The lipid kinase activity of the phosphatidylinositol 3-kinase is affected by its intrinsic protein kinase activity

1995 ◽  
Vol 23 (1) ◽  
pp. 14S-14S ◽  
Author(s):  
RüDIGER WOSCHOLSKI ◽  
TSUTOMU KODAKI ◽  
PETER J. PARKER
Keyword(s):  
Protein Kinase ◽  
Kinase Activity ◽  
Protein Kinase Activity ◽  
Phosphatidylinositol 3 Kinase ◽  
Lipid Kinase ◽  
Intrinsic Protein ◽  
Phosphatidylinositol 3

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 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 Comparison of the kinetic properties of the lipid- and protein-kinase activities of the p110α and p110β catalytic subunits of class-Ia phosphoinositide 3-kinases

2000 ◽  
Vol 350 (2) ◽  
pp. 353-359 ◽  
Author(s):  
Carolyn A. BEETON ◽  
Edwin M. CHANCE ◽  
Lazaros C. FOUKAS ◽  
Peter R. SHEPHERD
Keyword(s):  
Protein Kinase ◽  
Kinase Activity ◽  
Membrane Lipid ◽  
Kinetic Properties ◽  
Protein Kinase Activity ◽  
Lipid Kinase ◽  
Functional Roles ◽  
Wide Range ◽  
High Concentrations

Growth factors regulate a wide range of cellular processes via activation of the class-Ia phosphoinositide 3-kinases (PI 3-kinases). We directly compared kinetic properties of lipid- and protein-kinase activities of the widely expressed p110α and p110β isoforms. The lipid-kinase activity did not display Michaelis–Menten kinetics but modelling the kinetic data demonstrated that p110α has a higher Vmax and a 25-fold higher Km for PtdIns than p110β. A similar situation occurs with PtdIns(4,5)P2, because at low concentration of PtdIns(4,5)P2 p110β is a better PtdIns(4,5)P2 kinase than p110α, although this is reversed at high concentrations. These differences suggest different functional roles and we hypothesize that p110β functions better in areas of membranes containing low levels of substrate whereas p110α would work best in areas of high substrate density such as membrane lipid rafts. We also compared protein-kinase activities. We found that p110β phosphorylated p85 to a lower degree than did p110α. We used a novel peptide-based assay to compare the kinetics of the protein-kinase activities of p110α and p110β. These studies revealed that, like the lipid-kinase activity, the protein-kinase activity of p110α has a higher Km (550µM) than p110β (Km 8µM). Similarly, the relative Vmax towards peptide substrate of p110α was three times higher than that of p110β. This implies differences in the rates of regulatory autophosphorylation in vivo, which are likely to mean differential regulation of the lipid-kinase activities of p110α and p110β in vivo.

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