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

Zhanxiang Wang, Debbie C. Thurmond

Research output: Contribution to journalArticle

35 Citations (Scopus)

Abstract

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 ofRhoGDIresidue 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.

Original languageEnglish
Pages (from-to)6186-6197
Number of pages12
JournalJournal of Biological Chemistry
Volume285
Issue number9
DOIs
StatePublished - Feb 26 2010

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Phosphorylation
Chemical activation
Insulin
Glucose
RNA Interference
Islets of Langerhans
cdc42 GTP-Binding Protein
Mutation
Monomeric GTP-Binding Proteins
RNA
Exocytosis
Insulin-Secreting Cells
Guanosine Triphosphate
Immunoprecipitation
Serine
Tyrosine
Mass Spectrometry
Purification
Mass spectrometry
Proteins

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology
  • Medicine(all)

Cite this

Differential phosphorylation of RhoGDI mediates the distinct cycling of Cdc42 and Rac1 to regulate second-phase insulin secretion. / Wang, Zhanxiang; Thurmond, Debbie C.

In: Journal of Biological Chemistry, Vol. 285, No. 9, 26.02.2010, p. 6186-6197.

Research output: Contribution to journalArticle

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