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

Materials

Antibodies recognizing RhoGDI, phospho-RhoGDIα (Ser-101), Cdc42, Rac1, caveolin 1, ERK, phospho-ERK (Thr-202/Tyr-204), actin, rabbit and mouse IgG, and protein G Plus-agarose were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). PY20 antibody was obtained from BD Biosciences. The 4G10 mouse anti-phosphotyrosine monoclonal was purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Rabbit and mouse anti-EGFP antibodies were from Abcam Inc. (Cambridge, CA) and Clontech, respectively. Mouse anti-VAMP2 was purchased from Synaptic Systems (Gottingen, Germany). Goat anti-rabbit and anti-mouse horseradish peroxidase secondary antibodies were acquired from Bio-Rad. The EZ-Detect Cdc42 activation kit containing mouse anti-Cdc42 antibody and mammalian c-Myc tag IP/co-IP application set were purchased from Pierce. Lipofectamine 2000 and ECL reagents were purchased from Invitrogen and Amersham Biosciences, respectively. The sodium orthovanadate and hydrogen peroxide, RIA-grade bovine serum albumin, diazoxide, and d-glucose were obtained from Sigma. The human C-peptide and sensitive rat insulin RIA kits were purchased from Linco Research Inc. (St. Charles, MO). Three siRNA oligonucleotides targeting endogenous mouse RhoGDI (Ambion, Austin, TX) were used, designated as siA, siB, and siC, respectively; siA is GGAACUGGACAAGGACGAUtt; siB is GGUGUGGAGUACCGGAUAAtt; and siC is GGGUCAAUUGCCAAAACUCtt. A nontargeting siRNA (siCon) was obtained from Ambion. Purified recombinant His-Cdc42, His-Rac1, and RhoGDI-GST proteins for quantitative immunoblotting analysis were purchased from Cytoskeleton (Denver, CO).

Plasmids

The full-length human Cdc42 with N-terminal Myc tag was subcloned into the EcoRI and BamHI sites of pcDNA3. The full-length human RhoGDIα in pcDNA3.1 was a kind gift from Dr. Anjan Kowluru. pGEX4T3-RhoGDI-WT and -Y156E plasmids were kindly provided by Drs. G. M. Bokoch and C. DerMardirossian (Scripps Research Institute, La Jolla, CA) and used as templates to generate PCR products to subclone into BglII and HindIII sites of the pEGFP-C1 vector. The pEGFP-RhoGDI-S101A/S174A, pEGFP-RhoGDI-Y156F, and pEGFP-RhoGDI-Y156F/S101A/S174A mutants were made using the QuikChange® Multi Site-directed mutagenesis kit from Stratagene (Cedar Creek, TX). Adenoviral delivery plasmids were constructed by insertion of the annealed complementary oligonucleotide sequence of siA for RhoGDI or of siCon into the 5′-XhoI and 3′-SpeI sites of the pSilencer-Adeno-CMV vector (Ambion) using methods described previously (14). High purity cesium-chloride viral particles were obtained from Viraquest Inc. (North Liberty, IA), and viruses were packaged with GFP for use in identification of transduced islet cells in experiments. All constructs were verified by DNA sequencing analysis.

Tandem Affinity Purification Screen

Human Cdc42-WT cDNA was subcloned into the pNTAP shuttle vector for recombination as part of the InterPlay adenoviral tandem affinity purification system kit (Stratagene). MIN6 cells at 60% confluence were transduced with either NTAP-Cdc42WT-Ad or the control vector (TAP-CAT-Ad) CsCl-purified particles for 2 h at 37 °C (multiplicity of infection = 100) as described previously (14). Cells were then washed twice with phosphate-buffered saline and incubated 48 h longer in MIN6 culture medium at 37 °C, 5% CO₂. GFP fluorescence was visualized in greater than 95% of cells in all experiments. The TAP-CAT control and our TAP-Cdc42WT bait proteins were subsequently co-purified for identification of associated proteins using the Interplay tandem affinity purification kit (Stratagene) via LTQ linear ion-trap liquid chromatography-tandem mass spectrometry protein identification (INCAPS, Indianapolis, IN).

RNA Isolation and RT-PCR

Total RNA from MIN6 cells and isolated mouse pancreatic islets was obtained using the RNeasy mini kit (Qiagen, Valencia, CA). RNA (1 μg) was reverse-transcribed with TaqMan (Applied Biosystems, Foster City, CA), and 1% of the product was used for RT-PCR. The primers used for detection of RhoGDI isoforms (α, β, and γ) were as follows: RhoGDIα, forward 5′-cagaacaggaacccactgct and reverse 5′-cagtgctgcataccaaggtc; RhoGDIβ, forward 5′-acctggacagcaagctcaat and reverse 5′-gagatcgccagtaaggtcca; RhoGDIγ, forward 5′-gatgaggtgctggacgaaat and reverse 5′-catgatgataggccctggag. RT-PCR was performed with BioMix Red (Bioline, Taunton, MA) for 30 cycles: 94 °C for 1 min, 56 °C for 1 min, and 71 °C for 1 min with a final 10-min elongation at 71 °C. PCR products were visualized on 2% agarose gels.

Subcellular Fractionation

Subcellular fractions were isolated as described previously (25). Briefly, MIN6 cells were disrupted by 10 strokes through a 27-gauge needle prior to centrifugation at 900 × g for 10 min. Post-nuclear supernatants were centrifuged at 5500 × g for 15 min; supernatant was centrifuged at 25,000 × g for 20 min to pellet the storage granule fraction. The supernatant was further centrifuged at 100,000 × g for 1 h to obtain the cytosolic fraction. Plasma membrane fractions were obtained by mixing the postnuclear pellet with Buffers A and B for centrifugation at 113,000 × g for 1 h to obtain an interface, which was collected for centrifugation at 3000 × g for 10 min to pellet the plasma membrane. All pellets were resuspended in the 1% Nonidet P-40 lysis buffer.

Cell Culture, Transient Transfection, and Secretion Assays

MIN6 beta cells were cultured in Dulbecco's modified Eagle's medium (25 mm glucose) supplemented with 15% fetal bovine serum, 100 units/ml penicillin, 100 μg/ml streptomycin, 292 μg/ml l-glutamine, and 50 μm β-mercaptoethanol as described previously (26). Transfection of siRNA oligonucleotides into MIN6 cells was achieved as described previously (14). After 48 h of incubation, cells were washed and incubated in glucose-free modified Krebs-Ringer bicarbonate buffer (MKRBB: 5 mm KCl, 120 mm NaCl, 15 mm Hepes, pH 7.4, 24 mm NaHCO₃, 1 mm MgCl₂, 2 mm CaCl₂, and 1 mg/ml bovine serum albumin) for 2 h prior to stimulation with 20 mm d-glucose. Cells were harvested in 1% Nonidet P-40 lysis buffer (1% Nonidet P-40, 10% glycerol, 50 μm sodium fluoride, 10 mm sodium pyrophosphate, 1 mm sodium vanadate, 137 mm sodium chloride, 1 mm phenylmethylsulfonyl fluoride, 1 μg/ml pepstatin, 10 μg/ml aprotinin, 5 μg/ml leupeptin) and cleared by centrifugation at 14,000 × g for 10 min at 4 °C. For the secretion rescue assay, MIN6 cells at 50–60% confluence were co-transfected with 60 pmol of siCon or mouse siA (RhoGDI) oligonucleotides plus 0.5 μg of human proinsulin DNA (pCB6/INS, gift from Dr. Chris Newgard, Duke University, Durham, NC) and 0.5 μg of pcDNA3.1- human RhoGDI plasmid (pcDNA3.1 vector as negative control) using Lipofectamine 2000. After 48 h of incubation, cells were washed twice with and incubated for 2 h in freshly prepared MKRBB, followed by stimulation with 20 mm d-glucose for 1 h. MKRBB was then collected and centrifuged at 14,000 × g for 5 min for subsequent quantitation of human C-peptide secretion using a human C-peptide radioimmunoassay kit (Linco Research). MIN6 cells at 50–60% confluence were co-transfected with 1 μg of pEGFP-C1 vector (control), WT, or triple mutant RhoGDI plus 1 μg of human proinsulin cDNA using Transfectin. After 48 h of incubation, cells were washed and incubated in freshly prepared MKRBB for 2 h. Cells were then either stimulated with 40 mm KCl alone or incubated with 250 μm diazoxide for 5 min, followed by addition of 40 mm KCl and then stimulated with 20 mm d-glucose. MKRBB was then collected for subsequent human C-peptide quantitation as above.

Cdc42 Activation Assays

The EZ-Detect Cdc42 activation kit from Pierce was used as described previously (25). Freshly made cleared detergent cell lysate protein (500 μg) was combined with 20 μg of GST-Pak1-PDB-agarose. Following washes, proteins eluted were subjected to 12% SDS-PAGE for immunoblotting with mouse anti-Cdc42 antibody.

Mouse Islet Isolation, Adenoviral Transduction, and Perifusion

Pancreatic mouse islets were isolated, adenovirally transduced, and used for perifusion as described previously (14, 27). Briefly, pancreata from 10- to 14-week-old male mice (C57Bl/6J) were digested with collagenase and purified using a Ficoll density gradient. After isolation, islets were immediately transduced at an multiplicity of infection = 100 with either siRhoGDI-Ad or siCon-Ad CsCl-purified particles for 1 h at 37 °C, washed, and incubated overnight in RPMI 1640 medium at 37 °C, 5% CO₂. GFP fluorescence was visualized in greater than 95% of islets in all experiments. 50 GFP-positive (GFP⁺) islets were handpicked onto a column between two layers of Cytodex 3 beads (Amersham Biosciences), washed twice with Dulbecco's phosphate-buffered saline (magnesium-free), and preincubated for 30 min at 37 °C in KRBH Buffer (10 mm Hepes, pH 7.4, 134 mm NaCl, 5 mm NaHCO₃, 4.8 mm KCl, 1 mm CaCl₂, 1.2 mm MgSO₄, 1.2 mm KH₂PO₄ containing 0.5 mg/ml bovine serum albumin) containing 2.8 mm glucose. Islets were perifused at a flow rate of 0.3 ml/min for 10 min in KRBH buffer containing 2.8 mm glucose with eluted fractions captured at 1-min intervals, followed by 16.7 mm glucose stimulation for 35 min. Insulin secreted into eluted fractions was quantitated by a sensitive rat insulin RIA immunoassay kit (Linco Research, Inc.).

Co-immunoprecipitation and Immunoblotting

For each immunoprecipitation, 2 mg of cleared detergent lysate was combined with 2 μg of antibody for 2 h at 4 °C. Co-immunoprecipitation reactions from cytosolic fractions used 1 mg of protein with 2 μg of antibody. Protein G Plus-agarose beads were added and reactions rotated for an additional 2 h. Following three washes with 1% Nonidet P-40 lysis buffer, the resulting immunoprecipitates were subjected to 12% SDS-PAGE followed by transfer to PVDF membrane for immunoblotting and visualization by ECL using a Chemi-Doc system (Bio-Rad). QuantityOne software (Bio-Rad) was used for quantitative analysis of immunoblots.

Quantitation of RhoGDI, Cdc42, and Rac1 Proteins in MIN6 Cell Cytosolic Fraction

Cytosolic fractions of MIN6 cells were subjected to electrophoresis alongside known quantities of GST-RhoGDI, His-Cdc42, and His-Rac1 recombinant proteins on 12% SDS-PAGE followed by transfer to PVDF membranes and immunoblotting with anti-RhoGDI, Cdc42, or Rac1 antibodies, respectively. Proteins were detected using enhanced chemiluminescence, using exposure well within the linear range of the blot, and quantitated using the Bio-Rad Quantity One software package.

Statistical Analysis

All data are expressed as means ± S.E. Data were evaluated for statistical significance using Student's t test.

RhoGDI Associates with the Cytosolic Pool of Cdc42 in a Glucose-sensitive Manner

To identify Cdc42 cycling proteins functional in insulin-secreting beta cells, a tandem affinity purification screen using a dual-tagged form of Cdc42 was performed in clonal mouse MIN6 beta cells. MIN6 cells were transduced with control adenovirus expressing chloramphenicol acetyltransferase (TAP-CAT) or TAP-Cdc42. The TAP-Cdc42 protein was immunodetected at ∼30 kDa, although endogenous Cdc42 was seen at the expected 22-kDa size (Fig. 1A, lanes 1 and 2). An additional ∼26-kDa band was present only in crude lysates and is presumed to be a degradation product of TAP-Cdc42; further purification via the dual streptavidin-binding peptide and calmodulin-binding peptide tags resulted in elimination of this product and detection of the single 30-kDa band (Fig. 1A, lane 3). This purified fraction was then subjected to mass spectrometry, resulting in detection of 46 peptides of the guanine dissociation inhibitor protein RhoGDI. RhoGDI was confirmed as a specific endogenous binding partner of Cdc42 in MIN6 beta cells by co-immunoprecipitation (Fig. 1B). Both Cdc42 and RhoGDI proteins were detected in human islets, mouse islets, and MIN6 cells, validating use of the mouse islet and MIN6 cell model systems for subsequent studies (Fig. 1C). Of the three RhoGDI isoforms evaluated by RT-PCR analysis, RhoGDIα was predominant in both MIN6 cells and isolated mouse islets (Fig. 1D). Subcellular fractionation of MIN6 cells localized RhoGDI principally to the cytosol, similar to its localization in rat beta cells (24). Cdc42 localized to each of the three fractions, with ∼80% of total present in the cytosolic fraction. Caveolin 1 localized only to plasma membrane and storage granule fractions (Fig. 1E), consistent with its role as the GDI for the granule-localized pool of Cdc42 (22). The presence of VAMP2 in the plasma membrane and storage granule fractions and exclusion from cytosol validated the fraction integrities. Cdc42-RhoGDI complexes could be co-immunoprecipitated from the cytosolic fraction (Fig. 1F), implicating RhoGDI as the GDI for the cytosolic pool of Cdc42.

We next determined whether the RhoGDI-Cdc42 interaction was sensitive to glucose stimulation, as would be characteristic for a GDI-GTPase interaction in beta cells. Cdc42 activation in MIN6 cells has been shown previously to reach peak activation within 3 min of glucose stimulation (14, 18). As shown in Fig. 2A, anti-Cdc42 co-immunoprecipitated 45% less RhoGDI from cells stimulated with glucose for 3 min than from unstimulated cleared detergent MIN6 lysates (to 0.55 ± 0.09 of basal level of RhoGDI/Cdc42 ratio, p < 0.05). Reciprocal immunoprecipitation using anti-RhoGDI showed a similar response (Fig. 2B; 0.41 ± 0.06 of basal level of Cdc42/RhoGDI ratio, p < 0.05). This decrease was fully recapitulated using only the cytosolic fraction for co-immunoprecipitation (data not shown). These data suggested a role for RhoGDI as the glucose-sensitive cytosolic Cdc42 guanine nucleotide dissociation inhibitor.

RhoGDI Depletion Potentiates Glucose-induced Cdc42 Activation

Guanine nucleotide dissociation inhibitor proteins bind to inactive GTPases. To determine whether RhoGDI was required to maintain inactivation of cytosolic Cdc42, RNAi-mediated depletion of endogenous RhoGDIα was employed. Three different siRNA oligonucleotides (siA, siB, and siC) were capable of reducing endogenous RhoGDI levels, with siA exerting the most efficient protein knockdown (Fig. 3A); siA was thereafter utilized in all subsequent assays. In Cdc42 activation assays, RhoGDI depletion was without effect relative to that from nontargeted siCon-transfected cell lysates under basal, unstimulated conditions (Fig. 3B; 1.2 ± 0.1 versus 1.0, respectively). Conversely, activated Cdc42 levels in glucose-stimulated (3 min) RhoGDI-depleted cells were significantly elevated (Fig. 3B; 2.5 ± 0.2 versus 1.9 ± 0.1-fold increase of siCon-depleted cells, p < 0.05, n = 3). This is in contrast to caveolin-1 depletion, which induced a 2 ± 0.2-fold increase in activated Cdc42 under basal conditions compared with control (p < 0.05) but failed to exert a change with glucose stimulation (2 ± 0.3-fold, n = 3). These data indicated that RhoGDI functioned as a guanine nucleotide inhibitor of Cdc42 activation in MIN6 cells, in a manner distinct from that served by the Cdc42 granule-localized GDI protein, caveolin-1. Rac1 activation in either control- or RhoGDI-depleted cells was unaffected at either of these time points (data not shown).

RhoGDI Depletion Selectively Enhances Second-phase Insulin Release

To determine the requirement for RhoGDI in biphasic insulin secretion, islet perifusion was employed. RNAi-mediated RhoGDI depletion was used rather than islets isolated from RhoGDIα(−/−) knock-out mice because these mice are reported to exhibit severe metabolic abnormalities and infertility (28). As such, freshly isolated C57Bl/6J mouse islets were transduced with either RhoGDI siRNA (siRhoGDI-Ad) or control siRNA (siCon-Ad) and 40 h later were perifused, and insulin secretion was monitored. GFP expression from islets transduced with siRhoGDI was visualized within the majority of islet cells with high penetrance throughout the islet sphere (Fig. 4A). Knockdown efficiency and selectivity for RhoGDI in islets by adenoviral siRhoGDI was validated by immunoblotting (Fig. 4B). For analysis of biphasic insulin release, GFP-expressing islets were handpicked onto columns and perifused for 10 min in KRBH buffer containing 2.8 mm glucose (basal), followed by stimulation for 35 min at 16.7 mm glucose; islets were returned to basal for an additional 15 min to validate reversibility of response (Fig. 4C). Insulin release kinetics of siCon-Ad-transduced islets were similar to those of wild-type mice as reported previously (29). Similarly, siRhoGDI-Ad transduced islets exhibited a peak within 5 min of glucose stimulation, consistent with the occurrence of first-phase insulin secretion. However, islets transduced with siRhoGDI-Ad showed a significant augmentation of second-phase secretion compared with siCon-Ad islets, as quantified by the area under the curve between 18 and 45 min (area under the curve = 45 ± 7 versus siCon-Ad at 25 ± 3, p < 0.05). These data indicated RhoGDI selectively regulates the second phase of glucose-stimulated insulin secretion.

To verify that the enhancement of second-phase insulin release resulted from specific reduction in the RhoGDI protein, we replenished RhoGDI-depleted MIN6 cells with the human form of RhoGDI, because the human sequence is not targeted by this siRNA. A human C-peptide reporter assay was utilized to monitor secretion from transfectable MIN6 cells, wherein the human proinsulin plasmid was co-transfected with siRNA oligonucleotides and pcDNA3.1-RhoGDI. Human proinsulin is packaged and processed to human C-peptide and insulin in secretory granules in a manner similar to that of the mouse proinsulin present in the MIN6 cells, but the human C-peptide is immunologically distinct from that of the mouse C-peptide. Addition of human RhoGDI protein restored the stimulation index to that of siCon-transfected cells (Fig. 4D). Expression of the human RhoGDI protein was validated by immunoblotting, and its resistance to the siRNA was confirmed (data not shown). Thus, these data revealed that RhoGDI was required as a negative regulator of the second phase of insulin secretion, the same phase known to be regulated by its binding partner Cdc42.

Differential Phosphorylation of RhoGDI Promotes Its Dissociation from Either Cdc42 or Rac1

Kowluru and Veluthakal (24) have previously shown RhoGDI to interact with another small GTPase, Rac1, in rat beta cells. Rac1 activation is dependent upon Cdc42 and peaks 12–17 min after that of Cdc42 (14). Given this, RhoGDI depletion could have impacted activation of Cdc42 or Rac1, or both, to elicit the amplification of second-phase insulin release. Because GDI proteins bind to GTPases in a 1:1 stoichiometry (20), we tested the notion that in the beta cell cytosol, RhoGDI-Cdc42 and RhoGDI-Rac1 complexes exist and are differentially responsive to glucose. Anti-RhoGDI antibody co-immunoprecipitated both Cdc42 and Rac1 from unstimulated MIN6 cell lysates (Fig. 5A). Within 3 min of glucose stimulation, Cdc42 co-immunoprecipitation was reduced by ∼50% but recovered fully after 17 min more (Fig. 5, A and B). In contrast, Rac1 remained bound to RhoGDI at 3 min of glucose stimulation but dissociated by nearly 40% after 20 min of glucose stimulation (Fig. 5, A and C). These data suggested that glucose differentially impacted RhoGDI interactions with Cdc42 versus Rac1.

To address whether RhoGDI expressed in cytosol is sufficient to function as GDI for both Cdc42 and Rac1, two approaches were utilized. First, sequential IP from MIN6 cytosol was conducted. As shown in Fig. 5D, after three sequential immunoprecipitation reactions, co-immunoprecipitation of Cdc42 and Rac1 was undetectable, although RhoGDI could still be immunoprecipitated, suggesting that the amount of RhoGDI was not limiting. Second, quantitative immunoblotting was employed to evaluate the stoichiometric ratio of these proteins in the cytosolic fraction of MIN6 cells. RhoGDI was found to be present at 0.39 μmol/liter, whereas Cdc42 and Rac1 were present at 0.15 and 0.11 μmol/liter, respectively. These quantitative results are consistent with a previous report stating that GDI:GTPase associations occur at a 1:1 stoichiometric ratio in multiple cell types (20).

In other cell types, the GDI-GTPase complex dissociation can occur with stimulus-induced phosphorylation of the GDI protein (20, 30). Given that glucose stimulation of MIN6 beta cells triggers tyrosine phosphorylation of the exocytotic machinery required for second-phase insulin release (31, 32), we examined whether RhoGDI underwent tyrosine phosphorylation in MIN6 cells and whether this would impact its interaction with Cdc42 or Rac1. Fresh pervanadate (pV) treatment for 5 min was used to globally inhibit protein-tyrosine phosphatases and trap proteins in their tyrosine-phosphorylated forms. Phosphorylation of ERK was used to validate pV-inducible phosphorylation (Fig. 6A). As shown in Fig. 6B, anti-RhoGDI antibody co-precipitated significantly more phosphotyrosine-reactive (anti-4G10) RhoGDI (1.6 ± 0.1-fold increase in 4G10/total RhoGDI, versus control, p < 0.05). Reciprocal co-immunoprecipitation using anti-4G10 antibody demonstrated similar results (data not shown). The pV treatment induced a doublet recognized by both the anti-RhoGDI as well as the 4G10 antibodies, consistent with significant induction of phosphorylation. This doublet induced by pV treatment was also recognized by a second phosphotyrosine-specific antibody, PY20 (data not shown). The Cdc42-RhoGDI interaction was completely disrupted by treatment of MIN6 cells with pV. By contrast, pV failed to completely deter Rac1 binding to RhoGDI (69 ± 8% remained compared with untreated control, n = 3).

RhoGDI contains nine tyrosine residues, with Tyr-156 shown to be a major target for modification by Src kinase in other cell types (30). To determine whether phosphorylation of this particular residue might participate in disassociation of RhoGDI-Cdc42 complexes, exogenous EFGP-tagged RhoGDI-WT or a nonphosphorylatable Y156F mutant construct was expressed in MIN6 cells, and association with endogenous Cdc42 in response to glucose stimulus was evaluated. Anti-EGFP antibody effectively co-immunoprecipitated Cdc42 by both EGFP-RhoGDI-WT and Y156F mutant, with the Y156F coprecipitating slightly but not significantly less Cdc42 relative to WT under basal conditions (Fig. 7B). In the presence of glucose for 3 min, the EGFP-RhoGDI-WT precipitated ∼40% less Cdc42 compared with the basal condition, consistent with that of endogenous RhoGDI-Cdc42 complex disruption in response to acute glucose stimulation. By contrast, EGFP-RhoGDI-Y156F-Cdc42 complexes failed to respond to glucose stimulation. Distinct from Cdc42, Rac1 bound to both EGFP-RhoGDI-WT and the -Y156F mutant similarly in the absence or presence of glucose stimulation for 3 min (Fig. 7A). However, Rac1 dissociation from EGFP-RhoGDI-Y156F was blocked following a 20-min glucose stimulation (Rac1/RhoGDI-Y156F ratio = 1.1 ± 0.04, compared with control = 1.0, n = 3), consistent with the effect of pV. These results suggested that RhoGDI tyrosine phosphorylation was involved in interactions with both Cdc42 and Rac1.

Because the Y156F mutation in RhoGDI altered its interaction with Rac1 with only longer glucose stimulation periods, we examined the level of RhoGDI tyrosine phosphorylation at longer time points. Indeed, the level of RhoGDI tyrosine phosphorylation had peaked but did remain elevated for the 20-min time period assessed (Fig. 8, A and B). Unlike pV, glucose stimulation did not induce a visible doublet.

RhoGDI has also been demonstrated to undergo phosphorylation on serine residues 101 and 174 in non-beta cell types (33). To determine whether in beta cells this occurred in response to glucose stimulation, a phosphospecific S101-RhoGDI antibody was used to immunoblot co-immunoprecipitated RhoGDI. Indeed, pS101-RhoGDI detection was increased by 1.6-fold after 10 min of glucose stimulation (Fig. 8, A and C), albeit not within 3 min. The incidence of this RhoGDI serine phosphorylation event preceded both the dissociation of Rac1 from RhoGDI and the subsequent Rac1 activation, but it fell well after the peak of Cdc42 activation (3 min). This suggested serine phosphorylation as a possible mechanism for the dissociation of RhoGDI-Rac1 complexes in beta cells.

Similar to the approach used to delineate Tyr-156 as the site of action in RhoGDI-Cdc42 dissociation, an EGFP-RhoGDI-S101A/S174A double mutant was compared for its ability to co-immunoprecipitate endogenous Rac1 with that of EGFP-RhoGDI-WT. In the absence of stimulus, the double mutant and WT forms of RhoGDI bound equivalent levels of Rac1 (Fig. 9, A and B). However, although glucose induced the expected 50% loss of Rac1 association with EGFP-RhoGDI-WT, Rac1 failed to dissociate from the EGFP-RhoGDI-S101A/S174A mutant. Cdc42 binding to RhoGDI was unaffected by either mutation of the serine residues or glucose stimulation at this 20-min time point, and it did not alter the complex dynamic upon acute glucose stimulation for just 3 min (ratio of 0.7 ± 0.1 of basal WT for EGFP-RhoGDI-WT compared with 0.7 ± 0.2 for EGFP-RhoGDI-S101A/S174A, n = 3). These data indicated that phosphorylation of serine residues 101 and/or 174 of RhoGDI is required for its dissociation from Rac1 but not from Cdc42.

RhoGDI-Rac1 Disassociation Is Selectively Involved in Glucose-induced Amplification

Another method used to assess the second phase of glucose stimulation is by the diazoxide paradigm. This pharmacological paradigm is based upon the premise that the first-phase release is due to ATP-sensitive potassium channel (K_ATP)-dependent triggering, although the second phase/amplifying phase of insulin secretion is mainly regulated by K_ATP channel-independent effects (although a K_ATP channel-dependent signal is still required) (34–38). By subjecting beta cells to depolarizing KCl concentrations in the presence of the K_ATP channel opener diazoxide, low glucose simulates triggering, while high glucose simulates amplification. We used this paradigm as a second approach to ascertain the incidence of RhoGDI/Rac1 and RhoGDI/Cdc42 dissociation events within the amplification phase of glucose-stimulated insulin release. MIN6 cells stimulated with glucose for 20 min in the presence of diazoxide plus KCl secreted ∼2-fold more insulin compared with non-glucose-stimulated cells (Fig. 10A), which recapitulated the glucose-amplification effect seen otherwise in primary islets, indicating their utility as the model system for our protein-protein interaction studies, and consistent with other reports of the MIN6 line as possessing characteristics of glucose-induced amplification (39–41). Under this paradigm, glucose stimulation for 20 min decreased the amount of Rac1 that co-immunoprecipitated with RhoGDI by 40 ± 7% (Fig. 10B). Although the MIN6 cells maintained the expected triggering response to KCl in terms of insulin secretion (Fig. 10C), the ratio of Rac1/RhoGDI remained unchanged (Fig. 10D). These data indicated that RhoGDI-Rac1 dissociation occurs in response to a glucose-induced amplification signal but not the triggering signal.

KCl stimulation alone failed to separate Cdc42 from RhoGDI, consistent with our previous observation that KCl failed to activate Cdc42 in MIN6 cells. However, we did not observe RhoGDI-Cdc42 dissociation after 3 min of glucose stimulation in this pharmacological paradigm (diazoxide plus KCl ± glucose, data not shown).

A triple RhoGDI phosphorylation mutant (EGFP-RhoGDI-Y156Y/S101A/S174A) was expressed in MIN6 cells and assessed for functional impact upon the amplifying phase of glucose-stimulated insulin secretion using the diazoxide paradigm. Cells were transiently transfected to co-express human C-peptide to serve as a reporter of insulin release. In cells with glucose-induced triggering phase blocked by diazoxide plus KCl, overexpression of the WT form of EGFP-RhoGDI significantly inhibited glucose-induced human C-peptide release, as expected given the known inhibitory action of RhoGDI (Fig. 11A). Remarkably, the triple RhoGDI phosphorylation mutant inhibited the human C-peptide release even further. Neither the EGFP-RhoGDI-WT nor the triple phosphorylation mutant altered KCl-induced human C-peptide release from MIN6 cells, indicating specificity of RhoGDI action and importance of its phosphorylation status to the amplifying phase of glucose-stimulated insulin secretion (Fig. 11B).