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 ...