Protein kinase activity and substrates at the surface of intact HeLa cells.

D Kübler,W Pyerin,V Kinzel

doi:10.1016/s0021-9258(19)68365-1

Abstract

Evidence is presented for the location at the surface of HeLa cells of a protein kinase capable of phosphorylating surface as well as extracellular (foreign) proteins. The reaction products have been found to be proteins containing phosphoryl groups as monoesters of seryl and threonyl residues (but not of tyrosine). The enzyme is of the cyclic AMP-independent type, since neither cyclic AMP nor the heat- and acid-stable inhibitor protein (specific for cyclic AMP-dependent protein kinases) influenced its activity. Further, co-substrate ATP could in part be substituted by GTP, and the spectrum of proteins phosphorylated by the ecto-enzyme differed from that phosphorylated by cyclic AMP-dependent protein kinases. Evidence for the ecto-enzymic nature of this protein kinase includes (a) utilization of co-substrate and location of products at the surface of cells carefully controlled as being in an intact state and (b) phosphorylation of exogenous protein (phosvitin; specific serum proteins) by intact cells. Conclusive proof was gained by qualitative and quantitative comparative studies of phosphorylation in cultures with varying degrees of damaged cells either as a whole or after separation into groups of intact and damaged cells by electronic cell sorting. The results of experiments with cell sonicates excluded the possibility that either enzyme or substrates released from damaged cells were simply adsorbing to the cell surface.

Highlights

Of HeLa cells of a protein kinase capable of phospho- this has not been done in the studies made to rylating surfaceas well as extracellular pro- date
HeLa cells/dish were phosphorylated a t 37 "C with [y-32P]ATP(0.5 p ~ )A. t the times indicated, the reaction was terminated by removal of the supernatant and addition of cold5% trichloroacetic acid
The protein kinase inhibitor used was prepared from rat muscle and had been shown to inhibit both type I and type I1 cyclicAMP-dependent protein kinase isolated from HeLa cells[32].The amounts of protein kinase inhibitor applied were sufficientto suppress the activity of >lo" units of highly purified catalytic subunit of cyclic AMP-dependent protein kinase by 50%.These data classify the HeLa ecto-enzyme as belonging to the group of cyclic AMP-independent protein kinase

Summary

Introduction

Of HeLa cells of a protein kinase capable of phospho- this has not been done in the studies made to rylating surfaceas well as extracellular (foreign) pro- date. Conditions for and Characterization of the Endogenous Cell Surface Phosphorylation-On incubation of intact HeLa cells (monolayer) under isoosmotic conditions in presence of micromolar concentrations of [Y-~*P]ATwPe observed incorporation of radioactivity into material precipitable by trichloroacetic acid.

Results

Conclusion