Differential phosphorylation of RhoGDI mediates the distinct cycling of Cdc42 and Rac1 to regulate second-phase insulin secretion

J Biol Chem. 2010 Feb 26;285(9):6186-97. doi: 10.1074/jbc.M109.072421. Epub 2009 Dec 22.


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Glucose / pharmacology
  • Guanine Nucleotide Dissociation Inhibitors / metabolism*
  • Guanine Nucleotide Dissociation Inhibitors / physiology
  • Humans
  • Insulin / metabolism*
  • Insulin Secretion
  • Insulin-Secreting Cells / metabolism
  • Mass Spectrometry / methods
  • Mice
  • Mutation, Missense
  • Phosphorylation / physiology
  • Protein Binding / drug effects
  • cdc42 GTP-Binding Protein / metabolism*
  • rac1 GTP-Binding Protein / metabolism*
  • rho Guanine Nucleotide Dissociation Inhibitor alpha
  • rho-Specific Guanine Nucleotide Dissociation Inhibitors


  • ARHGDIA protein, human
  • Guanine Nucleotide Dissociation Inhibitors
  • Insulin
  • rho Guanine Nucleotide Dissociation Inhibitor alpha
  • rho-Specific Guanine Nucleotide Dissociation Inhibitors
  • cdc42 GTP-Binding Protein
  • rac1 GTP-Binding Protein
  • Glucose