 phosphorylation but did not appear to affect PI3K lipid kinase activity. The in vitro phosphorylation of p85 alpha or p110 alpha derived from thrombin-stimulated platelets was no different than that of resting platelets, but we confirm that in thrombin receptor-stimulated platelets enhanced levels of p85 alpha and PI3K lipid kinase activity were recovered in antiphosphotyrosine antibody immunoprecipitates. These results suggest PI3K alpha can autophosphorylate on serine and threonine, and both p85 alpha and p110 alpha are substrates for a constitutively-associated protein tyrosine kinase in platelets.

Bruton's tyrosine kinase (Btk) is essential for normal B lymphocyte development and function. The activity of Btk is partially regulated by transphosphorylation within its kinase domain by Src family kinases at residue Tyr-551 and subsequent autophosphorylation at Tyr-223. Activation correlates with Btk association with cellular membranes. Based on specific loss of function mutations, the Btk pleckstrin homology (PH) domain plays an essential role in this activation process. The Btk PH domain can bind in vitro to several lipid end products of the phosphatidylinositol 3-kinase (PI 3-kinase) family including phosphatidylinositol 3,4,5-trisphosphate. Activation of Btk as monitored by elevation of phosphotyrosine content and a cellular transformation response was dramatically enhanced by coexpressing a weakly activated allele of Src (E378G) and the two subunits of PI 3-kinase-gamma. This activation correlates with new sites of phosphorylation on Btk identified by two-dimensional phosphopeptide mapping. Activation of Btk was dependent on the catalytic activity of all three enzymes and an intact Btk PH domain and Src transphosphorylation site. These combined data define Btk as a downstream target of PI 3-kinase-gamma and Src family kinases.

We identified a novel phosphatidylinositol (PI) 3-kinase by screening human brain cDNA libraries with probes designed from the conserved kinase-domain sequence. Analysis of cDNAs indicated that two different forms of transcripts are present: one is the full-length form composed of 1,044 amino acid residues and the other is the short form that the N-terminal 216 amino acid residues including a putative p85 binding domain has been truncated (828 amino acid residues). Database search revealed the sequence of the full-length form to be identical to that recently registered by D. Chantry et al. (Accession No. U86453 in GenBank release, August 1997). Northern blot analysis showed this mRNA to be ubiquitously expressed in various tissues, with relatively higher expression was observed in spleen, thymus and leukocytes. Based on fluorescence in situ hybridization and PCR-based analyses with both human/rodent mono-chromosomal hybrid cell panels and radiation hybrid mapping panels, this gene was localized to chromosome region 1p36.2. This region is frequently lost in a variety of human malignancies, including neuroblastoma. The novel PI3K could be a candidate target of the 1p36 alteration that occurs in neuroendocrine tumors.

An 85-kD cytosolic complex (p62(cplx)), consisting of a 62-kD phosphoprotein (p62) and a 25-kD GTPase, has been shown to be essential for the cell-free reconstitution of polymeric IgA receptor (pIgA-R)-containing exocytic transport vesicle formation from the TGN (Jones, S.M., J.R. Crosby, J. Salamero, and K.E. Howell. 1993. J. Cell Biol. 122:775-788). Here the p62(cplx) is identified as a regulatory subunit of a novel phosphatidylinositol 3-kinase (PI3-kinase). This p62(cplx)-associated PI3-kinase activity is stimulated by activation of the p62(cplx)-associated GTPase, and is specific for phosphatidylinositol (PI) as substrate, and is sensitive to wortmannin at micromolar concentrations. The direct role of this p62(cplx)-associated PI3-kinase activity in TGN-derived vesicle formation is indicated by the finding that both lipid kinase activity and the formation of pIgA-R-containing exocytic vesicles from the TGN are inhibited by wortmannin with similar dose-response curves and 50% inhibitory concentrations (3.5 microM). These findings indicate that phosphatidylinositol-3-phosphate (PI[3]P) is required for the formation of TGN-derived exocytic transport vesicles, and that the p62(cplx)-associated PI3-kinase and an activated GTPase are the essential molecules that drive production of this PI(3)P.

Mammalian phosphatidylinositol 3-kinase (PI 3-kinase) plays an important role in the regulation of various cellular, receptor tyrosine kinase-mediated processes, such as mitogenesis and transformation. PI 3-kinase is composed of a 110-kDa catalytic subunit and a regulatory subunit of 85 kDa or 55 kDa. We have cloned a gene for a regulatory subunit from Drosophila melanogaster, named droPIK57, from head-specific cDNA libraries. The droPIK57 gene encodes a protein containing two SH2 domains with significant sequence homology to those in p85 and p55. Like the p55 subunits, DroPIK57 is missing the SH3 domain and the bcr homology region of the p85 subunit. The short N-terminus as well as the C-terminus of the DroPIK57 protein show no identity to the known PI 3-kinase subunits, suggesting that it is a new member in the family of regulatory subunits. In-situ hybridization and Northern blot analysis indicate a widespread function of this gene during embryogenesis and in the CNS.

The generation of phosphatidylinositide 3-phosphates has been observed in a variety of cellular responses. The enzymes that mediate synthesis are the phosphoinositide 3-kinases (PI3-Ks) that form a family of structurally diverse enzymes with distinct substrate specificities. In this paper, we describe the cloning of a novel human PI3-K, namely PI3-K-C2 alpha, which contains a C-terminal C2 domain. This enzyme can be assigned to the class II PI3-Ks, which was defined by characterization of the Drosophila 68D enzyme and includes the recently described murine enzymes m-cpk and p170. Despite the overall similarity in the amino acid sequence of the murine and human enzymes, which suggests that they are encoded by closely related genes, these molecules show marked sequence heterogeneity at their N-termini. Biochemical analysis of recombinant PI3-K-C2 alpha demonstrates a restricted lipid substrate specificity. As reported for other members of this class, the enzyme only phosphorylates PtdIns and PtdIns4P when the lipids are presented alone. However, when lipids were presented together with phosphatidylserine acting as a carrier, phosphorylation of PtdIns(4,5)P2 was also observed. The catalytic activity of PI3-K-C2 alpha is refractory to concentrations of wortmannin and LY294002 which inhibit the PI3-K activity of other family members. The comparative insensitivity of PI3-K-C2 alpha to these inhibitors suggests that their use should be reevaluated in the study of PI3-Ks.

Ligand-activated tyrosine kinase receptors of insulin superfamily peptides can realize the signal transduction to SH2-proteins (phosphatidylinositol 3-kinase, PI3K), protein phosphotyrosine phosphatase (PPTP), GRB2-adaptor protein in two pathways: 1) with participation of specific proteins--insulin receptor substrates 1 and 2 (IRS1/IRS2); and 2) direct interaction between receptors and SH2-proteins (without IRS-proteins). Consequently, structural related determinants, which are responsible for the interaction with SH2-proteins, must be present in the receptor and IRS molecules. The comparative analysis of amino acid sequences (AAS) of human receptors of insulin, insulin-like growth factor-I and insulin-related peptide and AAS of IRS1/IRS2 proteins allow one to identify for the first time the long homologous regions in their primary structures. After alignment of AAS of the regions, the sited-targets for tyrosine phosphorylation, most important for functional activity of tyrosine kinase receptors and IRS proteins, coincided with each other. These results show that some homologous regions can have similar function. Thus, the regions can involve in coupling the receptors and IRS-proteins with SH2-proteins, such as PI3K, PPTP, GRB2-adaptor protein. It is also possible that the homologous regions of tyrosine kinase receptors and IR1/IRS2 proteins mediate the interaction between their proteins.