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

Abstract

Class I phosphoinositide 3-kinases (PI3Ks) are bifunctional enzymes possessing lipid kinase activity and the capacity to phosphorylate their catalytic and/or regulatory subunits. In this study, in vitro autophosphorylation of the G protein-sensitive p85-coupled class I A PI3K␤ and p101-coupled class I B PI3K␥ was examined. Autophosphorylation sites of both PI3K isoforms were mapped to Cterminal serine residues of the catalytic p110 subunit (i.e. serine 1070 of p110␤ and serine 1101 of p110␥). Like other class I A PI3K isoforms, autophosphorylation of p110␤ resulted in down-regulated PI3K␤ lipid kinase activity. However, no inhibitory effect of p110␥ autophosphorylation on PI3K␥ lipid kinase activity was observed. Moreover, PI3K␤ and PI3K␥ differed in the regulation of their autophosphorylation. Whereas p110␤ autophosphorylation was stimulated neither by G␤␥ complexes nor by a phosphotyrosyl peptide derived from the platelet-derived growth factor receptor, autophosphorylation of p110␥ was significantly enhanced by G␤␥ in a time-and concentration-dependent manner. In summary, we show that autophosphorylation of both PI3K␤ and PI3K␥ occurs in a C-terminal region of the catalytic p110 subunit but differs in its regulation and possible functional consequences, suggesting distinct roles of autophosphorylation of PI3K␤ and PI3K␥.Class I phosphoinositide 3-kinases (PI3Ks) 1 are lipid kinases that are activated in response to a variety of extracellular stimuli including hormones, neurotransmitters, and growth factors, which act via G protein-coupled receptors or receptor tyrosine kinases. These lipid kinases phosphorylate the D3 position of the inositol ring of phosphoinositides, thus generating intracellular lipid second messengers (1, 2). PtdIns, PtdIns-4-P, and PtdIns-4,5-P 2 are in vitro substrates of class I PI3Ks, although these enzymes predominantly produce PtdIns-3,4,5-P 3 in vivo. Class I PI3K lipid products transmit signals by recruiting intracellular effector molecules to the membrane, which contain particular pleckstrin homology domain modules. Effectors include serine/threonine kinases like Akt/protein kinase B, Tec family tyrosine kinases, and guanine nucleotide exchange factors for monomeric GTP-binding proteins like Grp1 and Vav (3). Consequently, class I PI3Ks are involved in the regulation of a wide variety of cellular functions such as differentiation, proliferation, survival, migration, and metabolism (4, 5).All class I PI3Ks are heterodimers consisting of a p110 catalytic and a p85 or p101 type regulatory subunit. According to the type of their associated regulatory subunit, class I PI3Ks can be further distinguished. The class I A catalytic p110␣, -␤, and -␦ subunits complex with adaptor molecules containing two Src homology 2 domains, of which p85 is the prototype. Through interaction of the adaptor Src homology 2 domains with phosphotyrosines, class I A PI3Ks are activated by receptor tyrosine kinases. In contrast, the only class I B member, p110␥, associates with a p101 regulatory subunit and is ...

Regulation of Phosphoinositide 3-Kinase by Its Intrinsic Serine Kinase Activity In Vivo

“…It is also consistent with the finding that in an in vitro autokinase assay, recombinant p110␤ preferentially autophosphorylates instead of phosphorylating the p85 regulatory subunit (17). The site of p110␤ autophosphorylation has recently been mapped as Ser1070 (12). Also, when we coexpressed the p85␣ subunit with a catalytically inactive p110␣, recognition by the phosphospecific antibody was barely observed (Fig.…”

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

“…However, PI3Ks also possess inherent protein kinase activity that can lead to autophosphorylation and phosphorylation of diverse target proteins (24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35). To determine the enzymatic activities required for oncogenic transformation, we generated kinase-inactive mutations and determined their transforming activity.…”

Nuclear inositol lipid metabolism: More than just second messenger generation?

“…This phosphorylation step down-regulates the lipid kinase activity and occurs in vivo in response to stimulation with growth factors, including insulin and platelet-derived growth factor. In contrast, p110b and p110d autophosphorylate on Ser 1070 and Ser 1039 , respectively [Vanhaesebroeck et al, 1999;Czupalla et al, 2003]. Also in these cases, autophosphorylation results in a decreased lipid kinase activity.…”