scholarly journals Interaction of calmodulin with myosin light chain kinase and cAMP-dependent protein kinase in bovine brain.

1981 ◽  
Vol 256 (15) ◽  
pp. 8183-8189 ◽  
Author(s):  
D.R. Hathaway ◽  
R.S. Adelstein ◽  
C.B. Klee
Keyword(s):  
Protein Kinase ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Bovine Brain ◽  
Dependent Protein Kinase ◽  
Camp Dependent Protein Kinase ◽  
Dependent Protein

scholarly journals Regulation of actin microfilament integrity in living nonmuscle cells by the cAMP-dependent protein kinase and the myosin light chain kinase.

1988 ◽  
Vol 106 (6) ◽  
pp. 1955-1971 ◽  
Author(s):  
N J Lamb ◽  
A Fernandez ◽  
M A Conti ◽  
R Adelstein ◽  
D B Glass ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Complete Loss ◽  
Dependent Protein Kinase ◽  
Actin Microfilament ◽  
Camp Dependent Protein Kinase ◽  
Nonmuscle Cells ◽  
Dependent Protein

Microinjection of the catalytic subunit of cAMP-dependent protein kinase (A-kinase) into living fibroblasts or the treatment of these cells with agents that elevate the intracellular cAMP level caused marked alterations in cell morphology including a rounded phenotype and a complete loss of actin microfilament bundles. These effects were transient and fully reversible. Two-dimensional gel electrophoresis was used to analyze the changes in phosphoproteins from cells injected with A-kinase. These experiments showed that accompanying the disassembly of actin microfilaments, phosphorylation of myosin light chain kinase (MLCK) increased and concomitantly, the phosphorylation of myosin P-light chain decreased. Moreover, inhibiting MLCK activity via microinjection of affinity-purified antibodies specific to native MLCK caused a complete loss of microfilament bundle integrity and a decrease in myosin P-light chain phosphorylation, similar to that seen after injection of A-kinase. These data support the idea that A-kinase may regulate microfilament integrity through the phosphorylation and inhibition of MLCK activity in nonmuscle cells.

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

1980 ◽  
Vol 58 (4) ◽  
pp. 299-308 ◽  
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.

scholarly journals Polyamines inhibit phospholipid-sensitive and calmodulin-sensitive Ca2+-dependent protein kinases

1983 ◽  
Vol 213 (2) ◽  
pp. 281-288 ◽  
Author(s):  
D F Qi ◽  
R C Schatzman ◽  
G J Mazzei ◽  
R S Turner ◽  
R L Raynor ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinases ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Polymyxin B ◽  
Dependent Protein Kinase ◽  
Dependent Protein ◽  
Endogenous Proteins

Effects of polyamines on various protein kinases were investigated. It was found that both phospholipid-sensitive Ca2+-dependent protein kinase and myosin light-chain kinase (a calmodulin-sensitive species of Ca2+-dependent protein kinase) were inhibited to different degrees by polyamines, with an approximate order of inhibitory potency of spermine = 1, 12-diaminododecane greater than spermidine = 1, 10-diaminodecane much greater than cadaverine = putrescine. Kinetic analysis revealed that spermine inhibited the phospholipid-sensitive enzyme non-competitively with respect to Ca2+ (Ki = 0.84 mM) and phosphatidylserine (Ki = 0.90 mM); it also inhibited myosin light-chain kinase non-competitively with respect to Ca2+ (Ki = 1.82 mM) and calmodulin (Ki = 2.73 mM). 1, 12-Diaminododecane, in comparison, inhibited the phospholipid-sensitive enzyme competitively with respect to Ca2+ (Ki = 0.45 mM) and phosphatidylserine (Ki = 0.50 mM); it also inhibited myosin light-chain kinase competitively with respect to calmodulin (Ki = 0.63 mM) but non-competitively with respect to Ca2+ (Ki = 1.49 mM). Moreover, spermine (0.5 mM) was found to inhibit markedly phosphatidylserine/Ca2+- and calmodulin/Ca2+-stimulated phosphorylation of endogenous proteins in rat brain particulate fraction. All the polyamines tested were practically without effect on cyclic AMP-dependent and cyclic GMP-dependent protein kinases. Polyarginine, like spermine, was found to be a more selective inhibitor of Ca2+-dependent protein kinases, whereas polyglutamate preferentially inhibited the cyclic nucleotide-dependent enzymes. The present results indicated that, in addition to certain lipophilic compounds (such as trifluoperazine, palmitoylcarnitine, adriamycin and naphthalenesulphonamide) and polypeptides with hydrophobic regions (such as melittin and polymyxin B) previously reported, polycationic compounds (exemplified by polyamines) could also inhibit the two classes of Ca2+-dependent protein kinases requiring either phospholipid or calmodulin as a cofactor. Because of the high cellular concentration (up to 10 mM) and the differential effects of polyamines, it is suggested that spermine, and to smaller extents spermidine and putrescine, may be involved in the regulation of certain Ca2+-dependent protein-phosphorylation systems in vivo.

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