Purification and Characterization of Gβγ-responsive Phosphoinositide 3-Kinases from Pig Platelet Cytosol

Abstract

A G-protein ␤␥ subunit (G␤␥)-responsive phosphoinositide 3-kinase (PI 3-kinase) was purified approximately 5000-fold from pig platelet cytosol. The enzyme was purified by polyethylene glycol precipitation of the cytosol followed by column chromatography on Q-Sepharose fast flow, gel filtration, heparin-Sepharose, and hydroxyapatite. The major G␤␥-responsive PI 3-kinase is distinct from p85 containing PI 3-kinase as the activities can be distinguished chromatographically and immunologically and is related to p110␥ as it crossreacts with anti-p110␥-specific antibodies. The p110␥-related PI 3-kinase cannot be activated by G-protein ␣ i/o subunits, and it has an apparent native molecular mass of 210 kDa. The p110␥-related PI 3-kinase phosphorylates phosphatidylinositol (PtdIns), phosphatidylinositol 4-phosphate (PtdIns4P), and phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2 ). The apparent K m values for ATP were found to be 25 M with PtdIns, 44 M with PtdIns4P, and 37 M with PtdIns(4,5)P 2 as the substrate. G␤␥ subunits did not alter the K m of the enzyme for ATP; however, V max increased 2-fold with PtdIns as substrate, 3.5-fold with PtdIns4P, and 10-fold with PtdIns(4,5)P 2 . Under basal conditions the apparent K m values for lipid substrates were 64, 10, and 15 M for PtdIns, PtdIns4P, and PtdIns(4,5)P 2 , respectively. In the presence of G␤␥ subunits the dependence of PI 3-kinase activity on the concentrations of lipid substrates became complex with the highest level of stimulation occurring at high substrate concentration, suggesting that the binding of G␤␥ and lipid substrate (particularly PtdIns(4,5)P 2 ) may be mutually cooperative. Wortmannin and LY294002 inhibit the G␤␥-responsive PI 3-kinase activity with IC 50 values of 10 nM and 2 M, respectively. Unlike the p85 containing PI 3-kinase in platelets, the p110␥-related PI 3-kinase is not associated with a PtdIns(3,4,5)P 3 specific 5-phosphatase.The p85-associated PI 3-kinase was not activated by G␤␥ alone but could be synergistically activated by G␤␥ and phosphotyrosyl platelet-derived growth factor receptor peptides. This may represent a form of coincidence detection through which the effects of tyrosine kinase and G-protein-linked receptors might be coordinated.Phosphoinositide 3-kinases (PI 3-kinases, 1 EC 2.7.1.137) phosphorylate the D-3 position of the inositol ring in inositol phospholipids and were originally identified through their association with viral oncoproteins and activated tyrosine kinases (1, 2). Receptor-regulated forms of PI 3-kinase appear to prefer to phosphorylate phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2 ) in vivo (3-5). The product of this reaction, phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P 3 ), is a candidate second messenger that regulates a variety of cellular responses to growth factors perhaps through the activation of serine/threonine protein kinases such as Akt/PKB and/or certain protein kinase C isoforms and small GTP-binding proteins such as Rac 1 (6 -10).The first form of PI 3-kinase to be...

Ca2+-Evoked Serotonin Secretion by Parafollicular Cells: Roles in Signal Transduction of Phosphatidylinositol 3′-kinase, and the γ and ζ Isoforms of Protein Kinase C

“…These phosphorylated proteins bind to the Src homology 2 domains of p85, which in turn regulates the activity of p110 (␣, ␤, or ␦). A second mode of PI3Ј-kinase activation is via the ␤␥ subunits of G proteins, which activate the p110␥ monomer (Tang and Downes, 1997). A third mechanism of PI3Ј-kinase activation involves ␤␥ subunits working synergistically with a phosphotyrosyl peptide to activate the heterodimer enzyme p110␤/p85 (Thomason et al, 1994;Kurosu et al, 1997).…”

“…PI3Ј-kinase is a heterodimer composed of an 85 kDa regulatory subunit (p85␣) and a 110 kDa catalytic subunit (p110) (Carpenter et al, 1990;Shibasaki et al, 1991;Kapeller and Cantley, 1994). Several isoforms of this enzyme have been detected (␣, ␤, and ␥) (Thomason et al, 1994;Rordorf-Nikolic et al, 1995;Ptasznik et al, 1996;Kurosu et al, 1997;Tang and Downes, 1997). Immunoblot analysis demonstrated that subunits of PI3Ј-kinase, p85 and p110 (␣ϩ␤), are expressed in PF cells (Fig.…”

An Epidermal Growth Factor Receptor/Gab1 Signaling Pathway Is Required for Activation of Phosphoinositide 3-Kinase by Lysophosphatidic Acid

“…In addition, although p110␥ is not apparently involved in generation of PI3K lipid products, a recent report demonstrated that the protein kinase activity of p110␥ is essential for MAPK activation (18), in agreement with our observation that a catalytically dead mutant of p110␥ moderately inhibited cell growth without influencing PIP 3 levels. However, it is also important to consider that the expression of p110␥ in fibroblasts is marginal in comparison to blood platelets, 3 type where activation of PI3K by the thrombin GPCR has been reported to engage p110␥ (32,33). Similarly, in neutrophils where p110␥ is readily expressed (15), wortmannin has been shown to inhibit GPCR-induced signaling independently of p85 (34).…”

Phosphatidylinositol transfer proteins and protein kinase C make separate but non-interacting contributions to the phosphorylation state necessary for secretory competence in rat mast cells

“…Here we report that, after permeabilization, the secretory reaction becomes progressively more sensitive to inhibition by neomycin and that this can be reversed by providing, from the outset, phosphatidylinositol transfer proteins (PITPs) or PtdIns(4,5)P # in soluble form, -(j)-sn-1,2-di-O-octanoyl PtdIns(4,5)P # (3-O-phospho-linked) [diC ) -PtdIns(4,5)P # ]. Even after substantial run-down, the requirement for ATP can be saturated at 50 µM, which is sufficient to allow the operation of phosphoinositide 3-kinases (PI-3Ks) and phosphoinositide 4-kinases (PI-4Ks) [5,6] and also some isoforms of protein kinase C (PKC) [7]. Further experiments in which we applied neomycin, 1-O-hexadecyl-2-O-methyl-racglycerol (AMG-C "' ) (an inhibitor of PKC) and PITPs indicate that the phosphorylation state required for secretory competence in mast cells is composed of two distinct components.…”