Phosphorylation of Endogenous Membrane Proteins by Endogenous Protein Kinase at the Outer Surface of Ehrlich Cells

Abstract

An endogenous protein kinase at the surface of Ehrlich cells has been studied. Using exogenous (Y~~P)ATP as a phosphoryl group donator, a transfer was demonstrated into endogenous acceptor protein(s) as well as to exogenous phosvitin. Seryl-and threonyl-residues isolated from the endogenous and exogenous acceptor protein were found to be labeled. The ratio between the labeled phosphorylserine and phosphorylthreonine was about 3.5 : 1 for both the endogenous acceptor of the intact cells and the exogenous acceptor. In similar experiments with a membrane preparation from Ehrlich cells, this ratio increased to about 7 : 1 provided the exogenous acceptor protein was absent. The results were independent of whether l~l O -~ M dibutyryl cyclic AMP was used or not with intact cells and a membrane fraction mainly consisting of vesicles. Whether the regulatory subunit of the membrane-associated protein kinase was in cis-or trans-disposition to the catalytic subunit no binding and dependence of the cyclic nucleotide was observed. Since the purified membrane fraction was considered free from endogenous cyclic AMP, it was concluded that the membrane-associated protein kinase of Ehrlich cells is not dependent on cyclic AMP. The critical role of arginine for: the cyclic AMP dependence of the serinecontaining residue in the catalytic subunit is discussed.

Phosphorylation of endogenous proteins by endogenous protein kinase of density gradient—purified plasma membrane vesicles of Ehrlich cells

“…In accordance with previous findings on intact Ehrlich cells and a plasma membrane preparation from these cells a, Agren and Ronquist 1976, Ronquist et al 1977, cyclic AMP and cyclic GMP did not stimulate the protein kinase reaction in the membrane vesicles (Table IV).…”

“…This reaction was also stimulated to a limited degree by cyclic AMP. Protein kinase-catalyzed membrane phosphorylations have subsequently been published for many membrane systems 404 PROTEIN KINASE IN EHRLlCH CELL VESICLES 405 (for references see Agren and Ronquist 1976). These involved either the endogenous protein kinase and an extrinsic acceptor or cases where both the enzyme and the acceptor were endogenous.…”