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

Abstract

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 p110alpha and p110beta isoforms. The lipid-kinase activity did not display Michaelis-Menten kinetics but modelling the kinetic data demonstrated that p110alpha has a higher V(max) and a 25-fold higher K(m) for PtdIns than p110beta. A similar situation occurs with PtdIns(4,5)P(2), because at low concentration of PtdIns(4,5)P(2) p110beta is a better PtdIns(4,5)P(2) kinase than p110alpha, although this is reversed at high concentrations. These differences suggest different functional roles and we hypothesize that p110beta functions better in areas of membranes containing low levels of substrate whereas p110alpha would work best in areas of high substrate density such as membrane lipid rafts. We also compared protein-kinase activities. We found that p110beta phosphorylated p85 to a lower degree than did p110alpha. We used a novel peptide-based assay to compare the kinetics of the protein-kinase activities of p110alpha and p110beta. These studies revealed that, like the lipid-kinase activity, the protein-kinase activity of p110alpha has a higher K(m) (550 microM) than p110beta (K(m) 8 microgM). Similarly, the relative V(max) towards peptide substrate of p110alpha was three times higher than that of p110beta. This implies differences in the rates of regulatory autophosphorylation in vivo, which are likely to mean differential regulation of the lipid-kinase activities of p110alpha and p110beta in vivo.

Identification and Characterization of the Autophosphorylation Sites of Phosphoinositide 3-Kinase Isoforms β and γ

“…In contrast to the predominant autophosphorylation of p110␤ observed in this and previous studies (27), others have reported p85 phosphorylation as the major target of PI3K␤ autophosphorylation (25,26). In order to explain the apparent discrepancies, one must consider the experimental conditions used in these studies.…”

“…Recent reports have described autophosphorylation of both subunits of PI3K␤ (i.e. p85 and p110␤) (25)(26)(27). In order to analyze autophosphorylation of PI3K isoforms in vitro, we expressed recombinant heterodimeric PI3K␣ and PI3K␤ in insect cells and measured their protein kinase activities (Fig.…”

Experimental Evidence for the Limiting Role of Enzymatic Reactions in Chemoattractant-induced Pseudopod Extension in Human Neutrophils

“…Another estimate for the same concentration was 6.7 M from the calculated PtdIns(4,5)P 3 concentration from the measurement of 1.8 ng PtdIns(4,5)P 2 per 10 6 neutrophils (65), while taking into account the volume of a single neutrophil, which is on the order of 270 m 3 (66). These estimates are consistent with the relationship ͓S͔ Ͻ Ͻ K m from Equation 4, and the value of the Michaelis-Menten constant for PI3K on the order of K m ϭ 100 M (54). The total enzyme concentration, 30 nM PI3K, was calculated from the total number of available binding sites for wortmannin per neutrophil, which is on the order of 5,000 molecules (58).…”

Oncogenic transformation induced by the p110β, -γ, and -δ isoforms of class I phosphoinositide 3-kinase

“…The relative enzymatic activity of p110␣ is higher than that of other isoforms (49,50), suggesting that the inability of the wild type p110␣ to induce transformation is not due to low specific activity. Western analyses suggest that the non-␣ isoforms of p110 are expressed at higher levels than p110␣, assuming that the sensitivities of isoform detection by the different antibodies are roughly comparable (Fig.…”