UNIPROT:P67775 - FACTA Search

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Query: UNIPROT:P67775 (alpha isoform)
797 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Phosphatidylinositol-3 kinase (PI-3 kinase) has been implicated in cellular events such as mitogenic signaling, actin organization, and receptor sorting. The p85 subunit of PI-3 kinase contains multiple domains capable of protein-protein interactions that may contribute to mediate the multiple physiological functions of this enzyme. Here, we demonstrate that antibodies raised against the p85 subunit of PI-3 kinase immunoprecipitate a single tyrosine-phosphorylated protein of 120 kDa (pp120) from lysates of activated Jurkat T cells and A20 B cells. This protein is the only significant phosphotyrosine-containing protein in p85 immunoprecipitates from these cells, and it cannot be detected in immunoprecipitates of other signaling proteins such as PLC gamma. Furthermore, antibodies specific for the beta isoform of p85 but not antibodies specific for the alpha isoform immunoprecipitate this tyrosine-phosphorylated protein. pp120 completely comigrates with the proto-oncogene c-cbl, which is a 120 kDa protein product abundant in lymphoid cells. Furthermore, immunoblots of p85 immunoprecipitates using antibodies raised against c-cbl detect a band at exactly the position of pp120. In addition, p85 can be detected in immunoblots of c-cbl immunoprecipitates. Thus, pp120 appears to correspond to c-cbl. A direct association between c-cbl and p85 can be observed in vitro using a fusion protein comprising the Src homology 2 (SH2) domains of p85, and this binding is abolished by phenyl phosphate, suggesting that the interaction is mediated through phosphotyrosine-SH2 domain interactions. Thus, these results show important functional differences between the alpha and beta isoforms of p85 in vivo and point to c-cbl as a potentially important mediator of some of the functions of PI-3 kinase in intact cells.
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PMID:Specific association of the beta isoform of the p85 subunit of phosphatidylinositol-3 kinase with the proto-oncogene c-cbl. 762 44

The neu differentiation factors/heregulins (HRGs) comprise a family of polypeptide growth factors that activate p185(erbB-2) through direct binding to either erbB-3 or erbB-4 receptor tyrosine kinases. We have previously shown that HRG-beta is mitogenic for various human mammary epithelial cell lines that coexpress c-erbB-2 and c-erbB-3. Phosphatidylinositol 3-kinase (PI3K) is activated by p185(erbB-2) /erbB-3 heterodimers in cells stimulated by HRG, and PI3K is constitutively activated by p185(erbB-2) /erbB-3 in breast carcinoma cells that overexpress c-erbB-2. To better understand the relative abilities of HRGs, epidermal growth factor (EGF), or insulin to activate PI3K under normal physiological conditions, we compared the levels of recruitment of the 85-kDa regulatory subunit of PI3K when activated by the type I (erbB) or type II [insulin-like growth factor (IGF)] receptor tyrosine kinases in two different nontransformed human mammary epithelial cell lines. The nontransformed H16N-2 cells isolated from normal tissue express EGFR, p185(erbB-2), and erbB-3, and are highly responsive to the mitogenic effects of HRG-beta as well as to the combination of EGF and insulin in serum-free culture. We measured the stoichiometry of p85 recruited by tyrosine-phosphorylated proteins induced in H16N-2 cells by either the alpha or the beta isoform of HRG. HRG-beta was greater than 10-fold more potent in inducing p85 recruitment than was the less biologically active HRG-alpha isoform. HRG-beta was also a more potent inducer of p85 recruited by tyrosine-phosphorylated proteins than was either EGF, insulin, or EGF and insulin combined. Furthermore, erbB-3 principally mediated the direct recruitment of p85 in cells stimulated by HRG or EGF, indicating that, in addition to the high-level activation of PI3K by p185(erbB-2) / erbB-3, EGFR/erbB-3 heterodimer interaction is essential for the weak but significant level of PI3K activated by EGF in cells that express normal EGFR levels. Studies using the PI3K inhibitor wortmannin also indicated that PI3K activation was required for the proliferation of H16N-2 cells induced by either HRG-beta or EGF and insulin in serum-free culture. Finally, HRG-beta was also an especially potent inducer of PI3K in the nontransformed MCF-10A cells, which were derived spontaneously from normal reduction mammoplasty tissue. These data show, for the first time, a side-by-side quantitative comparison of the relative degree of PI3K activated by different growth factors in nontransformed growth factor-dependent cells under precisely defined conditions in culture.
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PMID:Heregulin-beta is especially potent in activating phosphatidylinositol 3-kinase in nontransformed human mammary epithelial cells. 1079 4

PP2A is a central regulator of eukaryotic signal transduction. The human catalytic subunit PP2Acalpha functionally replaces the endogenous yeast enzyme, Pph22p, indicating a conservation of function in vivo. Therefore, yeast cells were employed to explore the role of invariant PP2Ac residues. The PP2Acalpha Y127N substitution abolished essential PP2Ac function in vivo and impaired catalysis severely in vitro, consistent with the prediction from structural studies that Tyr-127 mediates substrate binding and its side chain interacts with the key active site residues His-118 and Asp-88. The V159E substitution similarly impaired PP2Acalpha catalysis profoundly and may cause global disruption of the active site. Two conditional mutations in the yeast Pph22p protein, F232S and P240H, were found to cause temperature-sensitive impairment of PP2Ac catalytic function in vitro. Thus, the mitotic and cell lysis defects conferred by these mutations result from a loss of PP2Ac enzyme activity. Substitution of the PP2Acalpha C-terminal Tyr-307 residue by phenylalanine impaired protein function, whereas the Y307D and T304D substitutions abolished essential function in vivo. Nevertheless, Y307D did not reduce PP2Acalpha catalytic activity significantly in vitro, consistent with an important role for the C terminus in mediating essential protein-protein interactions. Our results identify key residues important for PP2Ac function and characterize new reagents for the study of PP2A in vivo.
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PMID:Important role for phylogenetically invariant PP2Acalpha active site and C-terminal residues revealed by mutational analysis in Saccharomyces cerevisiae. 1097 72

The mitogen-activated protein kinases (MAPKs) and the cyclin-dependent kinases (CDKs) are key mediators of cell proliferation in response to extracellular signals. Recent additions to each of these families and the identification of kinases with structural features of both have provided insights into fundamental processes, such as cell division and differentiation. To identify novel serine kinases with features of MAPKs or CDKs, a degenerate PCR-based amplification approach was undertaken. The 57- and 52-kDa isoforms of a novel protein kinase, termed NKIATRE, were molecularly cloned from rat brain and jejunum cDNA libraries. Like the MAPKs, NKIATRE has a Thr-Xaa-Tyr motif in kinase subdomain VIII. NKIATRE also shows close homology to the cyclin-dependent kinase class of protein kinases and the cdc2-related kinases NKIAMRE, KKIALRE, and KKIAMRE, containing both conserved inhibitory phosphorylation sites and a putative cyclin-binding domain. Two isoforms of NKIATRE that differ in their carboxy-terminal ends have been identified. A functional nuclear localization signal is specific to the longer 57-kDa alpha isoform. Sequence similarity to the putative human tumor suppressor gene NKIAMRE, which is lost in leukemic patients with chromosome 5q deletions, suggests that NKIATRE may have a role in restricting cell growth or maintaining differentiation.
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PMID:NKIATRE is a novel conserved cdc2-related kinase. 1116 6

The oligomeric metalloenzymes protein phosphatases dephosphorylate OH groups of Ser/Thr or Tyr residues of proteins whose actions depend on the phosphorus signal. The catalytic units of Ser/Thr protein phosphatases 1, 2A and 2B (PP1c, PP2Ac and PP2Bc, respectively), which exhibit about 45% sequence similarity, have their active centers practically identical. This feature strongly suggests that the unknown structure of PP2Ac could be successfully homology-modeled from the known structures of PP1c and/or PP2Bc. Initially, a theoretical model of PP1c was built, including a phosphate and a metal dication in its catalytic site. The latter was modeled, together with a structural hydroxyl anion, as a triangular pseudo-molecule (Zno or Mno), composed of two metal cations (double Zn2+ or Mn2+, respectively) and the OH- group. To the free PP1c two inhibitor sequences R29RRRPpTPAMLFR40 of DARPP-32 and R30RRRPpTPATLVLT42 of Inhibitor-1, and two putative substrate sequences LRRApSVA and QRRQRKpRRTI were subsequently docked. In the next step, a free PP2Ac model was built via homology re-modeling of the PP1c template and the same four sequences were docked to it. Thus, together, 20 starting model complexes were built, allowing for combination of the Zno and Mno pseudo-molecules, free enzymes and the peptide ligands docked in the catalytic sites of PP1c and PP2Ac. All models were subsequently subjected to 250-300 ps molecular dynamics using the AMBER 5.0 program. The equilibrated trajectories of the final 50 ps were taken for further analyses. The theoretical models of PP1c complexes, irrespective of the dication type, exhibited increased mobilities in the following residue ranges: 195-200, 273-278, 287-209 for the inhibitor sequences and 21-25, 194-200, 222-227, 261, 299-302 for the substrate sequences. Paradoxically, the analogous PP2Ac models appeared much more stable in similar simulations, since only their "prosegment" residues 6-10 and 14-18 exhibited an increased mobility in the inhibitor complexes while no areas of increased mobility were found in the substrate complexes. Another general observation was that the complexes with Mn dications were more stable than those with Zn dications for both PP1c and PP2Ac units.
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PMID:Theoretical models of catalytic domains of protein phosphatases 1 and 2A with Zn2+ and Mn2+ metal dications and putative bioligands in their catalytic centers. 1144 Jan 82

We have previously reported that cyclic strain results in rapid phosphorylation of p38 mitogen activated protein kinase (MAPKs). The aim of this study was to examine the role of protein phosphatase type 2A (PP2A) in regulating p38 MAPK activation in bovine aortic endothelial cells exposed to cyclic strain. In this study, we demonstrate that the catalytic subunit of PP2A is tyrosine phosphorylated by cyclic strain, resulting in inhibition of phosphatase activity. Okadaic acid, an inhibitor of PP2A at lower concentrations increased phosphorylation of p-38. Phospho-p38 MAPK physically associated with the catalytic subunit, PP2Ac. Phospho-p38 MAPK was dephosphorylated by purified PP2Ac in cell lysates, but if pretreated with okadaic acid, phospho-p38 MAPK was maintained. Taken together, our result suggests that PP2A plays a regulatory role in p38 MAPK activation in endothelial cells exposed to cyclic strain.
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PMID:Role of PP2A in the regulation of p38 MAPK activation in bovine aortic endothelial cells exposed to cyclic strain. 1254 54

It has been previously shown that 5-HT uptake inhibition produced by tetanus toxin (TeTx) corresponds to a non-competitive inhibition, and it is preceded by phosphorylation of the tyrosine-kinase receptor trkA, phospholipase C activation and translocation of protein kinase C isoforms [FEBS Lett. 481 (2000) 177; FEBS Lett. 486 (2000) 136]. In the present work, it is shown that agonists of tyrosine-kinase receptors (NGF, EGF, basic FGF) enhance Na(+)-dependent, 5-hydroxytryptamine (serotonin, 5-HT) uptake in the synaptosomal-enriched P(2) fraction from rat-brain, suggesting a divergence in the intracellular signal pathways triggered by TeTx and by agonists of TyrK receptors. Co-applications of TeTx and agonists of TyrK receptors result in a mutual and partial reversion of their effects on 5-HT transport. In spite of their differences on transport, TeTx, TPA and NGF produce an increase in serotonin transporter phosphorylation in Ser separately, which is abolished by the PKC-inhibitor bisindolylmaleimide-1. Co-application of sodium vanadate, a tyrosine-phosphatase inhibitor, partially abolishes the effect produced by TeTx, whereas genistein, a tyrosine-kinase inhibitor, does not exert any variation of TeTx inhibition. Analyses by immunoblotting of the activation of specific PKC isoforms activation, determined as translocation to the membrane compartment, reveals differences in the pattern produced by NGF and TeTx. PKC gamma, delta, and epsilon isoforms are equally activated by both compounds, whereas the beta isoform is activated in a sustained manner only by TeTx, and the alpha isoform is only down-regulated by NGF. The aim of the present work was to explore whether NGF have the same effect on 5-HT transport than TeTx, since both compounds share the ability of activate part of the same transduction pathways. In spite of this, growth factors and TeTx show an opposite effect on 5-HT transport, even though SERT phosphorylation is enhanced in both cases. The differential effect on alpha- and beta-PKC isoenzymes found between NGF and TeTx action could explain this apparent discrepancy.
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PMID:Serotonin transport is modulated differently by tetanus toxin and growth factors. 1259 Sep 35

The class II PI 3-kinases are known to be activated by growth factors and chemokines but to date there are no reports of cytokine mediated regulation. Further, the intracellular signalling mechanisms regulating the class-II PI 3-kinases are poorly understood. We investigated the effects of the cytokines TNFalpha and leptin on the activity of the alpha isoform of the class II PI 3-kinase (PI3K-C2alpha) and find that these stimulate the enzyme 2-fold and 3-fold, in CHO cells and J774.2 macrophages, respectively. The stimulation by leptin was not accompanied by recruitment of any tyrosine phosphorylated proteins to PI3K-C2alpha and no shift in electrophoretic mobility was noted. Furthermore, we demonstrate that the actions of both cytokines are blocked by the MEK inhibitor PD98059. These findings indicate that the cytokines activate PI3K-C2alpha and do so by a mechanism that requires activation of the ERK pathway and thus differs from the mechanism used by insulin to activate the enzyme.
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PMID:TNF-alpha and leptin activate the alpha-isoform of class II phosphoinositide 3-kinase. 1278 79

The cellular localization of the 35 kDa, low molecular mass acid metallophosphatase (LMW AcPase) from the frog (Rana esculenta) liver and its activity towards P-Ser and P-Tyr phosphorylated peptides were studied. This enzyme was localized to the cytoplasm of hepatocytes but did not appear in other cells of liver tissue (endothelium, macrophages, blood cells). This LMW AcPase does not display activity towards (32)P-phosphorylase a under conditions standard for the enzymes of PPP family. Proteins containing P-Ser: rabbit (32)P-phosphorylasea and phosvitin are hydrolysed only at acidic pH and are poor substrates for this enzyme. The frog AcPase is not inhibited by okadaic acid and F(-) ions, the Ser/Thr protein phosphatase inhibitors. Moreover, the frog enzyme does not cross-react with specific antisera directed against N-terminal fragment of human PP2A and C-terminal conserved fragment of the eukaryotic PP2A catalytic subunits. These results exclude LMW AcPase from belonging to Ser/Thr protein phosphatases: PP1c or PP2Ac. In addition to P-Tyr, this enzyme hydrolyses efficiently at acidic pH P-Tyr phosphorylated peptides (hirudin and gastrin fragments). K(m) value for the hirudin fragment (7.55 +/- 1.59 x 10(-6) M) is 2-3 orders of magnitude lower in comparison with other substrates tested. The enzyme is inhibited competitively by typical inhibitors of protein tyrosine phosphatases (PTPases): sodium orthovanadate, molybdate and tungstate. These results may suggest that the LMW AcPase of frog liver can act as PTPase in vivo. A different cellular localization and different response to inhibition by tetrahedral oxyanions (molybdate, vanadate and tungstate) provide further evidence that LMW AcPase of frog liver is distinct from the mammalian tartrate-resistant acid phosphatases.
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PMID:The 35 kDa acid metallophosphatase of the frog Rana esculenta liver: studies on its cellular localization and protein phosphatase activity. 1283 81

Soy isoflavones, the focus of much research and controversy, are often referred to as "weak estrogens". In fact, genistein is a relatively potent agonist for the recently characterized beta isoform of the estrogen receptor (ERbeta). The low nanomolar serum concentrations of unconjugated free genistein achieved with high-nutritional intakes of soy isoflavones are near the binding affinity of genistein for this receptor, but are about an order of magnitude lower than genistein's affinity for the "classical" alpha isoform of the estrogen receptor (ERalpha). Moreover, these concentrations are far too low to inhibit tyrosine kinases or topoisomerase II, in vitro activities of genistein often cited as potential mediators of its physiological effects. The thesis that these physiological effects are in fact mediated by ERbeta activation provides a satisfying rationale for genistein's clinical activities. Hepatocytes do not express ERbeta; this explains why soy isoflavones, unlike oral estrogen, neither modify serum lipids nor provoke the prothrombotic effects associated with increased risk for thromboembolic disorders. The lack of uterotrophic activity of soy isoflavones reflects the fact that ERalpha is the exclusive mediator of estrogen's impact in this regard. Vascular endothelium expresses both ERalpha and ERbeta, each of which has the potential to induce and activate nitric oxide synthase; this may account for the favorable influence of soy isoflavones on endothelial function in postmenopausal women and ovariectomized rats. The ERbeta expressed in osteoblasts may mediate the reported beneficial impact of soy isoflavones on bone metabolism. Suggestive evidence that soy-rich diets decrease prostate cancer risk, accords well with the observation that ERbeta appears to play an antiproliferative role in healthy prostate. In the breast, ERalpha promotes epithelial proliferation, whereas ERbeta has a restraining influence in this regard - consistent with the emerging view that soy isoflavones do not increase breast cancer risk, and possibly may diminish it. Premenopausal women enjoy a relative protection from kidney failure; since ERbeta is an antagonist of TGF-beta signaling in mesangial cells, soy isoflavones may have nephroprotective potential. Estrogen also appears to protect women from left ventricular hypertrophy, and recent evidence suggests that this effect is mediated by ERbeta. In conjunction with reports that isoflavones may have a modestly beneficial impact on menopausal symptoms - perhaps reflecting the presence of ERbeta in the hypothalamus - these considerations suggest that soy isoflavone regimens of sufficient potency may represent a safe and moderately effective alternative to HRT in postmenopausal women. Further clinical research is required to characterize the impact of optimal genistein intakes on endothelial and bone function in men. Studies with ERbeta-knockout mice could be helpful for clarifying whether ERbeta does indeed mediate the chief physiological effects of low nanomolar genistein. S-equol, a bacterial metabolite of daidzein, has an affinity for ERbeta nearly as high as that of genistein; whether this compound contributes meaningfully to the physiological efficacy of soy isoflavones in some individuals is still unclear.
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PMID:Isoflavones made simple - genistein's agonist activity for the beta-type estrogen receptor mediates their health benefits. 1651 88

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