Akira Akatsuka

Phosphorylation of troponin and myosin light chain by cAMP-independent casein kinase-2 from rabbit skeletal muscle

Casein kinase-2 from rabbit skeletal muscle was found to phosphorylate, in addition to glycogen synthase, troponin from skeletal muscle, and myosin light chain from smooth muscle. Troponin T and the 20,000 Mr myosin light chain are phosphorylated by casein kinase-2 at much greater rates than glycogen synthase. The V values for the phosphorylation of troponin and myosin light chain are nearly an order of magnitude greater than that of glycogen synthase; however, the Km values for these two substrates are greater than that for glycogen synthase. The kinase activities with the various protein substrates are stimulated approximately three- and fivefold by 5 mM spermidine and 3 mM spermine, respectively. Heparin is a potent inhibitor of the kinase when casein, glycogen synthase, or myosin light chain is the substrate. However, with troponin as substrate the kinase is relatively insensitive to inhibition by heparin. The amount of heparin required for 50% inhibition with troponin as substrate is at least 10 times greater than with casein as substrate. The phosphorylation of troponin by casein kinase-2 results in the incorporation of phosphate into two major tryptic peptides, which are different from those phosphorylated by casein kinase-1. The site in myosin light chain phosphorylated by casein kinase-2 is different from that phosphorylated by myosin light chain kinase.

Phosphorylation of smooth muscle myosin light chain by five different kinases

Phosphorylation of the 20 kDa myosin light chain from smooth muscle by five different kinases was investigated. Three of the kinases (myosin light chain kinase, phosphorylase kinase, and cAMP‐dependent protein kinase) phosphorylate serine residues, the fourth (casein‐kinase‐2) mainly threonine, and the fifth (glycogen synthase (casein) kinase‐1) both serine and threonine. Isoelectric focusing analyses of 32P‐labelled chymotryptic peptides indicate that phosphorylase kinase and cAMP‐dependent protein kinase phosphorylate the same site as myosin light chain kinase. However, both casein kinase‐2 and glycogen synthase (casein) kinase‐1 phosphorylate different sites.

Cyclic nucleotide- and Ca2+-independent phosphorylation of tubulin and microtubule-associated protein-2 by glycogen synthase (casein) kinase-1.

MAP-2 and tubulin are both shown to be substrates for glycogen synthase (casein) kinase-1 (CK-1). Greater than 40 mol 32P is incorporated into MAP-2 by CK-1 compared to only 14 mol 32P observed when cyclic AMP-dependent protein kinase (A-kinase) is the catalyst. Peptide mapping shows that CK-1 and A-kinase recognize a few common sites; the majority of the sites phosphorylated on MAP-2 by CK-1 are quite distinct. Up to 4 mol 32P can be incorporated into the tubulin dimer by CK-1 compared to only 0.9 mol 32P by A-kinase. The preferred substrate for both kinases is beta-tubulin.

A multifunctional cyclic nucleotide- and Ca2+-independent protein kinase from rabbit skeletal muscle

Abstract A cyclic nucleotide- and Ca2+-independent protein kinase, initially identified as a glycogen synthase kinase (Itarte, E. and Huang, K.-P. (1979) J. Biol. Chem. 254 , 4052–4057), was also found to phosphorylate phosphorylase kinase and troponin from skeletal muscle as well as myosin light chain and myosin light chain kinase from both smooth and skeletal muscles. With the exception of myosin light chain from skeletal muscle, all the above-mentioned proteins are also substrates for the multifunctional cAMP-dependent protein kinase. The results suggest that this cyclic nucleotide- and Ca2+-independent protein kinase, like cAMP-dependent protein kinase, may have multiple cellular functions.

Comparison of the liver glycogen synthase from normal and streptozotocin-induced diabetic rats

Glycogen synthase in the liver extracts of short-term (3 days) streptozotocin-induced diabetic rats is poorly activated by the endogenous synthase phosphatase as well as phosphatase(s) from the liver extracts of normal animals. However, synthase in the liver extracts of diabetic rats is readily activated by the 35,000 Mr rabbit liver protein phosphatase (H. Brandt, F.L. Capulong, and E. Y. C. Lee (J. Biol. Chem. 250, 8038-8044 (1975)). The purified synthases from normal and diabetic animals respond differently after incubations with three different phosphatases. Both normal and diabetic synthase are activated by the 35,000 Mr protein phosphatase; however, the total activity of diabetic, but not the normal, synthase is significantly increased. Normal, but not the diabetic, synthase is activated by a synthase phosphatase from normal rats; this activation is accompanied by an increase in total synthase activity. Incubation of the diabetic synthase with calf intestinal alkaline phosphatase results in a reduction of the total synthase activity, whereas synthase activity of the normal is not significantly affected. The reduction in total activity of the diabetic synthase by treatment with alkaline phosphatase was prevented by prior dephosphorylation with 35,000 Mr rabbit liver protein phosphatase. In addition to their differences in responses to different phosphatases, the normal and diabetic synthases are also different in their molecular weights as determined by sucrose density gradient centrifugation (154,000 +/- 17,000 (n = 6) for the normal and 185,000 +/- 15,000 (n = 8) for the diabetic synthase) and their kinetic properties.

The regulatory role of Mg2+ on rabbit skeletal muscle phosphorylase kinase

Abstract The stimulation of phosphorylase kinase by Mg2+ was studied. Both the nonactivated and activated kinases are stimulated by Mg2+ at concentrations that are 100- to 200-fold greater than ATP. This stimulation is observed at both pH 6.8 and 8.2 and results in a 10-fold increase in the activity of the nonactivated kinase. Mg2+ stimulation is additive with that observed by calmodulin. Both the Ca2+-dependent and -independent activities of the kinase are stimulated by high [Mg2+]. Kinetically this stimulation can be explained by a decrease in the Km for both phosphorylase b and ATP or an increase in V. The pH 6.8 8.2 ratio (0.06) is unaffected by [Mg2+] between 5 and 20 m m , but increases when [Mg2+] is less than 5 m m or greater than 20 m m . The stimulation by high [Mg2+] is explained by a direct effect of this cation on the kinase molecule rather than on its protein substrate, phosphorylase. This activating effect of high [Mg2+] does not result in any permanent change in the kinase molecule and can be readily reversed by diluting [Mg2+] to a low value.

Phosphorylation and inactivation of rabbit skeletal muscle glycogen synthase: Distinction between kinase Fa-, phosphorylase kinase-, and glycogen synthase (casein) kinase-1-catalyzed reactions

Rabbit skeletal muscle glycogen synthase was phosphorylated by kinase Fa, phosphorylase kinase, and cAMP-independent synthase (casein) kinase-1 to determine the differences among these kinase-catalyzed reactions. The stoichiometry of phosphate incorporation, the extent of inactivation, and the sites of phosphorylation were compared. Synthase (casein) kinase-1 catalyzes the highest level of synthase phosphorylation (4 mol/subunit) and inactivation (reduction of the activity ratio to below 0.05). The sites, defined by characteristic tryptic peptides, phosphorylated by synthase (casein) kinase-1 are distinguishable from those by kinase Fa and phosphorylase kinase. In addition, synthase (casein) kinase-1, unlike kinase Fa, does not activate ATP X Mg2+-dependent protein phosphatase. These results demonstrate that synthase (casein) kinase-1 is a distinct glycogen synthase kinase.

Phosphorylation of rat liver glycogen synthase bound to the glycogen particle

Rat liver glycogen synthase bound to the glycogen particle was partially purified by repeated high-speed centrifugation. This synthase preparation was labeled with 32P by incubations with cAMP-dependent protein kinase and cAMP-independent synthase (casein) kinase-1 in the presence of [gamma-32P]ATP. The phosphorylated synthase was separated from other proteins in the glycogen pellet by immunoprecipitation with rabbit anti-rat liver glycogen synthase serum. Analysis of the immunoprecipitates by sodium dodecyl sulfate-gel electrophoresis showed that synthase subunits of Mr 85,000 and 80,000 were present in varying proportions. The 32P-labeled synthase in the immunoprecipitate was digested with trypsin, and the resulting peptides were analyzed by isoelectric focusing. Synthase bound to the glycogen particle was phosphorylated by cAMP-dependent protein kinase at more sites and by cAMP-independent synthase (casein) kinase-1 at less sites than when the homogeneous synthase was incubated with these kinases. Phosphorylation of synthase in the glycogen pellet by either cAMP-dependent protein kinase or cAMP-independent synthase (casein) kinase-1 did not cause a significant inactivation as has been observed when the synthase was incubated with these kinases. Inactivation of synthase in the glycogen pellet, however, can be achieved by the combination of both kinases. This inactivation appears to result from the phosphorylation of a new site by cAMP-independent synthase (casein) kinase-1 neighboring a site previously phosphorylated by cAMP-dependent protein kinase.