scholarly journals Regulation of the p85/p110 Phosphatidylinositol 3′-Kinase: Stabilization and Inhibition of the p110α Catalytic Subunit by the p85 Regulatory Subunit

1998 ◽  
Vol 18 (3) ◽  
pp. 1379-1387 ◽  
Author(s):  
Jinghua Yu ◽  
Yitao Zhang ◽  
James McIlroy ◽  
Tamara Rordorf-Nikolic ◽  
George A. Orr ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Kinase Activity ◽  
Thermal Inactivation ◽  
Insect Cells ◽  
Catalytic Subunit ◽  
Regulatory Subunit ◽  
Phosphatidylinositol 3 Kinase ◽  
Lipid Kinase ◽  
Phosphatidylinositol 3

ABSTRACT We propose a novel model for the regulation of the p85/p110α phosphatidylinositol 3′-kinase. In insect cells, the p110α catalytic subunit is active as a monomer but its activity is decreased by coexpression with the p85 regulatory subunit. Similarly, the lipid kinase activity of recombinant glutathione S-transferase (GST)-p110α is reduced by 65 to 85% upon in vitro reconstitution with p85. Incubation of p110α/p85 dimers with phosphotyrosyl peptides restored activity, but only to the level of monomeric p110α. These data show that the binding of phosphoproteins to the SH2 domains of p85 activates the p85/p110α dimers by inducing a transition from an inhibited to a disinhibited state. In contrast, monomeric p110 had little activity in HEK 293T cells, and its activity was increased 15- to 20-fold by coexpression with p85. However, this apparent requirement for p85 was eliminated by the addition of a bulky tag to the N terminus of p110α or by the growth of the HEK 293T cells at 30°C. These nonspecific interventions mimicked the effects of p85 on p110α, suggesting that the regulatory subunit acts by stabilizing the overall conformation of the catalytic subunit rather than by inducing a specific activated conformation. This stabilization was directly demonstrated in metabolically labeled HEK 293T cells, in which p85 increased the half-life of p110. Furthermore, p85 protected p110 from thermal inactivation in vitro. Importantly, when we examined the effect of p85 on GST-p110α in mammalian cells at 30°C, culture conditions that stabilize the catalytic subunit and that are similar to the conditions used for insect cells, we found that p85 inhibited p110α. Thus, we have experimentally distinguished two effects of p85 on p110α: conformational stabilization of the catalytic subunit and inhibition of its lipid kinase activity. Our data reconcile the apparent conflict between previous studies of insect versus mammalian cells and show that p110α is both stabilized and inhibited by dimerization with p85.

scholarly journals The p85 and p110 Subunits of Phosphatidylinositol 3-Kinase-α Are Substrates, In Vitro, for a Constitutively Associated Protein Tyrosine Kinase in Platelets

Blood ◽  
1998 ◽  
Vol 91 (3) ◽  
pp. 930-939 ◽  
Author(s):  
Norman R. Geltz ◽  
James A. Augustine
Keyword(s):  
Tyrosine Kinase ◽  
Protein Tyrosine Kinase ◽  
Kinase Activity ◽  
Protein Kinase Activity ◽  
Phosphatidylinositol 3 Kinase ◽  
Lipid Kinase ◽  
Protein Tyrosine ◽  
Dependent Protein ◽  
Phosphatidylinositol 3

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, Mn2+-dependent protein serine kinase activity capable of phosphorylating the 85-kD subunit. Here, we examine the Mn2+-dependent protein kinase activity of PI3Kα immunoprecipitated from normal resting or thrombin-stimulated platelets, and characterize p85/p110 phosphorylation, in vitro. Phosphoamino acid analysis of phosphorylated PI3Kα 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 p85α or p110α 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 p85α and PI3K lipid kinase activity were recovered in antiphosphotyrosine antibody immunoprecipitates. These results suggest PI3Kα can autophosphorylate on serine and threonine, and both p85α and p110α are substrates for a constitutively-associated protein tyrosine kinase in platelets.

scholarly journals Phosphatidylinositol 3–Kinase Is Required for the Formation of Constitutive Transport Vesicles from the TGN

1997 ◽  
Vol 139 (2) ◽  
pp. 339-349 ◽  
Author(s):  
Steven M. Jones ◽  
Kathryn E. Howell
Keyword(s):  
Kinase Activity ◽  
Pi3 Kinase ◽  
Regulatory Subunit ◽  
Vesicle Formation ◽  
Phosphatidylinositol 3 Kinase ◽  
Response Curves ◽  
Direct Role ◽  
Lipid Kinase ◽  
Transport Vesicles ◽  
Phosphatidylinositol 3

An 85-kD cytosolic complex (p62cplx), 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 p62cplx is identified as a regulatory subunit of a novel phosphatidylinositol 3–kinase (PI3-kinase). This p62cplx-associated PI3-kinase activity is stimulated by activation of the p62cplx-associated GTPase, and is specific for phosphatidylinositol (PI) as substrate, and is sensitive to wortmannin at micromolar concentrations. The direct role of this p62cplx-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 μM). These findings indicate that phosphatidylinositol-3-phosphate (PI[3]P) is required for the formation of TGN-derived exocytic transport vesicles, and that the p62cplx-associated PI3-kinase and an activated GTPase are the essential molecules that drive production of this PI(3)P.

scholarly journals The p85 and p110 Subunits of Phosphatidylinositol 3-Kinase-α Are Substrates, In Vitro, for a Constitutively Associated Protein Tyrosine Kinase in Platelets

Blood ◽  
1998 ◽  
Vol 91 (3) ◽  
pp. 930-939 ◽  
Author(s):  
Norman R. Geltz ◽  
James A. Augustine
Keyword(s):  
Tyrosine Kinase ◽  
Protein Tyrosine Kinase ◽  
Kinase Activity ◽  
Protein Kinase Activity ◽  
Phosphatidylinositol 3 Kinase ◽  
Lipid Kinase ◽  
Protein Tyrosine ◽  
Dependent Protein ◽  
Phosphatidylinositol 3

Abstract 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, Mn2+-dependent protein serine kinase activity capable of phosphorylating the 85-kD subunit. Here, we examine the Mn2+-dependent protein kinase activity of PI3Kα immunoprecipitated from normal resting or thrombin-stimulated platelets, and characterize p85/p110 phosphorylation, in vitro. Phosphoamino acid analysis of phosphorylated PI3Kα 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 p85α or p110α 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 p85α and PI3K lipid kinase activity were recovered in antiphosphotyrosine antibody immunoprecipitates. These results suggest PI3Kα can autophosphorylate on serine and threonine, and both p85α and p110α are substrates for a constitutively-associated protein tyrosine kinase in platelets.

scholarly journals Tyrosine 1101 of Tie2 Is the Major Site of Association of p85 and Is Required for Activation of Phosphatidylinositol 3-Kinase and Akt

1998 ◽  
Vol 18 (7) ◽  
pp. 4131-4140 ◽  
Author(s):  
Christopher D. Kontos ◽  
Thomas P. Stauffer ◽  
Wen-Pin Yang ◽  
John D. York ◽  
Liwen Huang ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Vascular Development ◽  
Pi3 Kinase ◽  
Pleckstrin Homology Domain ◽  
Phosphatidylinositol 3 Kinase ◽  
Lipid Kinase ◽  
Embryonic Vascular Development ◽  
Phosphatidylinositol 3

ABSTRACT 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.

Schizosaccharomyces pombe Vps34p, a phosphatidylinositol-specific PI 3-kinase essential for normal cell growth and vacuole morphology

1995 ◽  
Vol 108 (12) ◽  
pp. 3745-3756 ◽  
Author(s):  
K. Takegawa ◽  
D.B. DeWald ◽  
S.D. Emr
Keyword(s):  
Amino Acid ◽  
Schizosaccharomyces Pombe ◽  
Mammalian Cells ◽  
Membrane Fraction ◽  
Kinase Activity ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Phosphatidylinositol 3 Kinase ◽  
Acid Protein ◽  
Phosphatidylinositol 3

We have cloned the gene, vps34+, from the fission yeast Schizosaccharomyces pombe which encodes an 801 amino acid protein with phosphatidylinositol 3-kinase activity. The S. pombe Vps34 protein shares 43% amino acid sequence identity with the Saccharomyces cerevisiae Vps34 protein and 28% identity with the p110 catalytic subunit of the mammalian phosphatidylinositol 3-kinase. When the vps34+ gene is disrupted, S.pombe strains are temperature-sensitive for growth and the mutant cells contain enlarged vacuoles. Furthermore, while wild-type strains exhibit substantial levels of phosphatidylinositol 3-kinase activity, this activity is not detected in the vps34 delta strain. S.pombe Vps34p-specific antiserum detects a single protein in cells of -90 kDa that fractionates almost exclusively with the crude membrane fraction. Phosphatidylinositol 3-kinase activity also is localized mainly in the membrane fraction of wild-type cells. Immunoisolated Vps34p specifically phosphorylates phosphatidylinositol on the D-3 position of the inositol ring to yield phosphatidylinositol(3)phosphate. but does not utilize phosphatidylinositol(4)phosphate or phosphatidylinositol(4,5)bisphosphate as substrates. In addition, when compared to the mammalian p110 phosphatidylinositol 3-kinase, S. pombe Vps34p is relatively insensitive to the inhibitors wortmannin and LY294002. Together, these results indicate that S. pombe Vps34 is more similar to the phosphatidylinositol-specific 3-kinase, Vps34p from S. cerevisiae, and is distinct from the p110/p85 and G protein-coupled phosphatidylinositol 3-kinases from mammalian cells. These data are discussed in relation to the possible role of Vps34p in vesicle-mediated protein sorting to the S. pombe vacuole.

scholarly journals A Mammalian Ortholog of Saccharomyces cerevisiae Vac14 That Associates with and Up-Regulates PIKfyve Phosphoinositide 5-Kinase Activity

2004 ◽  
Vol 24 (23) ◽  
pp. 10437-10447 ◽  
Author(s):  
Diego Sbrissa ◽  
Ognian C. Ikonomov ◽  
Jana Strakova ◽  
Rajeswari Dondapati ◽  
Krzysztof Mlak ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Kinase Activity ◽  
Ectopic Expression ◽  
Multivesicular Body ◽  
Hek293 Cells ◽  
Lipid Kinase ◽  
Morphological Alterations ◽  
Protein Levels ◽  
Interfering Rna

ABSTRACT Multivesicular body morphology and size are controlled in part by PtdIns(3,5)P2, produced in mammalian cells by PIKfyve-directed phosphorylation of PtdIns(3)P. Here we identify human Vac14 (hVac14), an evolutionarily conserved protein, present in all eukaryotes but studied principally in yeast thus far, as a novel positive regulator of PIKfyve enzymatic activity. In mammalian cells and tissues, Vac14 is a low-abundance 82-kDa protein, but its endogenous levels could be up-regulated upon ectopic expression of hVac14. PIKfyve and hVac14 largely cofractionated, populated similar intracellular locales, and physically associated. A small-interfering RNA-directed gene-silencing approach to selectively eliminate endogenous hVac14 rendered HEK293 cells susceptible to morphological alterations similar to those observed upon expression of PIKfyve mutants deficient in PtdIns(3,5)P2 production. Largely decreased in vitro PIKfyve kinase activity and unaltered PIKfyve protein levels were detected under these conditions. Conversely, ectopic expression of hVac14 increased the intrinsic PIKfyve lipid kinase activity. Concordantly, intracellular PtdIns(3)P-to-PtdIns(3,5)P2 conversion was perturbed by hVac14 depletion and was elevated upon ectopic expression of hVac14. These data demonstrate a major role of the PIKfyve-associated hVac14 protein in activating PIKfyve and thereby regulating PtdIns(3,5)P2 synthesis and endomembrane homeostasis in mammalian cells.

scholarly journals Correlation of α-Fetoprotein Expression in Normal Hepatocytes during Development with Tyrosine Phosphorylation and Insulin Receptor Expression

1998 ◽  
Vol 9 (5) ◽  
pp. 1093-1105 ◽  
Author(s):  
Leila Khamzina ◽  
Pierre Borgeat
Keyword(s):  
Insulin Receptor ◽  
Tyrosine Phosphorylation ◽  
Receptor Expression ◽  
Regulatory Subunit ◽  
Β Subunit ◽  
Phosphatidylinositol 3 Kinase ◽  
Fetal Hepatocytes ◽  
In Cells ◽  
Phosphatidylinositol 3

The molecular mechanism of hepatic cell growth and differentiation is ill defined. In the present study, we examined the putative role of tyrosine phosphorylation in normal rat liver development and in an in vitro model, the α-fetoprotein-producing (AFP+) and AFP-nonproducing (AFP−) clones of the McA-RH 7777 rat hepatoma. We demonstrated in vivo and in vitro that the AFP+ phenotype is clearly associated with enhanced tyrosine phosphorylation, as assessed by immunoblotting and flow cytometry. Moreover, immunoprecipitation of proteins with anti-phosphotyrosine antibody showed that normal fetal hepatocytes expressed the same phosphorylation pattern as stable AFP+clones and likewise for adult hepatocytes and AFP− clones. The tyrosine phosphorylation of several proteins, including the β-subunit of the insulin receptor, insulin receptor substrate-1, p85 regulatory subunit of phosphatidylinositol-3-kinase, andras-guanosine triphosphatase-activating protein, was observed in AFP+ clones, whereas the same proteins were not phosphorylated in AFP− clones. We also observed that fetal hepatocytes and the AFP+ clones express 4 times more of the insulin receptor β-subunit compared with adult hepatocytes and AFP− clones and, accordingly, that these AFP+clones were more responsive to exogenous insulin in terms of protein tyrosine phosphorylation. Finally, growth rate in cells of AFP+ clones was higher than that measured in cells of AFP− clones, and inhibition of phosphatidylinositol-3-kinase by LY294002 and Wortmannin blocked insulin- and serum-stimulated DNA synthesis only in cells of AFP+ clones. These studies provide evidences in support of the hypothesis that signaling via insulin prevents hepatocyte differentiation by promoting fetal hepatocyte growth.

scholarly journals Phosphatidylinositol 3-kinase associates, via its Src homology 2 domains, with the activated erythropoietin receptor

Blood ◽  
1993 ◽  
Vol 81 (12) ◽  
pp. 3204-3210 ◽  
Author(s):  
JE Damen ◽  
AL Mui ◽  
L Puil ◽  
T Pawson ◽  
G Krystal
Keyword(s):  
Erythropoietin Receptor ◽  
Regulatory Subunit ◽  
Phosphatidylinositol 3 Kinase ◽  
Protein Substrates ◽  
Sh2 Domains ◽  
Src Homology 2 ◽  
Src Homology ◽  
Specific Association ◽  
Phosphatidylinositol 3

The erythropoietin receptor (EpR) belongs to a family of hematopoietin receptors whose members lack tyrosine kinase activity. Nonetheless, within minutes of binding Ep, a number of cellular proteins become transiently phosphorylated on tyrosine residues. One of these proteins, as we and others have shown previously, is the EpR itself. To identify the remaining protein substrates, we have examined the antiphosphotyrosine immunoprecipitates of lysates from Ba/F3 cells expressing high levels of cell surface EpRs. We now present data showing that, in response to Ep, the 85-Kd regulatory subunit of phosphatidylinositol 3-kinase (PI 3-kinase) becomes immunoprecipitable with antiphosphotyrosine antibodies. This appears to be due, in large part, to the specific association of PI 3-kinase with the tyrosine- phosphorylated EpR, either directly or through a 93- or 70-Kd tyrosine- phosphorylated intermediate. The activity of this EpR associated PI 3- kinase, assessed in anti-EpR immunoprecipitates, is maximal within 2 minutes of incubation with Ep and returns almost to baseline levels by 10 minutes. In vitro studies suggest that the interaction between PI 3- kinase and the activated EpR is mediated by the N- and C-terminal SH2 domains of p85 and tyrosine-phosphorylated motifs on the EpR.

Regulation of phosphatidylinositol 3-kinase by insulin in rat skeletal muscle

1993 ◽  
Vol 265 (5) ◽  
pp. E736-E742 ◽  
Author(s):  
K. S. Chen ◽  
J. C. Friel ◽  
N. B. Ruderman
Keyword(s):  
Skeletal Muscle ◽  
Soleus Muscle ◽  
Protein Tyrosine Kinase ◽  
Kinase Activity ◽  
Receptor Protein ◽  
Mammalian Skeletal Muscle ◽  
Phosphatidylinositol 3 Kinase ◽  
Phosphatidylinositol 3

The presence of phosphatidylinositol 3-kinase (PI 3-kinase) in mammalian skeletal muscle and its response to insulin stimulation were investigated. PI kinase, immunoprecipitated from rat soleus muscle with antibodies directed toward its 85-kDa subunit phosphorylated PI, phosphatidylinositol 4-phosphate [PI(4)P], and phosphatidylinositol 4,5,-bisphosphate [PI(4,5)P2] to yield phosphatidylinositol 3-phosphate [PI(3)P], phosphatidylinositol 3,4,-bisphosphate, and phosphatidylinositol trisphosphate in vitro. PI 3-kinase activity was also immunoprecipitated with antiphosphotyrosine [alpha-Tyr(P)] antibodies and with antibodies raised against IRS-1, a substrate of the insulin receptor protein tyrosine kinase that associates with and activates PI 3-kinase. Incubation of the soleus with insulin in vitro, or injection of insulin into rats in vivo, produced three- to fivefold increases in alpha-Tyr(P)- and alpha-IRS-1-immunoprecipitable PI 3-kinase activity. In nonstimulated soleus muscle, PI 3-kinase activity immunoprecipitated with alpha-IRS-1 or with alpha-Tyr(P) antibodies was evenly distributed between particulate (200,000-g pellet) and soluble fractions. Insulin treatment increased immunoprecipitable PI 5-kinase activity in both fractions, but the increase in alpha-Tyr-(P)-precipitable activity was greater in the particulate fraction, whereas the increase in alpha-IRS-1-precipitable activity was greater in the soluble fraction. In intact soleus muscles incubated with 32PO4, insulin increased the labeling of PI(3)P but did not affect the labeling of PI(4)P or PI(4,5)P2. Activation of PI 3-kinase by insulin was unaffected by prior denervation of the muscle, a manipulation that has been shown to cause both insulin resistance and hypersensitivity in muscles, depending on the parameter measured.(ABSTRACT TRUNCATED AT 250 WORDS)

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