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.
Kowluru
Anjaneyulu
A
Department of Pharmaceutical Sciences, College of Pharmacy and Allied Health Professions, Wayne State University, Detroit, MI 48201, USA. akowluru@wizard.pharm.wayne.edu
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