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

Gab1 has structural similarities with Drosophila DOS (daughter of sevenless), which is a substrate of the protein tyrosine phosphatase Corkscrew. Both Gab1 and DOS have a pleckstrin homology domain and tyrosine residues, potential binding sites for various SH2 domain-containing adapter molecules when they are phosphorylated. We found that Gab1 was tyrosine phosphorylated in response to various cytokines, such as interleukin-6 (IL-6), IL-3, alpha interferon (IFN-alpha), and IFN-gamma. Upon the stimulation of IL-6 or IL-3, Gab1 was found to form a complex with phosphatidylinositol (PI)-3 kinase and SHP-2, a homolog of Corkscrew. Mutational analysis of gp130, the common subunit of IL-6 family cytokine receptors, revealed that neither tyrosine residues of gp130 nor its carboxy terminus was required for tyrosine phosphorylation of Gab1. Expression of Gab1 enhanced gp130-dependent mitogen-activated protein (MAP) kinase ERK2 activation. A mutation of tyrosine 759, the SHP-2 binding site of gp130, abrogated the interactions of Gab1 with SHP-2 and PI-3 kinase as well as ERK2 activation. Furthermore, ERK2 activation was inhibited by a dominant negative p85 PI-3 kinase, wortmannin, or a dominant negative Ras. These observations suggest that Gab1 acts as an adapter molecule in transmitting signals to ERK MAP kinase for the cytokine receptor gp130 and that SHP-2, PI-3 kinase, and Ras are involved in Gab1-mediated ERK activation.
Mol Cell Biol 1998 Jul
PMID:Gab1 acts as an adapter molecule linking the cytokine receptor gp130 to ERK mitogen-activated protein kinase. 963 95

Tie2 is an endothelium-specific receptor tyrosine kinase that is required for both normal embryonic vascular development and tumor angiogenesis and is thought to play a role in vascular maintenance. However, the signaling pathways responsible for the function of Tie2 remain unknown. In this report, we demonstrate that the p85 subunit of phosphatidylinositol 3-kinase (PI3-kinase) associates with Tie2 and that this association confers functional lipid kinase activity. Mutation of tyrosine 1101 of Tie2 abrogated p85 association both in vitro and in vivo in yeast. Tie2 was found to activate PI3-kinase in vivo as demonstrated by direct measurement of increases in cellular phosphatidylinositol 3-phosphate and phosphatidylinositol 3, 4-bisphosphate, by plasma membrane translocation of a green fluorescent protein-Akt pleckstrin homology domain fusion protein, and by downstream activation of the Akt kinase. Activation of PI3-kinase was abrogated in these assays by mutation of Y1101 to phenylalanine, consistent with a requirement for this residue for p85 association with Tie2. These results suggest that activation of PI3-kinase and Akt may in part account for Tie2's role in both embryonic vascular development and pathologic angiogenesis, and they are consistent with a role for Tie2 in endothelial cell survival.
Mol Cell Biol 1998 Jul
PMID:Tyrosine 1101 of Tie2 is the major site of association of p85 and is required for activation of phosphatidylinositol 3-kinase and Akt. 963 97

Reorganization of the actin cytoskeleton is an early cellular response to a variety of extracellular signals. Dissection of pathways leading to actin rearrangement has focused largely on those initiated by growth factor receptors or integrins, although stimulation of G protein-coupled receptors also leads to cytoskeletal changes. In transfected Cos-7SH cells, activation of the chemoattractant formyl peptide receptor induces cortical actin polymerization and a decrease in the number of central actin bundles. In this report, we show that cytoskeletal reorganization can be transduced by G protein betagamma heterodimers (Gbetagamma), phosphoinositide 3-kinase gamma (PI3-Kgamma), a guanosine exchange factor (GEF) for Rac, and Rac. Expression of inactive variants of either PI3-Kgamma, the Rac GEF Vav, or Rac blocked the actin rearrangement. Neither wortmannin nor LY294002, pharmacologic inhibitors of PI3-K, could inhibit the actin rearrangement induced by a constitutively active Rac. The inhibition of cytoskeletal reorganization by the dominant negative Vav variants could be rescued by coexpression of a constitutively active form of Rac. In contrast, a Vav variant with its pleckstrin homology (PH) domain missing constitutively induced JNK activation and led to cytoskeletal reorganization, even without stimulation by PI3-Kgamma. This suggests that the PH domain of Vav controls the guanosine exchange activity of Vav, perhaps by a mechanism regulated by D3 phosphoinositides generated by PI3-K. Taken together, these findings delineate a pathway leading from activation of a G protein-coupled receptor to actin reorganization which sequentially involves Gbetagamma, PI3-Kgamma, a Rac GEF, and Rac.
Mol Cell Biol 1998 Aug
PMID:Cytoskeletal reorganization by G protein-coupled receptors is dependent on phosphoinositide 3-kinase gamma, a Rac guanosine exchange factor, and Rac. 967 84

Recruitment of intracellular proteins to the plasma membrane is a commonly found requirement for the initiation of signal transduction events. The recently discovered pleckstrin homology (PH) domain, a structurally conserved element found in approximately 100 signaling proteins, has been implicated in this function, because some PH domains have been described to be involved in plasma membrane association. Furthermore, several PH domains bind to the phosphoinositides phosphatidylinositol-(4,5)-bisphosphate and phosphatidylinositol-(3,4,5)-trisphosphate in vitro, however, mostly with low affinity. It is unclear how such weak interactions can be responsible for observed membrane binding in vivo as well as the resulting biological phenomena. Here, we investigate the structural and functional requirements for membrane association of cytohesin-1, a recently discovered regulatory protein of T cell adhesion. We demonstrate that both the PH domain and the adjacent carboxyl-terminal polybasic sequence of cytohesin-1 (c domain) are necessary for plasma membrane association and biological function, namely interference with Jurkat cell adhesion to intercellular adhesion molecule 1. Biosensor measurements revealed that phosphatidylinositol-(3,4,5)-trisphosphate binds to the PH domain and c domain together with high affinity (100 nM), whereas the isolated PH domain has a substantially lower affinity (2-3 microM). The cooperativity of both elements appears specific, because a chimeric protein, consisting of the c domain of cytohesin-1 and the PH domain of the beta-adrenergic receptor kinase does not associate with membranes, nor does it inhibit adhesion. Moreover, replacement of the c domain of cytohesin-1 with a palmitoylation-isoprenylation motif partially restored the biological function, but the specific targeting to the plasma membrane was not retained. Thus we conclude that two elements of cytohesin-1, the PH domain and the c domain, are required and sufficient for membrane association. This appears to be a common mechanism for plasma membrane targeting of PH domains, because we observed a similar functional cooperativity of the PH domain of Bruton's tyrosine kinase with the adjacent Bruton's tyrosine kinase motif, a novel zinc-containing fold.
Mol Biol Cell 1998 Aug
PMID:The PH domain and the polybasic c domain of cytohesin-1 cooperate specifically in plasma membrane association and cellular function. 969 61

A novel Xenopus insulin receptor substrate cDNA was isolated by hybridization screening using the rat insulin receptor substrate-1 (IRS-1) cDNA as a probe. The xIRS-u cDNA encodes an open reading frame of 1003 amino acids including a putative amino-terminal pleckstrin homology (PH) domain and phosphotyrosine-binding (PTB) domain. The carboxy terminus of xIRS-u contains several potential Src homology 2 (SH2)-binding sites, five of which are in the context of YM/LXM (presumptive binding sites for phosphatidylinositol 3-kinase). It also contains a putative binding site for Grb2 (YINID). Pair-wise amino acid sequence comparisons with the previously identified xIRS-1 and the four members of the mammalian IRS family (1 through 4) indicated that xIRS-u has similar overall sequence homology (33-45% identity) to all mammalian IRS proteins. In contrast, the previously isolated xIRS-1 is particularly similar (67% identical) to IRS-1 and considerably less similar (31-46%) to the other IRS family members (2 through 4). xIRS-u is also distinct from xIRS-1, having an overall sequence identity of 47%. These sequence analyses suggest that xIRS-u is a novel member of the IRS family rather than a Xenopus homolog of an existing member. Microinjection of mRNA encoding a Myc-tagged xIRS-u into Xenopus oocytes resulted in the expression of a 120-kDa protein (including 5 copies of the 13-amino acid Myc tag). The injection of xIRS-u mRNA accelerated insulin-induced MAP kinase activation with a concomitant acceleration of insulin-induced oocyte maturation. An aminoterminal deletion of the PH domain (xIRS-u deltaPH) significantly reduced the ability of xIRS-u to potentiate insulin signaling. In contrast to the full-length protein, injection of xIRS-u (1-299), which encoded the PH and PTB domain, or xIRS-u (1-170), which encoded only the PH domain, blocked insulin signaling in Xenopus oocytes. Finally, xIRS-u (119-299), which had a truncated PH domain and an intact PTB domain, had no effect on insulin signaling. This is the first report that the PH domain of an IRS protein can function in a dominant negative manner to inhibit insulin signaling.
Mol Endocrinol 1998 Aug
PMID:A novel insulin receptor substrate protein, xIRS-u, potentiates insulin signaling: functional importance of its pleckstrin homology domain. 971 35

Insulin and insulin-like growth factor 1 (IGF-1) evoke diverse biological effects through receptor-mediated tyrosine phosphorylation of insulin receptor substrate (IRS) proteins. We investigated the elements of IRS-1 signaling that inhibit apoptosis of interleukin 3 (IL-3)-deprived 32D myeloid progenitor cells. 32D cells have few insulin receptors and no IRS proteins; therefore, insulin failed to inhibit apoptosis during IL-3 withdrawal. Insulin stimulated mitogen-activated protein kinase in 32D cells expressing insulin receptors (32DIR) but failed to activate the phosphatidylinositol 3 (PI 3)-kinase cascade or to inhibit apoptosis. By contrast, insulin stimulated the PI 3-kinase cascade, inhibited apoptosis, and promoted replication of 32DIR cells expressing IRS-1. As expected, insulin did not stimulate PI 3-kinase in 32DIR cells, which expressed a truncated IRS-1 protein lacking the tail of tyrosine phosphorylation sites. However, this truncated IRS-1 protein, which retained the NH2-terminal pleckstrin homology (PH) and phosphotyrosine binding (PTB) domains, mediated phosphorylation of PKB/akt, inhibition of apoptosis, and replication of 32DIR cells during insulin stimulation. These results suggest that a phosphotyrosine-independent mechanism mediated by the PH and PTB domains promoted antiapoptotic and growth actions of insulin. Although PI 3-kinase was not activated, its phospholipid products were required, since LY294002 inhibited these responses. Without IRS-1, a chimeric insulin receptor containing a tail of tyrosine phosphorylation sites derived from IRS-1 activated the PI 3-kinase cascade but failed to inhibit apoptosis. Thus, phosphotyrosine-independent IRS-1-linked pathways may be critical for survival and growth of IL-3-deprived 32D cells during insulin stimulation.
Mol Cell Biol 1998 Nov
PMID:The pleckstrin homology and phosphotyrosine binding domains of insulin receptor substrate 1 mediate inhibition of apoptosis by insulin. 977 92

ARNO is a member of a family of guanine-nucleotide exchange factors with specificity for the ADP-ribosylation factor (ARF) GTPases. ARNO possesses a central catalytic domain with homology to yeast Sec7p and an adjacent C-terminal pleckstrin homology (PH) domain. We have previously shown that ARNO localizes to the plasma membrane in vivo and efficiently catalyzes ARF6 nucleotide exchange in vitro. In addition to a role in endocytosis, ARF6 has also been shown to regulate assembly of the actin cytoskeleton. To determine whether ARNO is an upstream regulator of ARF6 in vivo, we examined the distribution of actin in HeLa cells overexpressing ARNO. We found that, while expression of ARNO leads to disassembly of actin stress fibers, it does not result in obvious changes in cell morphology. However, treatment of ARNO transfectants with the PKC agonist phorbol 12-myristate 13-acetate results in the dramatic redistribution of ARNO, ARF6, and actin into membrane protrusions resembling lamellipodia. This process requires ARF activation, as actin rearrangement does not occur in cells expressing a catalytically inactive ARNO mutant. PKC phosphorylates ARNO at a site immediately C-terminal to its PH domain. However, mutation of this site had no effect on the ability of ARNO to regulate actin rearrangement, suggesting that phosphorylation of ARNO by PKC does not positively regulate its activity. Finally, we demonstrate that an ARNO mutant lacking the C-terminal PH domain no longer mediates cytoskeletal reorganization, indicating a role for this domain in appropriate membrane localization. Taken together, these data suggest that ARNO represents an important link between cell surface receptors, ARF6, and the actin cytoskeleton.
Mol Biol Cell 1998 Nov
PMID:Remodeling of the actin cytoskeleton is coordinately regulated by protein kinase C and the ADP-ribosylation factor nucleotide exchange factor ARNO. 980 2

Caenorhabditis elegans dynamin is expressed at high levels in neurons and at lower levels in other cell types, consistent with the important role that dynamin plays in the recycling of synaptic vesicles. Indirect immunofluorescence showed that dynamin is concentrated along the dorsal and ventral nerve cords and in the synapse-rich nerve ring. Green fluorescent protein (GFP) fused to the N terminus of dynamin is localized to synapse-rich regions. Furthermore, this chimera was detected along the apical membrane of intestinal cells, in spermathecae, and in coelomocytes. Dynamin localization was not affected by disrupting axonal transport of synaptic vesicles in the unc-104 (kinesin) mutant. To investigate the alternative mechanisms that dynamin might use for translocation to the synapse, we systematically tested the localization of different protein domains by fusion to GFP. Localization of each chimera was measured in one specific neuron, the ALM. The GTPase, a middle domain, and the putative coiled coil each contribute to synaptic localization. Surprisingly, the pleckstrin homology domain and the proline-rich domain, which are known to bind to coated-pit constituents, did not contribute to synaptic localization. The GFP-GTPase chimera was most strongly localized, although the GTPase domain has no known interactions with proteins other than with dynamin itself. Our results suggest that different dynamin domains contribute to axonal transport and the sequestration of a pool of dynamin molecules in synaptic cytosol.
Mol Biol Cell 1998 Nov
PMID:Contribution of the GTPase domain to the subcellular localization of dynamin in the nematode Caenorhabditis elegans. 980 8

Phosphoinositide (PI) 3-kinase contributes to a wide variety of biological actions, including insulin stimulation of glucose transport in adipocytes. Both Akt (protein kinase B), a serine-threonine kinase with a pleckstrin homology domain, and atypical isoforms of protein kinase C (PKCzeta and PKClambda) have been implicated as downstream effectors of PI 3-kinase. Endogenous or transfected PKClambda in 3T3-L1 adipocytes or CHO cells has now been shown to be activated by insulin in a manner sensitive to inhibitors of PI 3-kinase (wortmannin and a dominant negative mutant of PI 3-kinase). Overexpression of kinase-deficient mutants of PKClambda (lambdaKD or lambdaDeltaNKD), achieved with the use of adenovirus-mediated gene transfer, resulted in inhibition of insulin activation of PKClambda, indicating that these mutants exert dominant negative effects. Insulin-stimulated glucose uptake and translocation of the glucose transporter GLUT4 to the plasma membrane, but not growth hormone- or hyperosmolarity-induced glucose uptake, were inhibited by lambdaKD or lambdaDeltaNKD in a dose-dependent manner. The maximal inhibition of insulin-induced glucose uptake achieved by the dominant negative mutants of PKClambda was approximately 50 to 60%. These mutants did not inhibit insulin-induced activation of Akt. A PKClambda mutant that lacks the pseudosubstrate domain (lambdaDeltaPD) exhibited markedly increased kinase activity relative to that of the wild-type enzyme, and expression of lambdaDeltaPD in quiescent 3T3-L1 adipocytes resulted in the stimulation of glucose uptake and translocation of GLUT4 but not in the activation of Akt. Furthermore, overexpression of an Akt mutant in which the phosphorylation sites targeted by growth factors are replaced by alanine resulted in inhibition of insulin-induced activation of Akt but not of PKClambda. These results suggest that insulin-elicited signals that pass through PI 3-kinase subsequently diverge into at least two independent pathways, an Akt pathway and a PKClambda pathway, and that the latter pathway contributes, at least in part, to insulin stimulation of glucose uptake in 3T3-L1 adipocytes.
Mol Cell Biol 1998 Dec
PMID:Requirement of atypical protein kinase clambda for insulin stimulation of glucose uptake but not for Akt activation in 3T3-L1 adipocytes. 981 85

Membrane trafficking is regulated in part by small GTP-binding proteins of the ADP-ribosylation factor (Arf) family. Arf function depends on the controlled exchange and hydrolysis of GTP. We have purified and cloned two variants of a 130-kDa phosphatidylinositol 4, 5-biphosphate (PIP2)-dependent Arf1 GTPase-activating protein (GAP), which we call ASAP1a and ASAP1b. Both contain a pleckstrin homology (PH) domain, a zinc finger similar to that found in another Arf GAP, three ankyrin (ANK) repeats, a proline-rich region with alternative splicing and SH3 binding motifs, eight repeats of the sequence E/DLPPKP, and an SH3 domain. Together, the PH, zinc finger, and ANK repeat regions possess PIP2-dependent GAP activity on Arf1 and Arf5, less activity on Arf6, and no detectable activity on Arl2 in vitro. The cDNA for ASAP1 was independently identified in a screen for proteins that interact with the SH3 domain of the tyrosine kinase Src. ASAP1 associates in vitro with the SH3 domains of Src family members and with the Crk adapter protein. ASAP1 coprecipitates with Src from cell lysates and is phosphorylated on tyrosine residues in cells expressing activated Src. Both coimmunoprecipitation and tyrosine phosphorylation depend on the same proline-rich class II Src SH3 binding site required for in vitro association. By directly interacting with both Arfs and tyrosine kinases involved in regulating cell growth and cytoskeletal organization, ASAP1 could coordinate membrane remodeling events with these processes.
Mol Cell Biol 1998 Dec
PMID:ASAP1, a phospholipid-dependent arf GTPase-activating protein that associates with and is phosphorylated by Src. 981 91


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