Pyruvate kinase type M 2 is phosphorylated at tyrosine residues in cells transformed by Rous sarcoma virus

Abstract

Chicken embryo cells (CECs) contain pyruvate kinase (PK) type M 2 (M 2-PK). Transformation of CECs by Rous sarcoma virus (RSV) leads to a reduction in the affinity of PK for the substrate phospho enolpyruvate. In vitro, M 2-PK can be phosphorylated at tyrosine residues by pp60 v-src, the transforming protein of RSV. To study tyrosine phosphorylation of M 2-PK in intact RSV-transformed cells, the protein was immunoprecipitated from 32P-labeled normal and RSV-SR-A-transformed CECs. Phosphoamino acid analysis of immunoprecipitated M 2-PK revealed that M 2-PK of both normal and transformed CECs contained phosphoserine and small amounts of phosphothreonine. Only M 2-PK of transformed CECs contained phosphotyrosine in addition. For enzyme kinetic studies M 2-PK was partially purified by chromatography upon DEAE-Sephacel and hydroxyapatite. A decreased affinity for phospho enolpyruvate was observed 3 h after the onset of transformation using the temperature-sensitive mutant of RSV, ts-NY 68. The kinetic changes were correlated with tyrosine phosphorylation of M 2-PK, but there is no direct evidence that they are caused by post-translational modification of the enzyme.