A novel regulatory mechanism for trimeric GTP-binding proteins in the membrane and secretory granule fractions of human and rodent β cells

Abstract

Recently we described roles for heterotrimeric and low-molecular-mass GTP-binding proteins in insulin release from normal rat islets. During these studies, we observed that a protein with an apparent molecular mass (37 kDa) similar to that of the beta subunit of trimeric GTP-binding proteins underwent phosphorylation in each of five classes of insulin-secreting cells. Incubation of the beta cell total membrane fraction or the isolated secretory granule fraction (but not the cytosolic fraction) with [gamma-32P]ATP or [gamma-32P]GTP resulted in the phosphorylation of this protein, which was selectively immunoprecipitated by an anti-serum directed against the common beta subunit of trimeric G-proteins. Disruption of the alpha beta gamma trimer (by pretreatment with either fluoroaluminate or guanosine 5'(-)[gamma-thio]triphosphate) prevented beta subunit phosphorylation. Based on differential sensitivities to pH, heat and the histidine-selective reagent diethyl pyrocarbonate (and reversal of the latter by hydroxylamine), the phosphorylated amino acid was presumptively identified as histidine. Incubation of pure beta subunit alone or in combination with the exogenous purified alpha subunit of transducin did not result in the phosphorylation of the beta subunit, but addition of the islet cell membrane fraction did support this event, suggesting that membrane localization (or a membrane-associated factor) is required for beta subunit phosphorylation. Incubation of phosphorylated beta subunit with G alpha.GDP accelerated the dephosphorylation of the beta subunit, accompanied by the formation of G alpha-GTP. Immunoblotting detected multiple alpha subunits (of Gi, G(o) and Gq) and at least one beta subunit in the secretory granule fraction of normal rat islets and insulinoma cells. These data describe a potential alternative mechanism for the activation of GTP-binding proteins in beta cells which contrasts with the classical receptor-agonist mechanism: G beta undergoes transient phosphorylation at a histidine residue by a GTP-specific protein kinase; this phosphate, in turn, may be transferred via a classical Ping-Pong mechanism to G alpha.GDP (inactive), yielding the active configuration G alpha.GTP in secretory granules (a strategic location to modulate exocytosis).

The Stimulus-induced Tyrosine Phosphorylation of Munc18c Facilitates Vesicle Exocytosis

“…Subcellular Fractionation-Subcellular fractions of beta cells were isolated as described previously (48,49). Briefly, MIN6 beta cells at 80 -90% confluence were harvested in 1 ml of homogenization buffer (20 mM Tris-HCl (pH 7.4), 0.5 mM EDTA, 0.5 mM EGTA, 250 mM sucrose, 1 mM dithiothreitol, and 1 mM sodium orthovanadate containing the protease inhibitors leupeptin (10 g/ml), aprotinin (4 g/ml), pepstatin (2 g/ml), and phenylmethylsulfonyl fluoride (100 M)).…”

“…Although Munc18c is a soluble protein, it localizes to the plasma membrane through its interaction with Syntaxin 4 (16). To investigate whether this particular pool of Munc18c can be tyrosine-phosphorylated in a stimulus-dependent manner, MIN6 cells were subfractionated according to previously described methods (48), and plasma membrane fractions were used in immunoprecipitation experiments. Munc18c immunoprecipitated from plasma membrane fractions prepared from unstimulated cells showed some phosphotyrosine immunoreactivity (Fig.…”

YES, a Src Family Kinase, Is a Proximal Glucose-specific Activator of Cell Division Cycle Control Protein 42 (Cdc42) in Pancreatic Islet β Cells

“…Subcellular Fractionation-All fractionation steps were completed at 4°C using an established protocol (32). In brief, after glucose stimulation MIN6 cells were washed twice with cold phosphate-buffered saline (PBS) and harvested with 500 l of homogenization buffer (20 mM Tris-HCl (pH 7.4), 0.5 mM EDTA, 0.5 mM EGTA, 250 mM sucrose, 1 mM DTT, 100 M PMSF, 10 g/ml leupeptin, 4 g/ml aprotinin, and 2 g/ml pepstatin).…”

Interaction of nucleoside diphosphate kinase B with heterotrimeric G protein βγ dimers: consequences on G protein activation and stability

“…In contrast to the NDPK, the phosphohistidine intermediate is not formed by incubation of the protein with the required NTP but required a histidine kinase activity which phosphorylates Gβ within the Gβγ dimer. Intermediately occurring histidine phosphorylation of Gβ has been detected in a variety of mammalian tissues (Nürnberg et al 1996), including human platelets (Hohenegger et al 1996) and rat pancreatic β cells (Kowluru et al 1996). The identity of the proposed membrane-bound histidine kinase to catalyse this reaction remained, however, elusive (Nürnberg et al 1996, Kowluru 2002.…”

“…The identity of the proposed membrane-bound histidine kinase to catalyse this reaction remained, however, elusive (Nürnberg et al 1996, Kowluru 2002. Also, in this phosphotransfer reaction, the phosphorylation of GDP bound to Gα was a matter of debate (Wieland et al 1993;Kaldenberg-Stasch et al 1994;Hohenegger et al 1996;Kowluru et al 1996;Niroomand et al 1997). The same experimental constraints, which cannot exclude the spontaneous release of the Gα-bound GDP and re-binding of formed GTP, apply here, and some experimental data are clearly arguing against a direct transfer on Gα-bound GDP (Hohenegger et al 1996).…”