Activation of casein kinase II in response to insulin and to epidermal growth factor (protein phosphorylation/signal transduction)

Transcription

1 Proc. Nail. Acad. Sci. USA Vol. 84, pp , December 1987 Biochemistry Activation of casein kinase II in response to insulin and to epidermal growth factor (protein phosphorylation/signal transduction) JAMES SOMMERCORN, JENNY A. MULLIGAN, FRED J. LOZEMAN, AND EDWIN G. KREBS Howard Hughes Medical Institute and Department of Pharmacology, University of Washington, Seattle, WA Communicated by Edmond H. Fischer, August 21, 1987 ABSTRACT 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 3% to 15%. Activation of CKH was apparent after 5 min of exposure of 3T3-L1 cells to insulin, was maximal by 1 min, and persisted through 9 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'(ec5 =.15 nm), suggesting that the effect is a true insulin response and not one mediated through insulin-like growth factor receptors. Epidermal growth factor (1 ng/ml for 1 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 of cell extracts on Sephadex G-25, it is unlikely that the effect is mediated by a low-molecularweight activator of the 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), acetyl-coa carboxylase (8, 9), and an Mr 22, protein (1). These observations have led to intensive efforts to identify and characterize protein-serine or -threonine kinases that are activated by insulin because such information could contribute greatly to understanding the mechanism of insulin signal transduction. We became interested in the possibility that casein kinase II (CKII) might be activated by insulin on the basis of a report by Witters et al. (9) showing that, in vitro, CKII phosphorylates the apparent insulin-dependent phosphorylation site in acetyl-coa carboxylase. Distinguishing characteristics of CKII (reviewed in ref. 11) are that it phosphorylates acidic proteins such as casein and phosvitin, it uses GTP as a substrate nearly as well as ATP, it is inhibited by low The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C solely to indicate this fact concentrations of heparin and is activated by polyamines (11). 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 PROCEDURES Cell Culture. 3T3-L1 mouse cells were grown and induced to differentiate to adipocytes as previously described (14). Insulin experiments were done routinely with cells that had differentiated for 7 or 8 days. In several experiments, cells that had differentiated for 1 days were used, but there was no effect of insulin on CKII activity. H4-IIE rat hepatoma cells and A431 human carcinoma cells were cultured as described (14). Time courses of the insulin response were constructed as follows. In each experiment, extracts of cells from two plates were prepared as zero-time controls. Fresh medium with 1o fetal calf serum either without insulin or with 5 nm insulin was then added to other plates, which were incubated at 37C. At various times thereafter, extracts were prepared from two plates each of control and insulintreated cells. Examination of the dose-response relationship was conducted by adding fresh medium with 1% fetal calf serum either without or with various concentrations of insulin (three plates each) and incubating the cells at 37C for 3 min. The influence of cycloheximide on stimulation of CKII activity by insulin was tested by using a protocol similar to that of the dose-response experiments with the exception that the cells were preincubated with cycloheximide (15 gg/ml) for 15 min at 37C and all test media contained cycloheximide. The effect of epidermal growth factor was tested by using A431 human carcinoma cells, which were given fresh medium containing 1% fetal calf serum either without or with epidermal growth factor (1 ng/ml) and incubated at 37C. Soluble extracts of the cells were prepared after 1 min. Soluble Extracts. Medium was removed and the plates were washed twice with 5 ml of cold Dulbecco's phosphatebuffered saline (GIBCO catalogue no ). The cells were scraped in 1.5 ml of cold extraction buffer (8 mm Abbreviation: CKII, casein kinase II.

2 Biochemistry: Sommercorn et al. 3-glycerophosphate/2 mm EGTA/15 mm MgCl2, ph 7.3) and disrupted by 2 strokes of a tight Dounce homogenizer. The homogenates were centrifuged at 2, x g for 2 min at 4C and aliquots of the supernates were assayed immediately for CKII activity. The remainder of each supernate was frozen and stored in liquid nitrogen, which preserved insulinenhanced CKII activity. Supplementation of the extraction buffer with a mixture of protease inhibitors and additional phosphatase inhibitors (5 mm NaF and.2 mm Na3VO4) did not appear to influence the magnitude of the insulin effect. Protein was assayed as described (14). Kinase Assays. CKII was assayed by monitoring incorporation of radioactivity from [y-32p]atp into the synthetic peptide substrate Arg-Arg-Arg-Glu-Glu-Glu-Thr-Glu-Glu- Glu (14). Each extract was assayed with and without peptide in duplicate and the difference between the averages were used to calculate the specific activity of CKII (pmol/min per mg of protein). Assays were linear with both the amount of extract assayed (5-2 pul/5 gl assay) and with time up to 15 min. Gel Filtration of Extracts. Frozen extracts of cells were thawed and a.5-ml aliquot was applied to a 2-ml column of Sephadex G-25 equilibrated with extraction buffer at 4C. Protein was eluted from the column with cold extraction buffer and 12-drop fractions (about.2 ml) were collected and immediately assayed with CKII peptide substrate. Peak fractions were pooled and assayed with and without peptide substrate. It was verified that these chromatographic conditions were sufficient to separate protein from low molecular weight compounds such as ATP and ammonium sulfate. NaDodSO4/PAGE and Immunoblotting. Frozen extracts of 3T3-L1 cells were thawed and aliquots containing equal amounts of protein were subjected to NaDodSO4/PAGE, electrotransferred to nitrocellulose, and probed with antiserum to CKII followed by 125I-labeled staphyloccocal protein A (14). Radioactivity associated with subunits of CKII was determined by y-ray counting of excised bands. Materials. 3T3-L1 fibroblasts and H4-IIE hepatoma cells were obtained from American Type Culture Collection. A431 human carcinoma cells were provided by Byron Gallis of this laboratory. Insulin and epidermal growth factor were purchased from Collaborative Research and cycloheximide from Sigma. Antiserum to bovine CKII was provided by Michael Dahmus (University of California, Davis). Sources of other materials were as previously described (14). RESULTS Treatment of 3T3-L1 cells with 5 nm insulin for 3 min resulted in enhanced activity of CKII measured in cell extracts (Fig. 1). The magnitude of the effect was independent of the amount of extract assayed and, in this experiment, equalled 86% compared to the control. Among all experiments, the magnitude of the response varied between 3% and 15%. This variability may be due to several factors, including the possible lability of the mechanism by which CKII is activated and the variability in the fraction of 3T3-L1 cells that differentiate into adipocytes, since only the adipocytes have appreciable numbers of insulin receptors (14, 15). To verify that insulin-stimulated peptide phosphorylation reflected CKII activity, the influences of heparin and spermine on peptide kinase activity were determined. Low concentrations of heparin inhibited peptide phosphorylation in extracts from both control and insulin-treated 3T3-L1 cells (Fig. 2). Concentrations that gave half-maximal inhibition were 1 nm and 13 nm for control and insulin-treated extracts, respectively, which are values in the range of those reported for the K, of purified CKII (1-2 nm, refs. 11, 13). Peptide kinase activity in extracts of both control and Proc. Natl. Acad. Sci. USA 84 (1987) 8835 '5- X 4-2-., 2- 'O Time, min FIG. 1. Activation of CKII by insulin. 3T3-L1 cells were incubated for 3 min with fresh medium or medium with 5 nm insulin. CKII was assayed with a synthetic peptide substrate and activities were corrected for different protein concentrations in the extracts. Assay mixtures contained either 5,l (o, o) or 2,ul (n, o) of extract in a total volume of 5 Al. Closed symbols indicate control extracts and open symbols refer to insulin extracts. insulin-treated 3T3-L1 cells was also stimulated 5o by 1,uM spermine (data not shown). These results, along with previous findings (13, 14), are consistent with the conclusion that insulin-stimulated peptide phosphorylation reflects activation of CKII. The time course of CKII activation by insulin is shown in Fig. 3, which presents results from three experiments. Activation is apparent by 5 min of exposure of 3T3-L1 cells to 5 nm insulin, it is maximal by 1 min, and it persists in the presence of insulin for as long as 9 min (data not shown). The difference between average values of CKII activity from control cells (n = 16) and from cells treated with insulin for 1 min or more (n = 25) was assessed by Student's t test (16) and found to be statistically significant (P <.1). The activity of CKII is very sensitive to insulin, as shown in Fig. 4, which is composed of data from three experiments. The plateau was arbitrarily drawn as the mean of values for insulin concentrations greater than.15 nm. From this figure, the concentration giving 5% of maximal activation (EC5) was estimated to be about.15 nm insulin. Because of evidence that insulin and some peptide growth factors have common effects on cells (1, 17-19), the influence of epidermal growth factor on CKII activity was tested with A431 human carcinoma cells. Incubation of cells with epidermal growth factor (1 ng/ml) for 1 min increased CKII activity in cell extracts from 9.1 ± 1.6 to 16.6 ± 1.7 pmol/min per mg of protein (mean ± SEM, n = 3), an effect that was statistically significant (P <.5) as determined by Student's t test (16). The effect of epidermal growth factor was confirmed in subsequent experiments, although the magnitude of the activation was not as large (2-3%). 11._ ,/ [Heparin], nm FIG. 2. Inhibition of control and insulin-stimulated CKII activity by heparin. Extracts of control (o) and insulin-treated (5 nm for 6 min) (o) 3T3-L1 cells were assayed for CKII with a synthetic peptide substrate.

3 8836 Biochemistry: Sommercorn et al. Proc. Natl. Acad. Sci. USA 84 (1987) c.4- Irl Ej cm E Time, min FIG. 3. Time course of activation of CKII by 5 nm insulin. The figure represents three separate experiments. At various times, extracts were prepared from two plates each of control (e) and insulin-treated cells (o) cells. CKII was assayed immediately, and the data from each experiment were standardized by expressing each value as a percentage of the zero-time control. Error bars represent SEM, n = 3-6; data points without error bars are the mean of two values. Fig. 5 shows a Lineweaver-Burk plot of the relationship between peptide concentration and CKII activity in extracts of control and insulin-treated 3T3-L1 cells. Linear regression equations of these data revealed that the apparent Km for the peptide ( mm) was not changed appreciably by insulin treatment, but that the Vm. was increased by 76% from 62.5 to 19.9 pmol/min per mg of protein. The enhanced maximal velocity of CKII in response to insulin could result from several different mechanisms. One possibility is that insulin increases the amount of CKII protein by either increasing its rate of synthesis or decreasing its rate of degradation. The contribution of protein synthesis to the activation was assessed by testing the influence ofcycloheximide on the ability of insulin to stimulate CKII activity in H4-IIE cells (Table 1). In the absence of cycloheximide, exposure of cells to 5 nm insulin for 3 min caused a 26% increase in CKII activity and, in the presence of cycloheximide, insulin increased activity by 43%. Because cycloheximide failed to block the effect, it appears that protein synthesis is not required for insulin to activate CKII. Although the above results show that enhanced CKII E 4-2- OR - : -1-9 Log [insulin], M * FIG. 4. Influence of insulin concentration on CKII activity. 3T3-L1 cells were incubated for 3 min in the presence of fresh control medium or medium supplemented with various concentrations of insulin. CKII in freshly prepared cell extracts was assayed and the data within each experiment were standardized as follows. The activation of CKII was calculated as the difference between the average activity in extracts of cells treated with a certain concentration of insulin and the average activity of the control extracts. The largest activation was taken as the maximum and the other values were expressed as a percentage of the maximum, which was 3-15% of the control value. The plateau was arbitrarily drawn as the average activation produced by insulin at concentrations greater than.15 nm ll[peptide] (1/mM) FIG. 5. Effect of the concentration of peptide substrate on the activity of CKII from control and insulin-treated cells. CKII was assayed in extracts of control (e) and insulin-treated (5 nm for 6 min) (o) 3T3-L1 cells in the presence of 1,uM ATP and various concentrations of the peptide substrate Arg-Arg-Arg-Glu-Glu-Glu- Thr-Glu-Glu-Glu. The data are presented as a double-reciprocal plot and lines were fitted by linear regression. These assays were repeated twice with different sets of extracts, and similar results were obtained. activity in response to insulin does not result from synthesis of new CKII, they do not eliminate the possibility that the effect involves an increased amount of soluble CKII protein resulting from a lowered rate of degradation of CKII or by release of CKII protein into the cytosol. To determine the amount of CKII protein in extracts of 3T3-L1 cells, aliquots were subjected to immunoblot analysis (Fig. 6). Radioactivity associated with the a and (3 subunits of CKII averaged, respectively, 1872 ± 8 (SEM) and 13 ± 63 cpm for extracts from control cells and 198 ± 66 and cpm for extracts from insulin-treated cells. Thus, activation of CKII by insulin does not appear to involve an increased concentration of CKII. To verify that the blotting method could detect small differences in the amount of CKII protein, additional aliquots containing 1.3- and 1.6-fold more ofone of the control extracts were included in the analysis. As shown in Fig. 6, there was a linear relationship between the amount of CKII a-subunit protein and the volume of extract analyzed. Alternative mechanisms for the effect include activation of CKII by an effector molecule and covalent modification.of the enzyme. To determine if the effect was mediated by an activator of CKII, extracts of control and insulin-treated 3T3-L1 cells were subjected to gel filtration chromatography on Sephadex G-25. An elution profile, shown in Fig. 7, demonstrates that insulin-stimulated CKII activity is retained after chromatography. In three such experiments, the average insulin effect in extracts was 51% ± 24% (SEM), and it Table 1. Influence of cycloheximide on the activation of CKII by insulin CKII activity, pmol/min Cycloheximide, per mg protein Activation, jig/ml Control Insulin % Significance P < ± ± P <.1 Confluent cultures of H4IIE cells were given fresh medium either without insulin or with 5 nm insulin. After incubation at 37C for 3 min, cells were harvested and extracts were prepared and frozen in liquid nitrogen. Cells that received cycloheximide were treated similarly except that they were preincubated with cycloheximide (15,ug/ml) for 15 min and all media contained cycloheximide. CKII activity was assayed in soluble extracts of the cells. The data are presented as mean ± SEM, n = 5. Statistical significance of the difference between control and insulin treatment was assessed by Student's t test. 1

4 26- F co rr -b IX -~ Biochemistry: Sommercorn et al. c 2- E Amount of extract (relative) Control Insulin 1.6A 1.3A A B C A B C FIG. 6. Immunoblot analysis of casein kinase II protein in extracts of control- and insulin-treated cells. Frozen extracts from control and insulin-treated 3T3-L1 cells were thawed and aliquots were blotted as described in the text. A photograph of the autoradiogram is shown, indicating positions of molecular weight markers (Mr X 1-3) and the a, a', a", and f subunits of CKII on the left. Different letters above the lanes identify different extracts. Lanes labeled 1.3A and 1.6A refer to additional aliquots of control extract A containing 1.3- and 1.6-fold more extract. The amount of CKII a subunit was quantitated by y-ray counting of bands cut from the nitrocellulose. The standard curve consists of data from control extract A. The average CKII activities (±SEM) were 34.1 ± 1.3 and 19.9 ± 5.7 pmol/min per mg of protein for extracts of control and insulin-treated cells. was 46% ± 2% in pooled fractions (numbers 4-6) after gel filtration. These data argue against the involvement of a low-molecular-weight activator in the mechanism by which CKII activity is stimulated by insulin and are consistent with the possibility that activation of CKII is produced by covalent modification of the enzyme. DISCUSSION The results presented here show that the activity of CKII is rapidly enhanced in the cytosolic fraction of cells treated with insulin. The mechanism of the activation remains to be determined, but it does not appear to require protein synthesis, since activation of the kinase was not blocked by cycloheximide. Treatment of cells with insulin did not cause an increase in the cytosolic concentration ofckii, nor did it influence the relative amounts of the a and /3 subunits of the kinase (Fig. 6). The latter observation is relevant to the hypothesis that the X3 subunit of CKII may have a regulatory function and that insulin could cause changes in the ratio of the subunits. It is unlikely that insulin mediator molecules (2-22) or other soluble effectors contribute to the activation of CKII by insulin because the enhanced activity was retained after extracts were subjected to gel filtration chromatography by Sephadex G-25 (Fig. 7) and because both CKII activity and the magnitude of the activation of CKII by insulin were independent of the amount of extract assayed (Fig. 1). Although these findings do not rule out the potential involvement of a very tightly bound activator of the kinase, a more probable explanation is that the enzyme is activated as a result of covalent modification, possibly by phosphorylation. Proc. Natl. Acad. Sci. USA 84 (1987) x 6- E :,, 4- <i 2- Y v I I Fraction FIG. 7. Gel filtration chromatography of extracts of control- and insulin-treated cells. Extracts of control (e) and insulin-treated (5 nm for 6 min) (o) 3T3-L1 cells, which contained equal concentrations of protein, were thawed and.5-ml aliquots were filtered on a 2-ml column of Sephadex G-25 equilibrated with extraction buffer. Fractions (about.2 ml) were collected and assayed with a synthetic peptide substrate. The time course of activation of CKII by insulin differs, in some respects, from the time courses of its effects on other enzymes. For example, the stimulation of glycogen synthase activity in Swiss mouse 3T3 cells by insulin requires 3 min to reach a maximum and then declines to basal activity by 9 min (18). Similarly, insulin-stimulated S6 kinase activity in 3T3-L1 cells requires 2-6 min to reach a maximum (2-4) and, as with glycogen synthase, the activity subsequently declines (3). Similar to the effect on CKII, activation of a microtubule-associated protein 2 (MAP-2) kinase activity by insulin reaches a maximum at 1 min, but, in contrast to the effect on CKII, and consistent with the effects on glycogen synthase and on S6 kinase, the activity rapidly declines to a basal level (23). A major difference between the effect of insulin on CKII activity and effects on other kinases is the fact that CKII activity remains elevated in the presence of insulin rather than rapidly declining after attaining a maximum. Because CKII is activated by very low concentrations of insulin, it is likely that the effect is mediated through insulin receptors and not through receptors for insulin-like growth factors. The estimated EC5 (.15 nm) is a physiological concentration that agrees with values for the effects of insulin on glycogen synthase and glucose transport in rat adipocytes (24) and is lower than concentrations of insulin that are required for stimulation of S6 kinase and MAP-2 kinase. Insulin EC5 values for these effects are, respectively,.5 nm (4) and 5. nm (23). Our results suggest that activation of CKII may be a general response to insulin or other growth factors. The effect of insulin is not restricted to 3T3-L1 cells. In two experiments with H4-IIE cells, the insulin effect was observed (e.g., Table 1), although the magnitude of activation (26%) was at the lower end of the range found in experiments with 3T3-L1 adipocytes. The reason for the smaller magnitude of activation is unclear, but it may result from differences between the cell types in numbers of insulin receptors or from the fact that the H4-IIE cells are transformed whereas the 3T3-L1 adipocytes are not. Epidermal growth factor, which is known to have insulin-like effects on carbohydrate (18) and lipid (19) metabolism, was also found to activate CKII. Thus, it is possible that this kinase is part of a mechanism of signal transduction common to insulin and other growth factors. The function that CKII activity may have in signal transduction is, of course, a matter of speculation. Activation of CKII could be one of several potential intermediate steps in transduction, or it may directly pass the signal to some target proteins. Consistent with the latter possibility is the fact that 12

5 8838 Biochemistry: Sommercorn et al. CKII phosphorylates a variety of key enzymes and proteins that contribute to the regulation of numerous cellular processes (12). Although CKII phosphorylates many regulated enzymes, the consequences of phosphorylation by this kinase to activity of the substrate protein are not always readily apparent. In some cases, phosphorylation by CKII directly alters activity of the substrate protein (25), but in others, phosphorylation by CKII appears to have a permissive, rather than a direct, effect on regulation of the activity of the substrate. This has been shown with type II regulatory subunit of camp-dependent protein kinase (26), glycogen synthase (27), and inhibitor 2 of protein phosphatase 1 (28). Phosphorylation of these proteins by CKII enhances their subsequent rate of phosphorylation by glycogen synthase kinase 3. Whereas phosphorylation of glycogen synthase and of inhibitor 2 by CKII does not alter activities of these proteins, phosphorylation by glycogen synthase kinase 3 inhibits glycogen synthase and reduces the effectiveness of inhibitor 2, which leads to increased phosphatase 1 activity. Thus, phosphorylation by CKII facilitates the regulation of these two proteins by the action of another kinase. Recent results also support a potential permissive effect of CKII phosphorylation on the regulation of acetyl-coa carboxylase (29). Phosphorylation ofcarboxylase by CKII enhances the rate of dephosphorylation of the site in the enzyme that is phosphorylated by camp-dependent protein kinase. The effect on dephosphorylation of carboxylase was observed with both phosphatases 1 and 2A. As with glycogen synthase and inhibitor 2, phosphorylation of carboxylase by CKII in vitro does not directly alter carboxylase activity (9). However, phosphorylation of the enzyme by camp-dependent protein kinase inhibits its activity and dephosphorylation of this site with protein phosphatases reverses the inhibition (3, 31). Thus, phosphorylation by CKII may facilitate activation of carboxylase by promoting dephosphorylation of an inhibitory site. It is possible that insulin-stimulated CKII activity contributes to the activation of acetyl-coa carboxylase by phosphorylating inhibitor 2, which facilitates the activation of phosphatase 1, and by phosphorylating carboxylase, thereby making it a better substrate for the activated phosphatase. In contrast, the permissive effect of CKII does not appear to explain the activation of glycogen synthase by insulin, because phosphorylation by glycogen synthase kinase 3 inhibits synthase activity. The influence of phosphorylation by CKII on the rate of dephosphorylation of site(s) 3 in glycogen synthase will have to be determined before conclusions can be made concerning the contribution of phosphorylation by CKII to activation of glycogen synthase by insulin. CKII is primarly a cytosolic enzyme, but it is also found in the nucleus (11). The function of the nuclear enzyme is not understood, but there is some evidence that it could contribute to the regulation of gene expression and of the cell cycle. CKII phosphorylates several nuclear proteins, including DNA topoisomerases 1 (32) and II (25) and RNA polymerases I and II (12), all of which contribute to the regulation of RNA synthesis. CKII also phosphorylates high mobility group protein 14, and the phosphate content of this site varies with the cell cycle (33). Furthermore, CKII has considerable sequence homology with a yeast cell division control protein, CDC28 (34). Although in the present study, cytosolic CKII activity was measured, no precautions were taken to ensure nuclear integrity, and thus some of the enhanced activity in response to insulin or epidermal growth factor could have derived from the nucleus. Because of the potential involvement of CKII in regulation of the cell cycle, it will be of considerable interest to determine if nuclear CKII activity is stimulated by insulin or by other growth factors. Proc. Natl. Acad. Sci. USA 84 (1987) We thank Dr. Michael Dahmus for providing antiserum to CKII and Dr. Patrick Chou, Ms. Maria Harrylock, and Mr. Henry Zebroski for preparing the peptide substrate for CKII. J.S. was supported by a postdoctoral fellowship from the American Diabetes Association, Washington Affiliate. 1. Denton, R. M. (1986) Adv. Cyclic Nucleotide Protein Phosphorylation Res. 2, Smith, C. J., Rubin, C. S. & Rosen,. M. (198) Proc. Natl. Acad. Sci. USA 77, Cobb, M. H. (1986) J. Biol. Chem. 261, Tabarini, D., Heinrich, J. & Rosen,. M. (1985) Proc. Natl. Acad. Sci. USA 82, Alexander, M. C., Palmer, J. L., Pointer, R. H., Kowaloff, E. M., Koumjian, L. L. & Avruch, J. (1982) J. Biol. Chem. 257, Pucci, D. L., Ramakrishna, S. & Benjamin, W. B. (1983) J. Biol. Chem. 258, Marchmont, R. J. & Houslay, M. D. (1981) Biochem. J. 195, Brownsey, R. W. & Denton, R. M. (1982) Biochem. J. 22, Witters, L. A., Tipper, J. P. & Bacon, G. W. (1983) J. Biol. Chem. 258, Blackshear, P. J., Nemenoff, R. A. & Avruch, J. (1983) Biochem. J. 214, Hathaway, G. M. & Traugh, J. A. (1982) Curr. Top. Cell. Regul. 211, Sommercorn, J. & Krebs, E. G. (1987) in Post-Translational Modifications ofproteins and Ageing, ed. Zappia, V. (Plenum, New York), in press. 13. Kuenzel, E. A. & Krebs, E. G. (1985) Proc. Natl. Acad. Sci. USA 82, Sommercorn, J. & Krebs, E. G. (1987) J. Biol. Chem. 262, Rubin, C. S., Hirsch, A., Fung, C. & Rosen,. M. (1978) J. Biol. Chem. 253, Steel, R. G. D. & Torrie, J. H. (198) Principles and Procedures of Statistics: A Biometrical Approach (McGraw-Hill, New York), 2nd Ed., pp Stumpo, D. J. & Blackshear, P. J. (1986) Proc. Natl. Acad. Sci. USA 83, Chan, C. P. & Krebs, E. G. (1985) Proc. Natl. Acad. Sci. USA 82, Haystead, T. A. J. & Hardie, D. G. (1986) Biochem. J. 234, Cheng, K., Thompson, M., Schwartz, C., Malchoff, C., Tamura, S., Craig, J., Locher, E. & Larner, J. (1985) in Molecular Basis ofinsulin Action, ed. Czech, M. P. (Plenum, New York), pp Jarett, L., Kiechle, F. L., Macaulay, S. L., Parker, J. C. & Kelly, K. L. (1985) in Molecular Basis of Insulin Action, ed. Czech, M. P. (Plenum, New York), pp Saltiel, A. R. & Cuatrecasas, P. (1986) Proc. Natl. Acad. Sci. USA 83, Ray, L. B. & Sturgill, T. W. (1987) Proc. Natl. Acad. Sci. USA 84, Lawrence, J. C., Guinovart, J. J. & Larner, J. (1977) J. Biol. Chem. 252, Ackerman, P., Glover, C. V. C. & Osheroff, N. (1985) Proc. Natl. Acad. Sci. USA 82, Hemmings, B. A., Aitken, A., Cohen, P., Raymond, M. & Hofmann, F. (1982) Eur. J. Biochem. 127, Picton, C., Woodgett, J., Hemmings, B. & Cohen, P. (1982) FEBS Lett. 15, 191-1%. 28. DePaoli-Roach, A. A. (1984) J. Biol. Chem. 259, Sommercorn, J., McNall, S. J., Fischer, E. H. & Krebs, E. G. (1987) Fed. Proc. Fed. Am. Soc. Exp. Biol. 46, 452 (abstr.). 3. Hardie, D. G. & Guy, P. S. (198) Eur. J. Biochem. 11, Tipper, J. P. & Witters, L. A. (1982) Biochim. Biophys. Acta 715, Durban, E., Goodenough, M., Mills, J. & Busch, H. (1985) EMBO J. 4, Walton, G. M. & Gill, G. N. (1983) J. Biol. Chem. 258, Takio, K., Kuenzel, E. A., Walsh, K. A. & Krebs, E. G. (1987) Proc. NatI. Acad. Sci. USA 84,