Protein kinase activity that is independent of cAMP has been reported to exist on the surface of intact HeLa cells. Here we report that the protein kinase activity can be released by the use of casein or phosvitin within a short period of time. The discharge of the enzyme occurs from intact cells since (i) the cells do not release intracellular material and (ii) the cultures continue to grow without any morphological alteration. As (Heidelberg, Federal Republic of Germany), trypsin ("1:250") was from Difco. Heatand acid-stable inhibitor protein specific for catalytic subunits of cAMP-dependent protein kinases was isolated from rat skeletal muscle as described (13). 32P-Labeled phosvitin was obtained by incubation of phosvitin (5 mg/ml) and [ y-32P]ATP (0.5 ,uM; specific activity, 50 mCi/mmol; 1 Ci = 37 GBq) with intact HeLa cells for 15 min at 37°C. Cell supernatant was harvested and centrifuged at 2,500 x g for 10 min. Unbound [y-32P]ATP was removed from [32P]phosvitin by gel filtration (1.5 X 5 cm column; Ultrogel AcA 202 from LKB). Gel electrophoresis and autoradiography showed that phosvitin from different batches may possess either one or two major substrate components as evident from Figs. 1 and 3.HeLa cells were propagated in minimal essential medium/ 10% calf serum (Flow Laboratories) as described (5); embryo lung fibroblasts (HEL; human) were grown in basal medium Eagle's/10% fetal calf serum (Boehringer Mannheim). Hepatocytes from rat (provided by D. Mayer) were cultured in Ham's F12 medium/10% fetal calf serum; primary cultures from mouse cerebellum (provided by M. Schachner, Institute for Neurobiology, University of Heidelberg) were raised in basal medium Eagle's/10% horse serum (GIBCO Biocult). For experiments, cells were plated in plastic Petri dishes (Falcon; 3-, 5-, or 10-cm diameter). Synchronized HeLa cultures were obtained as described (14). Cell cycle traverse was monitored by flow cytometry (carried out by M. Stohr) as described (5). Cells were propagated free from mycoplasms.Preparation of cell supernatants was done at 37°C if not otherwise indicated. Cells were washed twice with prewarmed assay mixture (5 ml per 5-cm plate) consisting of 70 mM NaCl/ 30 mM TrisHOAc/5 mM Mg(OAc)2/5 mM potassium phosphate/0.5 mM EDTA/75 mM glucose, pH 7.2 (osmolarity, 290 ± 10 mOsm). Incubation was with 2 ml of assay mixture in the absence or presence of exogenous protein as given for the particular experiment. Cell incubation was done by moving the mixture gently over the cells (rocker platform; Bellco Glass) for various periods of time. Cell supernatants were centrifuged at 2,500 X g for 10 min and the flocculant was discarded. The remaining supernatant was kept in an ice bath until use. Control experiments in the absence of cells were carried out.For phosphorylation of cell surface protein in the absence or presence of exogenous protein substrate, intact monolayer cells were treated as described for the preparation of cell supernatants but incubation was in the presence of 0.5 ,uM [y_32p]_ ...

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