Substrates for cyclic AMP-dependent protein kinase in islets of Langerhans. Studies with forskolin and catalytic subunit

To investigate substrates for cyclic AMP-dependent protein kinase in intact islets of Langerhans, batches of islets were incubated with 32PP(i) for 1 h in the presence of 10 mM-glucose; the adenylate cyclase activator forskolin, which in parallel experiments was shown to increase islet cyclic AMP content and insulin release, was then added. Islets were homogenized and subcellular fractions prepared by differential centrifugation. Phosphopeptides were electrophoresed on sodium dodecyl sulphate/polyacrylamide gels and quantified by autoradiography and densitometry. Within 5 min forskolin caused increased labelling of M(r)-25,000 and -30,000 cytosolic and M(r)-23,000 and -32,000 particulate peptides; a rapid decrease in phosphorylation of M(r)-18,000 and -34,000 cytosolic peptides was also observed. In addition, rather slower phosphorylation occurred of the M(r)-15,000 peptide previously identified as histone H3 Christie & Ashcroft (1984) Biochem. J. 218, 87-99. When similar subcellular fractions were incubated with γ-32PATP and purified catalytic subunit of cyclic AMP-dependent protein kinase, peptides phosphorylated included cytosolic species of M(r) 25,000 and 30,000 and particulate species of M(r) 23,000 and 32,000. The distribution of RNA in the subcellular fractions suggested that the M(r)-32,000 species could be a ribosomal protein. The 24,000g pellet was heterogeneous, as judged by marker assays, and was therefore fractionated further by Percoll-density-gradient centrifugation. The peak containing the M(r)-23,000 peptide was resolved from marker enzymes for plasma membranes, mitochondria and endoplasmic reticulum and coincided with a peak for insulin: hence the M(r)-23,000 peptide is likely to be a secretory-granule component. The study demonstrates that the potentiation of insulin release that occurs when islet cyclic AMP is increased is accompanied by rapid phosphorylation of specific islet substrates for cyclic AMP-dependent protein kinase. The data are consistent with the hypothesis that protein phosphorylation is involved in the regulation of insulin secretion