Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2011 Dec;301(6):E1072-80.
doi: 10.1152/ajpendo.00312.2011. Epub 2011 Aug 9.

Cool-1/βPIX functions as a guanine nucleotide exchange factor in the cycling of Cdc42 to regulate insulin secretion

Erica M Kepner  1 , Stephanie M Yoder, Eunjin Oh, Michael A Kalwat, Zhanxiang Wang, Lawrence A Quilliam, Debbie C Thurmond
Affiliations

Cool-1/βPIX functions as a guanine nucleotide exchange factor in the cycling of Cdc42 to regulate insulin secretion

Erica M Kepner et al. Am J Physiol Endocrinol Metab. 2011 Dec.

Abstract

Second-phase insulin release requires the sustained mobilization of insulin granules from internal storage pools to the cell surface for fusion with the plasma membrane. However, the detailed mechanisms underlying this process remain largely unknown. GTP-loading of the small GTPase Cdc42 is the first glucose-specific activation step in the process, although how glucose triggers Cdc42 activation is entirely unknown. In a directed candidate screen for guanine nucleotide exchange factors (GEFs), which directly activate small GTPases, Cool-1/βPix was identified in pancreatic islet beta cells. In support of its role as the beta cell Cdc42 GEF, βPix coimmunoprecipitated with Cdc42 in human islets and MIN6 beta cells in a glucose-dependent manner, peaking just prior to Cdc42 activation. Furthermore, RNAi-mediated βPix reduction by 50% corresponded to full ablation of glucose-induced Cdc42 activation and significant attenuation of basal and glucose-stimulated insulin secretion. Of the two Cdc42 guanine nucleotide dissociation inhibitor (GDI) proteins identified in beta cells, βPix competed selectively with caveolin-1 (Cav-1) but not RhoGDI in coimmunoprecipitation and GST-Cdc42-GDP interaction assays. However, a phospho-deficient Cav-1-Y14F mutant failed to compete with βPix; Cav-1(Tyr14) is an established phosphorylation site for Src kinase. Taken together, these data support a new model, wherein glucose stimulates Cav-1 and induces its dissociation from Cdc42, possibly via Src kinase activation to phosphorylate Cav-1(Tyr14), to promote Cdc42-βPix binding and Cdc42 activation, and to trigger downstream signaling and ultimately sustain insulin release.

PubMed Disclaimer

Figures

Fig. 1.
Fig. 1.
βPix isoforms A and C are expressed in MIN6 beta cells and pancreatic islets and colocalize with Cdc42 in MIN6 subcellular fractions. A: βPix protein was detectable by immunoblotting in MIN6 lysates, human islets, and mouse islets. Mouse brain lysates were used as a positive control for the many isoforms of βPix expressed. Anti-actin immunoblot (IB) detection was evaluated for protein loading. B: RT-PCR was used to detect isoforms of βPix expressed in MIN6 β-cells (A isoform primers used in lanes 1 and 3; B and C primer sets used together in lanes 2 and 4). Mouse brain, which contains all three isoforms, was used as a positive control for the primer sets, and GAPDH was used as a positive control for the integrity of both lysate sets. C: clarified MIN6 cell lysates were partitioned into subcellular fractions: PM, plasma membrane; SG, storage granule pool; Cyt, remaining soluble fraction. βPix content in each fraction was assessed by immunoblot (IB). Cdc42, Syntaxin 4 (Syn4) and VAMP2 were used to validate the integrity of the Cyt, PM, and SG fractions, respectively. Reassembly of noncontiguous lanes from within the same gel is demarcated by black lines. Data are representative of at least three independent experiments. D: mouse pancreas sections were costained for βPix and insulin as described in materials and methods; representative of three pancreatic sections.
Fig. 2.
Fig. 2.
βPix associates rapidly with Cdc42 in response to glucose stimulation in human islets and MIN6 beta cells. βPix was immunoprecipitated (IP) from cleared detergent lysates prepared from either MIN6 cells preincubated in MKRBB for 2 h and then acutely stimulated with 20 mM glucose for 1, 2, or 3 min (A), or human islets preincubated 2 h in KRBH buffer (B) and then acutely stimulated with 16.7 mM glucose for 2 min. Proteins were resolved by 12% SDS-PAGE and immunoblotted (IB) with anti-βPix and anti-Cdc42 antibodies. Input lysates validated equal Cdc42 expression under all conditions, and IgG immunoprecipitation validated the specificity of the Cdc42/βPix association. Data are shown as the average ± SE of three independent experiments, quantified for the ratio of Cdc42/βPix; *P < 0.05 vs. basal for MIN6 cells. Human islet blots are representative of two independent coimmunoprecipitation experiments using two human donor batches of islets. Reassembly of noncontiguous lanes from within the same gel is demarcated by black lines.
Fig. 3.
Fig. 3.
Knockdown of βPix in MIN6 beta cells attenuates both basal and glucose-stimulated insulin secretion. A: schematic of βPix isoform organization and targeting location of the siRNA oligonucleotides. B: two siRNA oligonucleotides sets, designated βPix1 and βPix2, or control (Con) siRNA were transfected into MIN6 cells and knockdown efficiency determined by anti-βPix immunoblotting (IB). GAPDH was used for normalization of loading. Data represent the average ± SE of five independent experiments; *P < 0.05 vs. siCon. C: glucose-stimulated insulin secretion was assessed using an hGH reporter-based assay. MIN6 cells were cotransfected with hGH and either siCon or siβPix2; 48 h later, cells were preincubated in glucose-free MKRBB for 2 h and then left unstimulated or stimulated with 20 mM glucose for 60 min. Secretion of hGH into the MKRBB was quantified by ELISA and normalized to hGH content in the corresponding cell lysates. Insulin levels were adjusted for corresponding total cell protein content and normalized to unstimulated siCon set equal to 1.0 in each assay, and data are presented as the average ± SE of three independent experiments; *P < 0.05 vs. unstimulated siCon; #P < 0.05 vs. glucose-stimulated siCon. D: protein expression of Cav-1 and RhoGDI in cells transfected with siRNA oligonucleotides targeted for βPix was validated from lysates in B.
Fig. 4.
Fig. 4.
Knockdown of βPix in MIN6 beta cells abolishes glucose-stimulated Cdc42 activation. Cells transfected with Control (Con) or βPix (βPix2) siRNA oligonucleotides were preincubated in glucose-free MKRBB for 2 h and then left unstimulated or acutely stimulated (3 min) with 20 mM glucose. Cdc42-GTP captured on GST-Pak1-PBD beads was resolved by 12% SDS-PAGE and detected by anti-Cdc42 immunoblotting (IB). Reassembly of noncontiguous lanes from within the same gel is demarcated by black lines. Activation levels were adjusted for GST loading and normalized to unstimulated siCon set equal to 1.0 in each assay, and data are presented as the average ± SE of three independent experiments; *P < 0.05 vs. unstimulated siCon.
Fig. 5.
Fig. 5.
Cav-1 Tyr14 is required for Cdc42-Cav-1 and βPix-Cdc42 complex formation. A: MIN6 cells were transiently transfected with RhoGDI-HA or Myc-tagged Cav-1 DNAs (WT or Cav-Y14F). Cells were preincubated in MKRBB for 2 h, glucose-stimulated for 2 min, and the resultant cleared detergent cell lysates were immunoblotted to validate expression of recombinant proteins, relative to endogenous Cdc42. Lysates were otherwise subjected to anti-βPix immunoprecipitation (IP) reactions (B) or GST-Cdc42-GDP interaction assays (C), as described in materials and methods. Precipitates were resolved on 12% SDS-PAGE, transferred to PVDF, and immunoblotted (IB) for βPix, Cdc42, Myc (Cav-1), and GST. Reassembly of noncontiguous lanes from within the same gel is demarcated by black lines. Data shown are representative of 3–5 independent experiments.
See this image and copyright information in PMC

Comment in

  • Second-phase insulin secretion gets cool.
    Tsushima RG. Tsushima RG. Am J Physiol Endocrinol Metab. 2011 Dec;301(6):E1070-1. doi: 10.1152/ajpendo.00491.2011. Epub 2011 Sep 20. Am J Physiol Endocrinol Metab. 2011. PMID: 21934039 No abstract available.

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Audebert S, Navarro C, Nourry C, Chasserot-Golaz S, Lecine P, Bellaiche Y, Dupont JL, Premont RT, Sempere C, Strub JM, Van Dorsselaer A, Vitale N, Borg JP. Mammalian Scribble forms a tight complex with the betaPIX exchange factor. Curr Biol 14: 987–995, 2004 - PubMed
    1. Barber MA, Donald S, Thelen S, Anderson KE, Thelen M, Welch HC. Membrane translocation of P-Rex1 is mediated by G protein betagamma subunits and phosphoinositide 3-kinase. J Biol Chem 282: 29967–29976, 2007 - PubMed
    1. Bokoch GM. Biology of the P21-activated kinases. Annu Rev Biochem 72: 743–781, 2003 - PubMed
    1. Chahdi A, Sorokin A. The role of beta(1)Pix/caveolin-1 interaction in endothelin signaling through Galpha subunits. Biochem Biophys Res Commun 391: 1330–1335, 2010 - PMC - PubMed
    1. Cheng H, Straub SG, Sharp GW. Inhibitory role of Src family tyrosine kinases on Ca2+-dependent insulin release. Am J Physiol Endocrinol Metab 292: E845–E852, 2007 - PubMed

Publication types

MeSH terms