Catalytic and Molecular Properties of Highly Purified Phosvitin/Casein Kinase Type II from Human Epithelial Cells in Culture (HeLa) and Relation to Ecto Protein Kinase

Abstract

Phosvitin/casein type II kinase was purified from HeLa cell extracts to homogeneity and characterized. The kinase prefers phosvitin over casein (Vmax phosvitin > Vmax casein; apparent Km 0.5 .mu.M phosvitin and 3.3 .mu.M casein) and utilizes as cosubstrate ATP (apparent Km 3-4 .mu.M), GTP (apparent Km 4-5 .mu.M) and other purine nucleoside triphosphates, including dATP and dGTP but not pyrimidine nucleoside triphosphates. Enzyme reaction is optimal at pH 6-8 and at 10-25 mM .**GRAPHIC**. .**GRAPHIC**. cannot be replaced by, but is antagonized by other divalent metal ions. The kinase is stimulated by polycations (spermine) and monovalent cations .**GRAPHIC**. and is inhibited by fluoride, 2,3-diphosphoglycerate, and low levels of heparin (50% inhibition at 0.1 .mu.g/ml). The HeLa enzyme is composed of three subunits with Mr of approximately 43,000 (.alpha.), 38,000 (.alpha.''), and 28,000 (.beta.) forming .alpha..alpha.''.beta.2 and .alpha.''2.beta.2 structures with obvious sequence homology of .alpha. with .alpha.'' but not with .beta.. Photoaffinity labeling with [.alpha.-32P]- and [.gamma.-32P]8-azido-ATP revealed high affinity binding sites on subunits .alpha. and .alpha.'' but not on subunit .beta.. The kinase autophosphorylates subunit .beta. and, much weaker, subunits .alpha. and .alpha.''. Ecto protein kinase, detectable only by its enzyme activity but not yet as a protein (J. Biol. Chem. 257, 322-329), was characterized in cell-bound form and in released form, and the released form both with and without prior separation from phosvitin which was employed to induce the kinase release from intact HeLa cells (Proc. Natl. Acad. Sci. U.S.A. 80, 4021-4025). Ratios of phosvitin/casein phosphorylation (> 2) and of ATP/GTP utilization (1.5-2.1), inhibition by heparin (50% inhibition at 0.1 .mu.g/ml), and amino-acid side chains phosphorylated in phosvitin and casein (serine, threonine) are comparable for cell-bound and released form. These properties resemble those of type II kinase as does Mr of released ecto kinase (120-150,000). Consistently, a protein with Mr 125,000 in calf serum and a protein (possibly two) with Mr > 300,000 in calf plasma which are selectively phosphorylated by the ecto kinase are also substrates of the type II kinase. Thus, nearly all properties examined of the ecto kinase are characteristic for a type II kinase.