Protein phosphorylation in the regulation of insulin secretion: the use of site-directed inhibitory peptides in electrically permeabilised islets of Langerhans.
We have used electrically permeabilised rat islets of Langerhans to investigate the role of protein phosphorylation in the regulation of insulin secretion using pseudosubstrate inhibitory peptides for cyclic AMP-dependent protein kinase (PKA) and for protein kinase C (PKC). The protein kinase inhibitor (PKI) peptide, PKI(6-22), completely inhibited the effects of cyclic AMP on islet PKA activity in vitro, on endogenous protein phosphorylation and on insulin secretion. This peptide had no significant effect on islet PKC activity in vitro, on Ca(2+)-induced protein phosphorylation and on secretory responses to Ca2+ or to the PKC activator, 4 beta-phorbol myristate acetate (PMA). The PKC pseudosubstrate inhibitory peptide, PKC(19-36), caused a marked inhibition of islet PKC activity in vitro and inhibite PMA-induced insulin secretion without affecting secretory responses to cyclic AMP and Ca2+. These results demonstrate that PKA- and PKC-induced protein phosphorylation is obligatory for cyclic AMP- and PMA-stimulated insulin secretion, respectively, and suggest that there is little "crosstalk" between the response elements of the secretory pathways to the different second messengers, at least after the generation of the messengers within the beta-cells.
Basudev
H
H
Biomedical Sciences Division, King's College London, Kensington, UK.
Jones
P M
PM
Howell
S L
SL
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001345:Autoradiography; D002118:Calcium / Q000494:pharmacology; D002352:Carrier Proteins / Q000494:pharmacology*; D002463:Cell Membrane Permeability; D000242:Cyclic AMP / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D018274:Electroporation; D006706:Homeostasis; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D047908:Intracellular Signaling Peptides and Proteins; D007515:Islets of Langerhans / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D007700:Kinetics; D010446:Peptide Fragments / Q000494:pharmacology*; D010750:Phosphoproteins / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000494:pharmacology; D051381:Rats; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D002352:Carrier Proteins; D007328:Insulin; D047908:Intracellular Signaling Peptides and Proteins; D010446:Peptide Fragments; D010750:Phosphoproteins; C072093:protein kinase C (19-36); C094636:protein kinase inhibitor (6-22); D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium