Stimulation by insulin of a serine kinase in human platelets that phosphorylates and activates the cGMP-inhibited cAMP phosphodiesterase.
We previously reported that insulin stimulation of human platelets induces serine phosphorylation and activation of the cGMP-inhibited cAMP phosphodiesterase (cGI-PDE). Here, we describe methods to detect and partially purify an insulin-stimulated cGI-PDE kinase (cGI-PDE ISK) from lysates of platelets incubated with insulin. Incubation of human platelets with 10(-8) M insulin increased cGI-PDE ISK activity two-fold. The DEAE-Sephacel-purified cGI-PDE ISK phosphorylated the cGI-PDE on serine in a time- and concentration-dependent manner resulting in an increased incorporation of about 0.2 mol of [32P]/mol of cGI-PDE and 15-20% increase in cGI-PDE activity. The phosphorylation of cGI-PDE was not affected by 10 microM PKI, 1 microgram/ml of heparin, 3 mM CaCl2 or 1 mM MnCl2. cGI-PDE ISK did not adsorb to antiphosphotyrosine antibodies. To maintain its activation it was necessary to add protein phosphatase inhibitors to the lysate-buffers. All of these findings are consistent with the conclusion that a serine/threonine phosphorylation of the cGI-PDE ISK is involved in its activation by insulin.
López-Aparicio
P
P
Department of Medical and Physiological Chemistry, University of Lund, Sweden.
Belfrage
P
P
Manganiello
V C
VC
Kono
T
T
Degerman
E
E
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000037:antagonists & inhibitors / Q000097:blood* / Q000302:isolation & purification; D001792:Blood Platelets / Q000201:enzymology*; D002122:Calcium Chloride / Q000494:pharmacology; D002712:Chlorides; D002852:Chromatography, Ion Exchange; D006152:Cyclic GMP / Q000494:pharmacology*; D004789:Enzyme Activation; D006493:Heparin / Q000494:pharmacology; D006801:Humans; D007328:Insulin / Q000494:pharmacology*; D007700:Kinetics; D008345:Manganese / Q000494:pharmacology; D017895:Manganese Compounds; D010766:Phosphorylation; D019000:Phosphotyrosine; D017346:Protein Serine-Threonine Kinases / Q000097:blood*; D014443:Tyrosine / Q000031:analogs & derivatives / Q000032:analysis
D002712:Chlorides; D007328:Insulin; D017895:Manganese Compounds; D019000:Phosphotyrosine; D014443:Tyrosine; D008345:Manganese; D006493:Heparin; D017346:Protein Serine-Threonine Kinases; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D006152:Cyclic GMP; D002122:Calcium Chloride; C025340:manganese chloride