H7-resistant protein kinase C substrates in two-dimensional gels of proestrous rat anterior pituitary gland.
The presence of Ca(2+)-dependent and -independent protein kinase C (PKC) activities which were stimulated by phorbol-12,13-dibutyrate (PDBu) and sensitive to the kinase inhibitor staurosporine (IC50 values approx. 100 nM) was demonstrated in proestrous rat anterior pituitary gland. These PDBu-induced activities were completely abolished by the PKC-specific inhibitors Ro31-8220 and GF109203X (3 microM). The Ca(2+)-independent activity was more resistant (IC50 = 61 microM) to the kinase inhibitor H7 than the Ca(2+)-dependent activity (IC50 approx. 20 microM), however, this (unusual) resistance to H7 was not observed in the brain regions, hypothalamus and hippocampus. Possible substrates for the Ca(2+)-independent PKC in anterior pituitary were identified by two-dimensional gel electrophoresis and autoradiography following incubation in vitro with [32P]phosphate and 300 nM PDBu +/- 300 nM staurosporine or 30 microM H7. The phosphorylation of six proteins (16, 16, 25, 36, 65 and 69 kDa) was found to be stimulated by PDBu and inhibited by staurosporine, but not H7, in whole tissue, and another two such phosphorylated proteins (each 76 kDa) were observed in microsomal subcellular fractions. These phosphoproteins may be substrates for an H7-resistant PKC isoform previously shown to mediate a number of cellular responses in rat anterior pituitary gland.
Simpson
J
J
Department of Pharmacology, University of Edinburgh, UK.
Johnson
M S
MS
Mitchell
R
R
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D000470:Alkaloids / Q000494:pharmacology; D000818:Animals; D015180:Electrophoresis, Gel, Two-Dimensional; D004971:Estrus; D005260:Female; D007546:Isoquinolines / Q000494:pharmacology*; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D010879:Piperazines / Q000494:pharmacology*; D010903:Pituitary Gland, Anterior / Q000201:enzymology*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D019311:Staurosporine; D013379:Substrate Specificity
D000470:Alkaloids; D007546:Isoquinolines; D010879:Piperazines; D015240:Phorbol 12,13-Dibutyrate; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D011493:Protein Kinase C; D019311:Staurosporine