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.

scholarly journals Inhibition by melittin of phospholipid-sensitive and calmodulin-sensitive Ca2+ -dependent protein kinases

1982 ◽  
Vol 202 (1) ◽  
pp. 217-224 ◽  
Author(s):  
N Katoh ◽  
R L Raynor ◽  
B C Wise ◽  
R C Schatzman ◽  
R S Turner ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinases ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Dependent Protein Kinase ◽  
Calmodulin Binding ◽  
Substrate Protein ◽  
Dependent Protein ◽  
A Site

Effects of melittin, an amphipathic polypeptide, on various species of protein kinases were investigated. It was found that melittin inhibited the newly identified phospholipid-sensitive Ca2+-dependent protein kinase (from heart, brain, spleen and neutrophils) and the cardiac myosin light-chain kinase, a calmodulin-sensitive Ca2+-dependent enzyme. In contrast, melittin had little or no effect on either the holoenzymes of the cardiac cyclic AMP-dependent and cyclic GMP-dependent protein kinases or the catalytic subunit of the former. Kinetic analysis indicated that melittin inhibited phospholipid-sensitive Ca2+-dependent protein kinase non-competitively with respect to ATP (Ki = 1.3 microM); although exhibiting complex kinetics, its inhibition of the enzyme was overcome by phosphatidylserine (a phospholipid cofactor), but not by protein substrate (histone H1) or Ca2+. On the other hand, melittin inhibited myosin light-chain kinase non-competitively with respect to ATP (Ki = 1.4 microM) or Ca2+ (Ki = 1.9 microM), and competitively with respect to calmodulin (Ki = 0.08 microM); although exhibiting complex kinetics, its inhibition of the enzyme was reversed by myosin light chains (substrate protein). The present findings indicate the presence of functionally important hydrophobic or hydrophilic loci on the Ca2+-dependent protein kinases, but not on the cyclic nucleotide-dependent class of protein kinase, with which melittin can interact. Moreover, the kinetic data suggest that melittin inhibited myosin light-chain kinase by interacting with a site on the enzyme the same as, or proximal to, the calmodulin-binding site, thus interfering with the formation of active enzyme-calmodulin-Ca2+ complex.

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.

scholarly journals Naphthalenesulphonamides block neutrophil superoxide production by intact cells and in a cell-free system: is myosin light chain kinase responsible for these effects?

1995 ◽  
Vol 311 (1) ◽  
pp. 81-87 ◽  
Author(s):  
P G Heyworth ◽  
R W Erickson ◽  
J Ding ◽  
J T Curnutte ◽  
J A Badwey
Keyword(s):  
Protein Kinase ◽  
Protein Kinases ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Intact Cells ◽  
Free System ◽  
Cell Free System ◽  
A Cell

Selective antagonists of myosin light chain kinase (MLCK) [e.g. ML-7; 1-(5-iodonaphthalene-1-sulphonyl)-1H-hexahydro-1,4-diazepine hydrochloride] were found to inhibit superoxide (O2-) release from stimulated neutrophils. The concentrations of ML-7 that were inhibitory were substantially lower than those reported for a selective antagonist of protein kinase C [i.e. H-7; 1-(5-isoquinolinesulphonyl)-2-methylpiperazine dihydrochloride]. ML-7 also reduced the phosphorylation of the 47 kDa subunit of the NADPH-oxidase system (p47-phox) and blocked translocation of this protein to the Triton X-100-insoluble fraction in stimulated cells. Interestingly, ML-7 also inhibited O2- production in a cell-free system derived from neutrophils at concentrations similar to those that were effective in vivo. This cell-free system does not require ATP and is insensitive to all other inhibitors of protein kinases tested, including some highly effective against MLCK (i.e. staurosporine). Thus, the data suggest that ML-7 does not block O2- release by inhibiting a protein kinase but instead may interact directly with a subunit of the oxidase. The binding site for ML-7 may provide a valuable target for inhibiting the inflammatory properties of phagocytic leucocytes by naphthalenesulphonamides designed to lack activity against protein kinases.

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