Insulin treatment enhances casein kinase II (CKII) activity in 3T3-L1 mouse adipocytes and H4-IIE rat bepatoma cells, the magnitude of the activation varying from 30% to 150%. Activation of CKH was apparent after 5 min of exposure of 3T3-L1 cells to insulin, was maximal by 10 min, and persisted through 90 min. The insulin-stimulated activity was inhibited by low concentrations of heparin and was stimulated by spermine. Activation of CKII was effected by physiological concentrations of insulin'(EC50 = 0.15 nM), suggesting that the effect is a true insulin response and not one mediated through insulin-like growth factor receptors. Epidermal growth factor (100 ng/ml for 10 min) also activated CKII in A431 human carcinoma cells, which is consistent with other observations that insulin and epidermal growth factor may have some common effects. Insulin stimulation of CKII activity was due to an increase in the maximal velocity of the kinase; the apparent Km for peptide substrate was not altered. Enhanced activity did not appear to result from increased synthesis of CKII protein, because cycloheximide did not block the effect and because an immunoblot developed with antiserum to CKII showed no effect of insulin on the cytosolic'concentration of CKII. Because insulin-stimulated CKII activity was maintained after chromatography ofcell extracts on Sephadex G-25, it is unlikely that the effect is mediated by a low-molecularweight activator ofthe kinase. Rather, the results are consistent with the possibility that insulin activates CKII by promoting a covalent modification of the kinase.There is considerable evidence that reversible protein phosphorylation contributes to the mechanism of insulin action (reviewed in ref. 1). The f3 subunit of the insulin receptor is a protein-tyrosine kinase that is activated by insulin, and the hormone promotes phosphorylation of several proteins on tyrosine residues (1). Insulin also enhances phosphorylation of sefine and threonine residues in proteins, including the insulin receptor (1), ribosomal protein S6 (2-4), ATP-citrate lyase (5, 6), membrane-bound cAMP phosphodiesterase (7) Although CKII has been implicated in the regulation of a wide variety of cellular processes, including the synthesis of glycogen, fatty acids, RNA, and protein (11, 12), there have been few studies of its regulation. Such studies have been facilitated, however, by the development of a specific peptide substrate for CKII (13) that was useful for estimating changes in kinase activity during differentiation of 3T3-L1 cells (14). Because CKII appears to phosphorylate an insulin-stimulated phosphorylation site and because it has been implicated in the regulation of a variety of fundamental cellular processes, we undertook studies of the short-term regulation of CKII activity and found that the kinase is rapidly activated by insulin (12). In this paper, we report in detail the characteristics of the response of CKII to insulin and that the kinase is also activated by epidermal growth factor. EXPERIMENTAL PROCE...