Stimulation by insulin of a serine kinase in human platelets that phosphorylates and activates the cGMP-inhibited cAMP phosphodiesterase

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Stimulation by insulin of a serine kinase in human platelets that phosphorylates and activates the cGMP-inhibited cAMP phosphodiesterase. / Lopez-Aparicio, Pilar; Belfrage, Per; Manganiello, Vincent C; Kono, Tetsuro; Degerman, Eva.

In: Biochemical and Biophysical Research Communications, Vol. 193, No. 3, 1993, p. 1137-1144.

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T1 - Stimulation by insulin of a serine kinase in human platelets that phosphorylates and activates the cGMP-inhibited cAMP phosphodiesterase

AU - Lopez-Aparicio, Pilar

AU - Belfrage, Per

AU - Manganiello, Vincent C

AU - Kono, Tetsuro

AU - Degerman, Eva

PY - 1993

Y1 - 1993

N2 - 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.

AB - 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.

U2 - 10.1006/bbrc.1993.1744

DO - 10.1006/bbrc.1993.1744

M3 - Article

VL - 193

SP - 1137

EP - 1144

JO - Biochemical and Biophysical Research Communications

JF - Biochemical and Biophysical Research Communications

SN - 1090-2104

IS - 3

ER -