Identification and characterization of a novel protein histidine kinase in the islet beta cell: evidence for its regulation by mastoparan, an activator of G-proteins and insulin secretion.
Using insulin-secreting cells, we previously demonstrated that specific proteins associated with the cytosolic, secretory granule, and mitochondrial fractions undergo a novel type of phosphorylation on their histidine residues. Subsequently, we identified these proteins as the nucleoside diphosphate kinase (NDPK) [Kowluru and Metz, Biochemistry 1994;33:12495-503], the beta subunit of trimeric GTP-binding proteins [Kowluru et al., Biochem J 1996;313:97-107], and the alpha subunit of succinyl-CoA synthetase [Kowluru, Diabetologia 2001;44:89-94], respectively. Since several other enzymes of intermediary metabolism (e.g. ATP-citrate lyase and glucose-6-phosphatase) also undergo histidine phosphorylation, these initial findings may have a more generalized significance to beta cells. Herein, we characterized a novel protein histidine kinase in pancreatic beta cells, and determined it to be acid- and heat-labile as well as alkali-resistant in its phosphorylation of histone 4. Such an activity was detected in normal rat islets, human islets, and clonal beta (HIT-T15 and INS-1) cells, and could utilize either ATP or GTP as a phosphoryl donor (with K(m) values in the range of 60-100 microM). On a size-exclusion column, its molecular mass was estimated to be in the range of 60-70 kDa. It was stimulated by divalent cations (Mg(2+)>Mn(2+)>control=Ca(2+)=Zn(2+)=Co(2+)), but was resistant to polyamines. It was inactivated by known in vitro inhibitors of protein histidine phosphorylation (e.g. UDP or cromoglycate). Mastoparan, a global activator of G-proteins and insulin secretion from isolated beta cells, but not mastoparan-17, its inactive analog, stimulated histidine kinase activity and histidine phosphorylation of G(beta) subunit and insulin secretion from isolated rat islets. These studies identify, for the first time, a protein kinase activity in the pancreatic beta cell that does not act on traditional -Ser, -Tyr, or -Thr residues. They also establish a possible link between histidine kinase activity and G(beta) phosphorylation in isolated beta cells.
Department of Pharmaceutical Sciences, College of Pharmacy and Allied Health Professions, Wayne State University, Detroit, MI 48201, USA. akowluru@wizard.pharm.wayne.edu
Anjaneyulu
A
Kowluru
D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006639:Histidine / Q000378:metabolism; D000071677:Histidine Kinase; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D036341:Intercellular Signaling Peptides and Proteins; D007515:Islets of Langerhans / Q000187:drug effects* / Q000201:enzymology; D008970:Molecular Weight; D010455:Peptides; D010766:Phosphorylation; D011494:Protein Kinases / Q000302:isolation & purification* / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D013347:Subcellular Fractions; D013379:Substrate Specificity; D014862:Wasp Venoms / Q000494:pharmacology*
D004791:Enzyme Inhibitors; D007328:Insulin; D036341:Intercellular Signaling Peptides and Proteins; D010455:Peptides; D014862:Wasp Venoms; D006639:Histidine; C037500:mastoparan; D011494:Protein Kinases; D000071677:Histidine Kinase; D019204:GTP-Binding Proteins