Dhand R, Hiles I, Panayotou G, Roche S, Fry M J, Gout I, Totty N F, Truong O, Vicendo P, Yonezawa K
Ludwig Institute for Cancer Research, London, UK.
EMBO J. 1994 Feb 1;13(3):522-33. doi: 10.1002/j.1460-2075.1994.tb06290.x.
Phosphatidylinositol 3-kinase (PI 3-kinase) has a regulatory 85 kDa adaptor subunit whose SH2 domains bind phosphotyrosine in specific recognition motifs, and a catalytic 110 kDa subunit. Mutagenesis of the p110 subunit, within a sequence motif common to both protein and lipid kinases, demonstrates a novel intrinsic protein kinase activity which phosphorylates the p85 subunit on serine at a stoichiometry of approximately 1 mol of phosphate per mol of p85. This protein-serine kinase activity is detectable only upon high affinity binding of the p110 subunit with its unique substrate, the p85 subunit. Tryptic phosphopeptide mapping revealed that the same major peptide was phosphorylated in p85 alpha both in vivo in cultured cells and in the purified recombinant enzyme. N-terminal sequence and mass analyses were used to identify Ser608 as the major phosphorylation site on p85 alpha. Phosphorylation of the p85 subunit at this serine causes an 80% decrease in PI 3-kinase activity, which can subsequently be reversed upon treatment with protein phosphatase 2A. These results have implications for the role of inter-subunit serine phosphorylation in the regulation of the PI 3-kinase in vivo.
磷脂酰肌醇3激酶（PI 3激酶）有一个85 kDa的调节性衔接子亚基，其SH2结构域在特定识别基序中结合磷酸酪氨酸，还有一个110 kDa的催化亚基。对p110亚基在蛋白质激酶和脂质激酶共有的序列基序内进行诱变，显示出一种新的内在蛋白激酶活性，该活性以约每摩尔p85 1摩尔磷酸盐的化学计量比使p85亚基的丝氨酸磷酸化。这种蛋白丝氨酸激酶活性只有在p110亚基与其独特底物p85亚基高亲和力结合时才能检测到。胰蛋白酶磷酸肽图谱分析表明，在培养细胞的体内和纯化的重组酶中，p85α中的同一主要肽段都被磷酸化。通过N端序列和质谱分析确定Ser608是p85α上的主要磷酸化位点。p85亚基在该丝氨酸处的磷酸化导致PI 3激酶活性降低80%，随后用蛋白磷酸酶2A处理可使其逆转。这些结果对亚基间丝氨酸磷酸化在体内PI 3激酶调节中的作用具有启示意义。
