Casein kinase II is a widely distributed protein serine/threonine kinase. The holoenzyme appears to be a tetramer, containing two alpha or alpha' subunits (or one of each) and two beta subunits. Complementary DNA clones encoding the subunits of casein kinase II were isolated from a human T-cell lambda gt10 library using cDNA clones isolated from Drosophila melanogaster [Saxena et al. (1987) Mol. Cell. Biol. 7, 3409-3417]. One of the human cDNA clones (hT4.1) was 2.2 kb long, including a coding region of 1176 bp preceded by 156 bp (5' untranslated region) and followed by 871 bp (3' untranslated region). The hT4.1 clone was nearly identical in size and sequence with a cDNA clone from HepG2 human hepatoma cultured cells [Meisner et al. (1989) Biochemistry 28, 4072-4076]. Another of the human T-cell cDNA clones (hT9.1) was 1.8 kb long, containing a coding region of 1053 bp preceded by 171 bp (5' untranslated region) and followed by 550 bp (3' untranslated region). Amino acid sequences deduced from these two cDNA clones were about 85% identical. Most of the difference between the two encoded polypeptides was in the carboxy-terminal region, but heterogeneity was distributed throughout the molecules. Partial amino acid sequence was determined in a mixture of alpha and alpha' subunits from bovine lung casein kinase II. The bovine sequences aligned with the 2 human cDNA-encoded polypeptides with only 2 discrepancies out of 535 amino acid positions. This confirmed that the two human T-cell cDNA clones encoded the alpha and alpha' subunits of casein kinase II. Microsequence data determined from separated preparations of bovine casein kinase II alpha subunit and alpha' subunit [Litchfield et al. (1990) J. Biol. Chem. 265, 7638-7644] confirmed that hT4.1 encoded the alpha subunit and hT9.1 encoded the alpha' subunit. These studies show that there are two distinct catalytic subunits for casein kinase II (alpha and alpha') and that the sequence of these subunits is largely conserved between the bovine and the human.

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...

Prostaglandins E1 and E2 increased the sensitivity of glycolysis to insulin in the isolated stripped soleus muscle of the rat, but prostaglandin F2 alpha had no effect. Indomethacin, which inhibits prostaglandin formation, markedly decreased the sensitivity of glycolysis to insulin. These findings suggest that prostaglandins of the E series increase the sensitivity of muscle glycolysis to insulin in vivo.

The deposition of i.m. fat, or marbling, in cattle is recognized as a desirable carcass trait in North American beef grading schemes. In order to investigate the relationship between degree of marbling and fatty acid composition of whole bovine muscle, we extracted the total lipid from pars costalis diaphragmatis (PCD) (n = 23) and longissimus (n = 36) muscles from Wagyu crossbred cattle that were assigned Canadian Grading Agency marbling scores ranging from 1 to 8 on an inverse 10-point scale (i.e., a score of 1 indicated "very abundant" marbling and a score of 10 would be assigned to a carcass "devoid" of marbling). Fatty acid methyl esters (FAME) of the total lipid and triacylglycerol fractions were resolved and quantified through GLC. Marbling scores were negatively associated with total lipid from both PCD (r = -.57, P < .01) and longissimus (r = -.80, P < .001). Differences between PCD and longissimus were found for almost all FAME studied from both lipid fractions, but no differences (P > .05) were seen when the monounsaturated:saturated fatty acid (MUFA/SFA) ratios were compared. Heifers had higher (P < .05) oleic acid content and lower (P < .05) palmitic acid content in lipid extracted from both muscles, resulting in higher (P < .05) MUFA/SFA ratios than those for steers. The relative amount of myristic acid increased as the lipid content (total lipid and triacylglycerol) increased in either longissimus (r values from .48 to .55; n = 36; P < .01) or PCD muscles (r from .67 to .76; n = 23; P < .001). The relative amount of linoleic acid (cis-9, cis-12 isomer) from total lipid was negatively associated with all chemical measurements of lipid from the longissimus (r from -.52 to -.64; n = 36; P < .001) and PCD muscles (r from -.75 to -.85; n = 23; P < .001). This association was not significant (P > .1) for either muscle when linoleic acid from the triacylglycerol fraction was examined, suggesting the negative association between this fatty acid and lipid content was due to a dilution of membrane phospholipids with increasing triacylglycerol. Indices of fatty acid elongase activity, calculated from FAME data, implicated the balance between this enzyme activity and fatty acid synthase as a source of variation between animals displaying various degrees of marbling and worthy of further investigation to better understand the process of marbling fat deposition in beef cattle.