Taming platelets with cyclic nucleotides11Abbreviations: ABP, actin binding protein; AC, adenylyl cyclase; cAMP, cyclic AMP; cAMP-PK, cAMP-dependent protein kinase; cGMP, cyclic GMP; cGMP-PK, cGMP-dependent protein kinase; DAG, 1,2-diacylglycerol; EDRF, endothelium-derived relaxing factor; GC, guanylyl cyclase; GP, glycoprotein; Hsp27, heat shock protein 27; IP3, inositol 1,4,5- trisphosphate; IRAG, IP3 receptor-associated cGMP-PK substrate; MAPK, mitogen-activated protein kinase; MAPKAPK-2, MAPK-activated protein kinase-2; MARCKS, myristoylated alanine-rich C kinase substrate; MLC, myosin light chain; MLCK, myosin light chain kinase; PDE, phosphodiesterase; PG-E1, prostaglandin E1; PG-I2, prostacyclin; PIP2, phosphatidylinositol 4,5-bisphosphate; PKC, protein kinase C; PLC, phospholipase C; TxA2, thromboxane A2, and VASP, vasodilator-stimulated phosphoprotein.

In Xenopus oocytes, initiation of maturation is dependent on reduction of cyclic AMP-dependent protein kinase (PKA) activity and the synthesis of the mos proto-oncogene product. Mos is required during meiosis I for the activation of both maturation-promoting factor (MPF) and mitogen-activated protein kinase (MAPK). Here we show that injection of the catalytic subunit of PKA (PKAc) prevented progesterone-induced synthesis of endogenous Mos as well as downstream MPF and MAPK activation. However, PKAc did not prevent injected soluble Mos product from activating MAPK. While MAPK is activated during Mos-PKAc coinjection, attendant MPF activation is blocked. Additionally, PKAc caused a potent block in the electrophoretic mobility shift of cdc25 that is associated with phosphatase activation. This inhibition of cdc25 activity was not reversed by progesterone, Mos, or MPF. We conclude that PKAc acts as a negative regulator at several points in meiotic maturation by preventing both Mos translation and MPF activation.

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Myosin light chain phosphorylation by Ca stimulated-phospholipid dependent protein kinase

The Japanese Journal of Pharmacology ◽

10.1016/s0021-5198(19)54256-1 ◽

1982 ◽

Vol 32 ◽

pp. 280

Author(s):

Michiko Naka ◽

Hiroyoshi Hidaka

Keyword(s):

Protein Kinase ◽

Light Chain ◽

Myosin Light Chain ◽

Myosin Light Chain Phosphorylation ◽

Dependent Protein Kinase ◽

Dependent Protein

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Calmodulin-dependent myosin light chain kinases from cardiac and smooth muscle: a comparative study

Canadian Journal of Biochemistry ◽

10.1139/o80-040 ◽

1980 ◽

Vol 58 (4) ◽

pp. 299-308 ◽

Cited By ~ 24

Author(s):

Michael P. Walsh ◽

Jean-Claude Cavadore ◽

Bernard Vallet ◽

Jacques G. Demaille

Keyword(s):

Smooth Muscle ◽

Protein Kinase ◽

Light Chain ◽

Myosin Light Chain ◽

Phosphorylation Site ◽

Dependent Protein Kinase ◽

Proteolytic Fragment ◽

Cardiac Enzyme ◽

Camp Dependent Protein Kinase ◽

Dependent Protein

Various properties of cardiac and smooth muscle calmodulin-dependent myosin light chain kinases (MLCKs) have been compared. The enzymes exhibit the same isoelectric point (6.5) but differ markedly in molecular weight (Mr = 72 000 for both canine and bovine cardiac MLCK, and Mr = 130 000 for smooth muscle MLCK). Comparison of the tryptic peptide maps of bovine cardiac and turkey gizzard MLCKs indicates that the cardiac enzyme is a fragment of a protein homologous to the smooth muscle kinase. While the smooth muscle kinase can be phosphorylated by the catalytic subunit of cAMP-dependent protein kinase, the same is not true for either bovine or canine cardiac MLCK. Controlled tryptic hydrolysis of phosphorylated smooth muscle MLCK, followed by affinity chromatography on a column of calmodulin–Sepharose, enables separation of a phosphopeptide (Mr = 22 000) from a mixture of peptides of Mr = 50 000 and 24 000 which are bound to the column in the presence of Ca2+ and eluted with ethylene glycol bis(β-aminoethyl ether)-N,N′-tetraacetic acid. The phosphorylation site, therefore, is distinct from the calmodulin-binding site. It appears that cardiac MLCK is proteolyzed during the isolation procedure. The purified cardiac enzyme represents a proteolytic fragment which retains Ca2+ and calmodulin dependence but only a fraction of the specific activity of the native enzyme, and has lost the site of phosphorylation by cAMP-dependent protein kinase. A protease is shown to exist in myocardium which is capable of digesting smooth muscle MLCK rapidly at low temperature, and which is resistant to classical antiproteases.

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