Identification and characterization of a novel protein histidine kinase in the islet β cell: evidence for its regulation by mastoparan, an activator of G-proteins and insulin secretion

Abstract

Listen

Translate article

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 β subunit of trimeric GTP-binding proteins [Kowluru et al., Biochem J 1996;313:97–107], and the α 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 β cells. Herein, we characterized a novel protein histidine kinase in pancreatic β 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 β (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 μM). 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 β cells, but not mastoparan-17, its inactive analog, stimulated histidine kinase activity and histidine phosphorylation of G β subunit and insulin secretion from isolated rat islets. These studies identify, for the first time, a protein kinase activity in the pancreatic β cell that does not act on traditional -Ser, -Tyr, or -Thr residues. They also establish a possible link between histidine kinase activity and G β phosphorylation in isolated β cells.