Differential phosphorylation of RhoGDI mediates the distinct cycling of Cdc42 and Rac1 to regulate second-phase insulin secretion.
Cdc42 cycling through GTP/GDP states is critical for its function in the second/granule mobilization phase of insulin granule exocytosis in pancreatic islet beta cells, although the identities of the Cdc42 cycling proteins involved remain incomplete. Using a tandem affinity purification-based mass spectrometry screen for Cdc42 cycling factors in beta cells, RhoGDI was identified. RNA interference-mediated depletion of RhoGDI from isolated islets selectively amplified the second phase of insulin release, consistent with the role of RhoGDI as a Cdc42 cycling factor. Replenishment of RhoGDI to RNA interference-depleted cells normalized secretion, confirming the action of RhoGDI to be that of a negative regulator of Cdc42 activation. Given that RhoGDI also regulates Rac1 activation in beta cells, and that Rac1 activation occurs in a Cdc42-dependent manner, the question as to how the beta cell utilized RhoGDI for differential Cdc42 and Rac1 cycling was explored. Co-immunoprecipitation was used to determine that RhoGDI-Cdc42 complexes dissociated upon stimulation of beta cells with glucose for 3 min, correlating with the timing of glucose-induced Cdc42 activation and the onset of RhoGDI tyrosine phosphorylation. Glucose-induced disruption of RhoGDI-Rac1 complexes occurred subsequent to this, coincident with Rac1 activation, which followed the onset of RhoGDI serine phosphorylation. RhoGDI-Cdc42 complex dissociation was blocked by mutation of RhoGDI residue Tyr-156, whereas RhoGDI-Rac1 dissociation was blocked by RhoGDI mutations Y156F and S101A/S174A. Finally, expression of a triple Y156F/S101A/S174A-RhoGDI mutant specifically inhibited only the second/granule mobilization phase of glucose-stimulated insulin secretion, overall supporting the integration of RhoGDI into the activation cycling mechanism of glucose-responsive small GTPases.
Wang
Zhanxiang
Z
Herman B Wells Center for Pediatric Research, Basic Diabetes Research Group, Department of Pediatrics, Indiana University School of Medicine, Indianapolis, Indiana 46202, USA.
Thurmond
Debbie C
DC
D000818:Animals; D005947:Glucose / Q000494:pharmacology; D020730:Guanine Nucleotide Dissociation Inhibitors / Q000378:metabolism* / Q000502:physiology; D006801:Humans; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D050417:Insulin-Secreting Cells / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods; D051379:Mice; D020125:Mutation, Missense; D010766:Phosphorylation / Q000502:physiology; D011485:Protein Binding / Q000187:drug effects; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism*; D020830:rac1 GTP-Binding Protein / Q000378:metabolism*; D062948:rho Guanine Nucleotide Dissociation Inhibitor alpha; D062947:rho-Specific Guanine Nucleotide Dissociation Inhibitors
C569638:ARHGDIA protein, human; D020730:Guanine Nucleotide Dissociation Inhibitors; D007328:Insulin; D062948:rho Guanine Nucleotide Dissociation Inhibitor alpha; D062947:rho-Specific Guanine Nucleotide Dissociation Inhibitors; D020764:cdc42 GTP-Binding Protein; D020830:rac1 GTP-Binding Protein; D005947:Glucose