Abstract
Phosphorylation of the insulin receptor beta-subunit on serine/threonine residues by protein kinase C reduces both receptor kinase activity and insulin action in cultured cells. Whether this mechanism regulates insulin action in intact animals was investigated in rats rendered insulin-resistant by 3 days of starvation. Insulin-stimulated autophosphorylation of the partially purified hepatic insulin receptor beta-subunit was decreased by 45% in starved animals compared to fed controls. This autophosphorylation defect was entirely reversed by removal of pre-existing phosphate from the receptor with alkaline phosphatase, suggesting that increased basal phosphorylation on serine/threonine residues may cause the decreased receptor tyrosine kinase activity. Tryptic removal of a C-terminal region of the receptor beta-subunit containing the Ser/Thr phosphorylation sites similarly normalized receptor autophosphorylation. To investigate which kinase(s) may be responsible for such increased Ser/Thr phosphorylation in vivo, protein kinase C and cAMP-dependent protein kinase A in liver were studied. A 2-fold increase in protein kinase C activity was found in both cytosol and membrane extracts from starved rats as compared to controls, while protein kinase A activity was diminished in the cytosol of starved rats. A parallel increase in protein kinase C was demonstrated by immunoblotting with a polyclonal antibody which recognizes several protein kinase C isoforms. These findings suggest that in starved, insulin-resistant animals, an increase in hepatic protein kinase C activity is associated with increased Ser/Thr phosphorylation which in turn decreases autophosphorylation and function of the insulin receptor kinase.
Highlights
Increased Protein Kinase C Activity Is Linked to Reduced Insulin Receptor Autophosphorylation in Liver of Starved Rats*
A parallel increase in protein kinase C was demonstrated by immunoblotting with a polyclonal antibody which recognizes several protein kinase C isoforms. These findings suggest that in starved, insulin-resistant animals, an increase in hepatic protein kinase C activity is associated with increased Ser/Thr phosphorylation which in turn decreases autophosphorylation and function of the insulin receptor kinase
Probing was done using polyclonal antibody against peptide 0442 of bovine brain protein kinase C [19], and visualization was with ““I-protein A and Regulation of transmembrane signalling by receptor phosphorylation is a mechanism common to several different receptor systems including the family of receptors which are tyrosine kinases
Summary
[Y-~*P]ATP, and Triton X-100 were obtained from Du Pont-New England Nuclear; porcine insulin was from Elanco; and wheat germ agglutinin-agarose was from Vector. 7.5, 2 mM EDTA, 2 mM EGTA, and 10 mM dithiothreitol were eluted in the same buffer containing 0.2 M NaCl. Kinase (Protein Kinase Cl-The activity of protein kinase C was determined by measurement of the enzymatic transfer of “P from [32P]ATP to histone (Hl) substrate. The standard assay consisted of the following components in a final volume of 100 ~1: 25 mM PIPES, pH 6.8, 10 mM magnesium acetate, 800 pg/ml histone Hl (type IIIs, Sigma), 25 mM 2-mercaptoethanol, 0.1 mM [y-“‘P]ATP (200-300 cpm/pmol), 20 mM NaF, 0.75 mM CaC&, 250 pg/ml phosphatidylserine, 10 pg/ml diolein, and a suitable amount of enzyme. Protein kinase C activity was determined by subtracting the basal 32P incorporation into histone obtained in the presence of 0.5 mM EGTA from 32P incorporation into histone obtained in the presence of CaCl, phosphatidylserine, and diolein.
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