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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The interaction between SH2 domains and phosphotyrosine-containing sequences was examined by real-time measurements of kinetic parameters. The SH2 domains of the p85 subunit of the phosphatidylinositol 3-kinase as well as of other signaling molecules were expressed in bacteria as glutathione S-transferase fusion proteins. Phosphotyrosine-containing peptides, corresponding to two autophosphorylation sites on the human platelet-derived growth factor beta-receptor that are responsible for phosphatidylinositol 3-kinase binding, were synthesized and used as capturing molecules, immobilized on a biosensor surface. The association and dissociation rate constants for binding to both sites were determined for intact p85 and the recombinant SH2 domains. High association rates were found to be coupled to very fast dissociation rates for all interactions studied. A binding specificity was observed for the two SH2 domains of p85, with the N-terminal SH2 binding with high affinity to the Tyr-751 site but not to the Tyr-740 site, and the C-terminal SH2 interacting strongly with both sites. This approach should be generally applicable to the study of the specificity inherent in the assembly of signaling complexes by activated protein-tyrosine kinase receptors.
Mol Cell Biol 1993 Jun
PMID:Interactions between SH2 domains and tyrosine-phosphorylated platelet-derived growth factor beta-receptor sequences: analysis of kinetic parameters by a novel biosensor-based approach. 838 38

Phosphatidylinositol (PI) 3-kinase is a heterodimer consisting of an 85-kDa subunit (p85) and 110-kDa subunit (p110). The 85-kDa noncatalytic subunit, which contains two Src homology 2 (SH2) domains, one SH3 domain, and a domain homologous to the carboxy terminus of the breakpoint cluster region gene product, is known to mediate the association of the PI 3-kinase complex with activated growth factor receptors. We previously demonstrated that the C-terminal SH2 domain of p85 is responsible for the interaction of PI 3-kinase with phosphorylated platelet-derived growth factor receptor. To define the region in p85 that directs the complex formation with the PI 3-kinase catalytic subunit, a series of truncated p85 mutants was analyzed for association with p110 in vivo. We found that a fragment of p85 containing the region between the two SH2 domains was sufficient to promote the interaction with p110 in vivo. The complex between the fragment of p85 and p110 had PI 3-kinase activity that was comparable in magnitude to the activity of p110 associated with full-length p85. The binding with p110 was abolished when this domain in p85 was disrupted. These results identify a novel structural and functional element that is responsible for localizing the catalytic subunit of PI 3-kinase.
Mol Cell Biol 1993 Sep
PMID:A region of the 85-kilodalton (kDa) subunit of phosphatidylinositol 3-kinase binds the 110-kDa catalytic subunit in vivo. 839 6

The -300 region of the interleukin 1 beta (IL-1 beta) promoter contains a functional NF-kappa B binding site composed of the decamer sequence 5'-GGGAAAATCC-3'. Probes representing the -300 region or the NF-kappa B site alone interacted with NF-kappa B proteins present in phorbol myristate acetate-, lipopolysaccharide-, or Sendai virus-induced myeloid cell extracts as well as recombinant NFKB1 (p50) and RelA (p65); furthermore, NF-kappa B protein-DNA complex formation was dissociated in vitro by the addition of recombinant I kappa B alpha. Mutation of the NF-kappa B site in the context of the IL-1 beta promoter reduced the responsiveness of the IL-1 beta promoter to various inducers, including phorbol ester, Sendai virus, poly(rI-rC), and IL-1 beta. A 4.4-kb IL-1 beta promoter fragment linked to a chloramphenicol acetyltransferase reporter gene was also preferentially inducible by coexpression of individual NF-kappa B subunits compared with a mutated IL-1 beta promoter fragment. When multiple copies of the IL-1 beta NF-kappa B site were linked to an enhancerless simian virus 40 promoter, this element was able to mediate phorbol ester- or lipopolysaccharide-inducible gene expression. In cotransfection experiments, RelA (p65) and c-Rel (p85) were identified as the main subunits responsible for the activation of the IL-1 beta NF-kappa B site; also, combinations of NFKB1 (p50) and RelA (p65) or c-Rel and RelA were strong transcriptional activators of reporter gene activity. The presence of a functional NF-kappa B binding site in the IL-1 beta promoter suggests that IL-1 positively autoregulates its own synthesis, since IL-1 is a strong inducer of NF-kappa B binding activity. Thus, the IL-1 beta gene may be considered as an important additional member of the family of cytokine genes regulated in part by the NF-kappa B/rel family of transcription factors.
Mol Cell Biol 1993 Oct
PMID:Characterization of a functional NF-kappa B site in the human interleukin 1 beta promoter: evidence for a positive autoregulatory loop. 841 23

Xenopus oocytes from unprimed frogs possess insulin-like growth factor I (IGF-I) receptors but lack insulin and IGF-I receptor substrate 1 (IRS-1), the endogenous substrate of this kinase, and fail to show downstream responses to hormonal stimulation. Microinjection of recombinant IRS-1 protein enhances insulin-stimulated phosphatidylinositol (PtdIns) 3-kinase activity and restores the germinal vesicle breakdown response. Activation of PtdIns 3-kinase results from formation of a complex between phosphorylated IRS-1 and the p85 subunit of PtdIns 3-kinase. Microinjection of a phosphonopeptide containing a pYMXM motif with high affinity for the src homology 2 (SH2) domain of PtdIns 3-kinase p85 inhibits IRS-1 association with and activation of the PtdIns 3-kinase. Formation of the IRS-1-PtdIns 3-kinase complex and insulin-stimulated PtdIns 3-kinase activation are also inhibited by microinjection of a glutathione S-transferase fusion protein containing the SH2 domain of p85. This effect occurs in a concentration-dependent fashion and results in a parallel loss of hormone-stimulated oocyte maturation. These inhibitory effects are specific and are not mimicked by glutathione S-transferase fusion proteins expressing the SH2 domains of ras-GAP or phospholipase C gamma. Moreover, injection of the SH2 domains of p85, ras-GAP, and phospholipase C gamma do not interfere with progesterone-induced oocyte maturation. These data demonstrate that phosphorylation of IRS-1 plays an essential role in IGF-I and insulin signaling in oocyte maturation and that this effect occurs through interactions of the phosphorylated YMXM/YXXM motifs of IRS-1 with SH2 domains of PtdIns 3-kinase or some related molecules.
Mol Cell Biol 1993 Nov
PMID:Insulin-stimulated oocyte maturation requires insulin receptor substrate 1 and interaction with the SH2 domains of phosphatidylinositol 3-kinase. 841 61

In somatic cells, phosphatidylinositol 3-kinase (PI3 kinase) is a critical intermediary in growth factor-induced mitogenesis. We have examined the role of this enzyme in meiotic maturation of Xenopus laevis oocytes. PI3 kinase activity was present in immunoprecipitates of the p85 subunit of PI3 kinase from immature oocytes and markedly increased following progesterone stimulation. Injection of bacterially expressed protein corresponding to the C-terminal SH2 domain of p85 (SH2-C) inhibited progesterone-induced PI3 kinase activation and meiotic maturation. Injection of protein corresponding to the N-terminal SH2 domain or the SH3 domain of p85 did not inhibit PI3 kinase activation or maturation. SH2-C did not inhibit oocyte maturation induced by c-mos RNA injection. In addition, radiolabelled SH2-C was used to probe oocyte lysates, revealing that a novel 200-kDa protein bound to SH2-C. This protein may be an important mediator of progesterone-induced lipid metabolism in oocytes.
Mol Cell Biol 1993 Nov
PMID:Phosphatidylinositol 3-kinase activity is important for progesterone-induced Xenopus oocyte maturation. 841 62

The mitogen response of p70/p85 S6 kinase (S6K) parallels that of mitogen-activated protein kinases (MAPK). However, S6K lies on a discrete signaling pathway from MAPK, since the immunosuppressant drug rapamycin inactivates S6K without affecting the MAPK cascade. Phosphatidylinositol 3-kinase operates upstream of S6K, but the intermediate effectors in this signaling pathway are unknown. We have identified an autoinhibitory domain in S6K that overrides the requirement of the amino terminus for the activation of S6K. The region between codons 58 and 77 is highly inhibitory, and its deletion results in constitutive kinase activation. Additionally, deletion of the first 77 codons confers mitogen independence and insensitivity to rapamycin. Rat1 cells expressing delta N77 S6K exhibit a distinctly abnormal morphology. This constitutively active mutant will provide a useful means of studying the effects of expressing unregulated S6K in cells. Subdeletion analysis of the amino terminus has defined two discrete domains in the N terminus of S6K--a domain between codons 1 and 58 is essential for the mitogen activation of S6K and confers rapamycin sensitivity; a second domain between codons 58 and 77 confers autoinhibition. We propose a model for the activation of S6 kinase in which mitogen-stimulated cellular factors interact with the amino terminus to negate the effects of the autoinhibitory domain.
Mol Cell Biol 1996 Jan
PMID:Constitutive activation of S6 kinase by deletion of amino-terminal autoinhibitory and rapamycin sensitivity domains. 852 22

We have investigated the effects of wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI 3-kinase), on antigen-mediated signaling in the RBL-2H3 mast cell model. In RBL-2H3 cells, the cross-linking of high affinity IgE receptors (Fc epsilon R1) activates at least two cytoplasmic protein tyrosine kinases, Lyn and Syk, and stimulates secretion, membrane ruffling, spreading, pinocytosis, and the formation of actin plaques implicated in increased cell-substrate adhesion. In addition, Fc epsilon R1 cross-linking activates PI 3-kinase. It was previously shown that wortmannin causes a dose-dependent inhibition of PI 3-kinase activity and also inhibits antigen-stimulated degranulation. We report that the antigen-induced synthesis of inositol(1,4,5)P3 is also markedly inhibited by wortmannin. Consistent with evidence in other cell systems implicating phosphatidylinositol(3,4,5)P3 in ruffling, pretreatment of RBL-2H3 cells with wortmannin inhibits membrane ruffling and fluid pinocytosis in response to Fc epsilon R1 cross-linking. However, wortmannin does not inhibit antigen-induced actin polymerization, receptor internalization, or the actin-dependent processes of spreading and adhesion plaque formation that follow antigen stimulation in adherent cells. Wortmannin also fails to inhibit either of the Fc epsilon R1-coupled tyrosine kinases, Lyn or Syk, or the activation of mitogen-activated protein kinase as measured by in vitro kinase assays. Strikingly, there is substantial in vitro serine/threonine kinase activity in immunoprecipitates prepared from Fc epsilon R1-activated cells using antisera to the p85 subunit of PI 3-kinase. This activity is inhibited by pretreatment of the cells with wortmannin or by the direct addition of wortmannin to the kinase assay, suggesting that PI 3-kinase itself is capable of acting as a protein kinase. We conclude that Fc epsilon R1 cross-linking activates both lipid and protein kinase activities of PI 3-kinase and that inhibiting these activities with wortmannin results in the selective block of a subset of Fc epsilon R1-mediated signaling responses.
Mol Biol Cell 1995 Sep
PMID:Wortmannin blocks lipid and protein kinase activities associated with PI 3-kinase and inhibits a subset of responses induced by Fc epsilon R1 cross-linking. 853 12

The receptor insulin substrate 1 protein (IRS-1) is a specific substrate for insulin receptor tyrosine kinase. Expression and tyrosyl phosphorylation of IRS-1 play an important role during normal hepatocyte growth, and the gene is overexpressed in hepatocellular carcinoma tissue. We determined if IRS-1 overexpression directly contributes to cellular transformation. The human IRS-1 gene was subcloned into a mammalian expression vector driven by the cytomegalovirus early promoter. NIH 3T3 cells transiently transfected with this vector subsequently developed transformed foci. Several stably transfected cell lines were established, and they grew efficiently under low-serum conditions and formed colonies when plated in soft agar. Cell lines overexpressing IRS-1 displayed increased tyrosyl phosphorylation of IRS-1 and association with Grb2 but not with the p85 subunit of phosphatidylinositol 3' kinase. Since Grb2 is a component of the son-of-sevenless-Ras pathway and upstream in the mitogen-activated protein kinase (MAPK) cascade, enzymatic activities of the major components of this cascade, such as MAPK kinase and MAPK were evaluated and found to be substantially increased in three independent cell lines with IRS-1 protein overexpression. Such cells, when injected into nude mice, were highly tumorigenic, and there may be a correlation between the degree of MAPK activation and tumor growth rate. This report describes the generation of a transformed phenotype by overexpression of a molecule without a catalytic domain far upstream in the signal transduction cascade and suggests that prolonged activation of MAPKs by this mechanism may be one of the molecular events related to hepatocellular transformation.
Mol Cell Biol 1996 Mar
PMID:Overexpression of human insulin receptor substrate 1 induces cellular transformation with activation of mitogen-activated protein kinases. 862 97

We have identified two novel alternatively spliced forms of the p85alpha regulatory subunit of phosphatidylinositol (PI) 3-kinase by expression screening of a human skeletal muscle library with phosphorylated baculovirus- produced human insulin receptor substrate 1. One form is identical to p85alpha throughout the region which encodes both Src homology 2 (SH2) domains and the inter-SH2 domain/p110 binding region but diverges in sequence from p85alpha on the 5' side of nucleotide 953, where the entire break point cluster gene and SH3 regions are replaced by a unique 34-amino-acid N terminus. This form has an estimated molecular mass of approximately 53 kDa and has been termed p85/AS53. The second form is identical to p85 and p85/AS53 except for a 24-nucleotide insert between the SH2 domains that results in a replacement of aspartic acid 605 with nine amino acids, adding two potential serine phosphorylation sites in the vicinity of the known serine autophosphorylation site (Ser-608). Northern (RNA) analyses reveal a wide tissue distribution of p85alpha, whereas p85/AS53 is dominant in skeletal muscle and brain, and the insert isoforms are restricted to cardiac muscle and skeletal muscle. Western blot (immunoblot) analyses using an anti-p85 polyclonal antibody and a specific anti-p85/AS53 antibody confirmed the tissue distribution of p85/AS53 protein and indicate a approximately 7-fold higher expression of p85/AS53 protein than of p85 in skeletal muscle. Both p85 and p85/AS53 bind to p110 in coprecipitation experiments, but p85alpha itself appears to have preferential binding to insulin receptor substrate 1 following insulin stimulation. These data indicate that the gene for the p85alpha regulatory subunit of PI 3-kinase can undergo tissue-specific alternative splicing. Two novel splice variants of the regulatory subunit of PI 3-kinase are present in skeletal muscle, cardiac muscle, and brain; these variants may have important functional differences in activity and may play a role in tissue-specific signals such as insulin-stimulated glucose transport or control of neurotransmitter secretion or action.
Mol Cell Biol 1996 May
PMID:Insulin receptor substrate 1 binds two novel splice variants of the regulatory subunit of phosphatidylinositol 3-kinase in muscle and brain. 862 86

The P13K SH3 domain, residues 1 to 85 of the P1-3 kinase p85 subunit, has been characterized by X-ray diffraction. Crystals belonging to space group P4(3)2(1)2 diffract to 2.0 angstroms resolution and the structure was phased by single isomorphous replacement and anomalous scattering (SIRAS). As expected, the domain is a compact beta barrel with an over-all confirmation very similar to the independently determined NMR structures. The X-ray structure illuminates a discrepancy between the two NMR structures on the conformation of the loop region unique to P13K SH3. Furthermore, the ligand binding pockets of P13K SH3 domain are occupied by amino acid residues from symmetry-related P13K SH3 molecules: the C-terminal residues I(82) SPP of one and R18 of another. The interaction modes clearly resemble those observed for the P13K SH3 domain complexed with the synthetic peptide RLP1, a class 1 ligand, although there are significant differences. The solid-state interactions suggest a model of protein-protein aggregation that could be mediated by SH3 domains.
J Mol Biol 1996 Apr 05
PMID:Crystal structure of P13K SH3 domain at 20 angstroms resolution. 864 29


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