Differential Phosphorylation of RhoGDI Mediates the Distinct Cycling of Cdc42 and Rac1 to Regulate Second-phase Insulin Secretion

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 interferencedepleted 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.

Dynamin 2 regulates biphasic insulin secretion and plasma glucose homeostasis

“…Both a negative role as a physical fusion barrier and a positive role of promoting vesicle transport and exocytosis have been proposed (86,87). In insulin-secreting cells, a fairly selective role of actin remodeling in the second phase of GSIS has been reported (11,13,14,16,61). Similarly, we observed a selective decrease in the second phase of GSIS along with strong actin reorganization in Dnm2 KO β cells.…”

“…The pronounced impairment of the second phase of GSIS in Dnm2 KO mice is strikingly similar to the effect of perturbations in actin regulatory proteins, such as Cdc42 (13), PAK1 (15), RAC1 (14), and N-WASP (16). We therefore examined the actin organization in Dnm2 KO β cells.…”

“…The selective reduction of the second phase of GSIS in Dnm2 KO mice shows striking parallel with the reduced insulin secretion after perturbations of actin remodeling (13)(14)(15)81), a key factor that selectively contributes to the second phase (10,11). Accordingly, enhanced actin disassembly by PPARβ/δ (17) or the actin-capping protein gelsolin (81) increases the second phase.…”

“…Moreover, increasing evidence supports an emerging model in which the actin cytoskeleton plays a critical role in biphasic GSIS (10,11) through granule mobilization and recruitment from the reserve pool to the PM. Many actin remodeling molecules, such as the Rho GTPase family proteins Cdc42 (13) and RAC1 (14), PAK1 (15), N-WASP (16), and PPARβ/δ (17), selectively regulate the second phase of GSIS, and perturbations of microtubules (18) and kinesin-1 (19) disrupt the second phase.…”

Tyrosine 397 phosphorylation is critical for FAK-promoted Rac1 activation and invasive properties in oral squamous cell carcinoma cells

“…3 In skeletal muscle, Rac1 is activated by insulin, and knockout of Rac1 results in insulin resistance and reduced insulin-stimulated glucose uptake. 4,5 Given that muscle makes up 40% of the body's volume, muscle has an essential role in post-prandial lowering of blood glucose.…”