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Activation and Translocation of Rho (and ADP Ribosylation Factor) by Insulin in Rat Adipocytes

APPARENT INVOLVEMENT OF PHOSPHATIDYLINOSITOL 3-KINASE*
  • Purushotham Karnam
    Affiliations
    J. A. Haley Veterans' Hospital Research Service and the Departments of Internal Medicine and Biochemistry/Molecular Biology, University of South Florida, Tampa, Florida 33612
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  • Mary L. Standaert
    Affiliations
    J. A. Haley Veterans' Hospital Research Service and the Departments of Internal Medicine and Biochemistry/Molecular Biology, University of South Florida, Tampa, Florida 33612
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  • Lamar Galloway
    Affiliations
    J. A. Haley Veterans' Hospital Research Service and the Departments of Internal Medicine and Biochemistry/Molecular Biology, University of South Florida, Tampa, Florida 33612
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  • Robert V. Farese
    Correspondence
    To whom correspondence should be addressed
    Affiliations
    J. A. Haley Veterans' Hospital Research Service and the Departments of Internal Medicine and Biochemistry/Molecular Biology, University of South Florida, Tampa, Florida 33612
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  • Author Footnotes
    * The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
    1 The abbreviations used are: PLDphospholipase DPCphosphatidylcholinePIphosphatidylinositolKRPKrebs Ringer phosphate bufferBSAbovine serum albuminPAGEpolyacrylamide gel electrophoresisPIP2phosphatidylinositol-4-5-(PO4)2IRS-1insulin receptor substrate-1PKCprotein kinase CPRK1protein kinase C-related kinase-1PKNprotein kinase NGTPγSguanosine 5′-3-O-(thio)triphosphate2-DOG[3H]2-deoxyglucoseARFADP ribosylation factor.
Open AccessPublished:March 07, 1997DOI:https://doi.org/10.1074/jbc.272.10.6136
      Insulin reportedly (Standaert, M. L., Avignon, A., Yamada, K., Bandyopadhyay, G., and Farese, R. V. (1996) Biochem. J. 313, 1039-1046) activates phospholipase D (PLD)-dependent hydrolysis of phosphatidylcholine (PC) in plasma membranes of rat adipocytes by a mechanism that may involve wortmannin-sensitive phosphatidylinositol (PI) 3-kinase. Because Rho and ADP ribosylation factor (ARF) activate PC-PLD, we questioned whether these small G-proteins are regulated by insulin and PI 3-kinase. We found that insulin provoked a rapid translocation of both Rho and ARF to the plasma membrane and increased GTP loading of Rho. Wortmannin and LY294002 inhibited Rho translocation in intact adipocytes, and the polyphosphoinositide, PI 4,5-(PO4)2, stimulated Rho translocation in adipocyte homogenates. On the other hand, wortmannin did not block GTP loading of Rho. Guanosine 5′-3-O-(thio)triphosphate stimulated both Rho and ARF translocation and activated PC-PLD in homogenates. C3 transferase, which inhibits and depletes Rho, inhibited PC-PLD activation by insulin in intact adipocytes. C3 transferase also inhibited insulin stimulation of [3H]2-deoxyglucose uptake. Our findings suggest that: (a) insulin translocates Rho by a PI 3-kinase-dependent mechanism, but another factor is responsible for GTP loading of Rho; (b) both Rho and ARF may contribute to PC-PLD activation during insulin action; and (c) Rho may be required for insulin stimulation of glucose transport.

      INTRODUCTION

      Phospholipase D (PLD)-mediated
      The abbreviations used are: PLD
      phospholipase D
      PC
      phosphatidylcholine
      PI
      phosphatidylinositol
      KRP
      Krebs Ringer phosphate buffer
      BSA
      bovine serum albumin
      PAGE
      polyacrylamide gel electrophoresis
      PIP2
      phosphatidylinositol-4-5-(PO4)2
      IRS-1
      insulin receptor substrate-1
      PKC
      protein kinase C
      PRK1
      protein kinase C-related kinase-1
      PKN
      protein kinase N
      GTPγS
      guanosine 5′-3-O-(thio)triphosphate
      2-DOG
      [3H]2-deoxyglucose
      ARF
      ADP ribosylation factor.
      hydrolysis of phosphatidylcholine (PC) is a major signaling system for agonists that activate tyrosine kinases. Insulin activates PC-PLD in rat adipocytes (
      • Standaert M.L.
      • Avignon A.
      • Yamada K.
      • Bandyopadhyay G.
      • Farese R.V.
      ) and other cells (
      • Baldini P.M.
      • Zannetti A.
      • Donchenko V.
      • Dini L.
      • Luly P.
      ,
      • Donchenko V.
      • Zannetti A.
      • Baldini P.M.
      ,
      • Standaert M.L.
      • Musunuru K.
      • Yamada K.
      • Cooper D.R.
      • Farese R.V.
      ), and this may be important for activation of signaling and targeting processes in the plasma membrane (
      • Standaert M.L.
      • Avignon A.
      • Yamada K.
      • Bandyopadhyay G.
      • Farese R.V.
      ,
      • Standaert M.L.
      • Musunuru K.
      • Yamada K.
      • Cooper D.R.
      • Farese R.V.
      ,
      • Hoffman J.M.
      • Standaert M.L.
      • Nair G.P.
      • Farese R.V.
      ). In adipocytes, insulin-induced activation of PC-PLD is inhibited by wortmannin (
      • Standaert M.L.
      • Avignon A.
      • Yamada K.
      • Bandyopadhyay G.
      • Farese R.V.
      ), an inhibitor of phosphatidylinositol (PI) 3-kinase, which is activated through its SH2 domains by specific phosphotyrosine motifs in proteins, such as insulin receptor substrate-1 (IRS-1) (
      • White M.F.
      • Kahn C.R.
      ); thus, PI 3-kinase may be required for PC-PLD activation. Accordingly, polyphosphoinositides, which are increased by insulin (
      • Farese R.V.
      • Larson R.E.
      • Sabir M.A.
      ,
      • Farese R.V.
      • Davis J.S.
      • Barnes D.E.
      • Standaert M.L.
      • Babischkin J.S.
      • Hock R.
      • Rosic N.K.
      • Pollet R.J.
      ,
      • Ruderman N.B.
      • Kapeller R.
      • White M.F.
      • Cantley L.C.
      ) through PI 3-kinase action (
      • Ruderman N.B.
      • Kapeller R.
      • White M.F.
      • Cantley L.C.
      ) and which may be required for PC-PLD activation (
      • Brown H.A.
      • Gutowski S.
      • Kahan R.A.
      • Sternweis P.C.
      ,
      • Terui T.
      • Kahn R.A.
      • Randazzo P.A.
      ,
      • Pertile P.
      • Liscovitch M.
      • Chalifa V.
      • Cantley L.C.
      ,
      • Siddiqi A.R.
      • Smith J.L.
      • Ross A.H.
      • Qiu R.G.
      • Symons M.
      • Exton J.H.
      ), may contribute directly to the stimulation of PLD by insulin. However, PC-PLD is also activated by Rho and ARF (
      • Brown H.A.
      • Gutowski S.
      • Kahan R.A.
      • Sternweis P.C.
      ,
      • Terui T.
      • Kahn R.A.
      • Randazzo P.A.
      ,
      • Pertile P.
      • Liscovitch M.
      • Chalifa V.
      • Cantley L.C.
      ,
      • Siddiqi A.R.
      • Smith J.L.
      • Ross A.H.
      • Qiu R.G.
      • Symons M.
      • Exton J.H.
      ,
      • Cockcroft S.
      • Thomas G.M.
      • Fensome A.
      • Geny B.
      • Cunningham E.
      • Gout I.
      • Hiles I.
      • Totty N.F.
      • Truong O.
      • Hsuan J.J.
      ,
      • Bowman E.P.
      • Uhlinger D.J.
      • Lambeth J.D.
      ), and we presently questioned whether these small G-proteins are regulated by insulin and PI 3-kinase. In addition, because Rho and PI 3-kinase are thought to be involved in vesicle trafficking and because PI 3-kinase appears to play an important role in insulin-stimulated glucose transport (
      • White M.F.
      • Kahn C.R.
      ), we questioned whether Rho may also be required for the latter process.

      EXPERIMENTAL PROCEDURES

      Adipocytes were prepared from epididymal fat pads (see Ref.
      • Standaert M.L.
      • Avignon A.
      • Yamada K.
      • Bandyopadhyay G.
      • Farese R.V.
      ), equilibrated in glucose-free Krebs Ringer phosphate (KRP) buffer containing 1% bovine serum albumin (BSA), and treated with wortmannin (Sigma), LY294002 (BioMol), and/or insulin (Elanco) as described in the text.
      To study Rho/ARF translocation in intact cells, after incubation, the cells were chilled and sonicated in Buffer I, which contained 250 mM sucrose, 20 mM Tris-HCl (pH 7.5), 1.2 mM EGTA, 20 μg/ml aprotinin, 20 μg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, and 20 mMβ-mercaptoethanol. Homogenates were centrifuged first at 500 × g for 10 min to remove nuclei, debris, and the fat cake and then for 30 min at 100,000 × g to obtain membrane and cytosol fractions. Membranes were suspended in Buffer I supplemented with 5 mM EGTA, 2 mM EDTA, and 1% Triton X-100, and insoluble (cytoskeleton) substances were removed by centrifugation. Plasma membranes, microsomes, and mixed nuclear/mitochondrial fractions were obtained as described (
      • Standaert M.L.
      • Avignon A.
      • Yamada K.
      • Bandyopadhyay G.
      • Farese R.V.
      ). Routinely, 60 μg of cytosolic protein and 80 μg of membrane protein were used for immunoblotting (note that cytosolic protein is three or four times more abundant than membrane protein).
      To study Rho/ARF translocation in vitro, post-nuclear homogenates were prepared in Buffer I containing 1 mM EDTA and incubated first for 20 min at room temperature to release GDP and then for 20 min at 37°C after adding 10 mM MgCl2 with or without GTPγS (Sigma) or PI 4,5-(PO4)2 (PIP2; Fluka). Subcellular fractions were then obtained as described above.
      Subcellular fractions were resolved by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, and immunoblotted, using chemiluminescence (ECL, Amersham Corp.) as described (
      • Standaert M.L.
      • Avignon A.
      • Yamada K.
      • Bandyopadhyay G.
      • Farese R.V.
      ). Immunoblots were quantified with a Bio-Rad Molecular Analyst Chemiluminescence/32P Imaging System. All results were expressed relative to controls (set at 100%) that were developed simultaneously on the same blots.
      Mouse monoclonal antibodies raised against synthetic peptide corresponding to amino acids 120-150 in human RhoA were purchased from Santa Cruz Biotechnology, Inc.; these antibodies do not cross-react with RhoC, RhoG, Rac1, Rac2, or Cdc42Hs. Mouse monoclonal antibodies raised against human ARF1 proteins (ID9) were kindly provided by Dr. Richard Kahn. Antibodies for Cdc42HS, Rac1, and Rac2 were obtained from Santa Cruz. Antibodies for PI 3-kinase were from Upstate Biotechnology, Inc.
      In some experiments, 30-40 ml of adipocytes were incubated in batches for 2 h in 2-3 volumes of low phosphate (0.12 mM NaH2PO4) Krebs Ringer buffer supplemented with 10 mM HEPES, 2.5 mM glucose, 1% BSA, and 10 mCi of 32PO4 (DuPont NEN). Aliquots were then treated with insulin, and Rho was quantitatively immunoprecipitated from total cell lysates in buffer containing 20 mM Tris-HCl (pH 7.4), 250 mM sucrose, 150 mM NaCl, 2 mM EGTA, 10 mM MgCl2, 1 mM Na4P2O7, 1 mM NaF, 1 mM Na3VO4, 1% Triton X-100, 0.5% Nonidet, 1 mM phenylmethylsulfonyl fluoride, 20 μg/ml leupeptin, and 20 μg/ml aprotinin. Precipitates were collected on protein AG-Sepharose beads, washed with lysis buffer and phosphate-buffered saline, and then heated for 20 min at 68°C in buffer containing 5 mM EDTA, 2 mM dithiothreitol, 0.2% SDS, 0.5 mM GTP, and 0.5 mM GDP. GDP and GTP were separated on polyethyleneimine-cellulose plates (Merck) developed with 1 M KH2PO4 (pH 3.4). Plates were scanned and quantified in the Bio-Rad 32P Imaging System.
      PLD activation was measured both in situ in intact adipocytes and in vitro in post-nuclear homogenates of adipocytes after overnight labeling with[3H]oleic acid as described (
      • Standaert M.L.
      • Avignon A.
      • Yamada K.
      • Bandyopadhyay G.
      • Farese R.V.
      ). For in situ assays, cells were electroporated as described (
      • Cooper D.R.
      • Watson J.E.
      • Hernandez H.
      • Yu B.
      • Standaert M.L.
      • Ways D.K.
      • Arnold T.
      • Ishizuka T.
      • Farese R.V.
      ) in Dulbecco's modified Eagle's medium buffer with or without 1 μg/ml Clostridium botulinum C3 transferase (List), a selective inhibitor of Rho (
      • Aktories K.
      • Just I.
      ). After overnight labeling (with or without C3 transferase), cells were equilibrated in KRP buffer containing 1% BSA and 2% ethanol and treated for 0-20 min with 10 nM insulin (see Ref.
      • Standaert M.L.
      • Avignon A.
      • Yamada K.
      • Bandyopadhyay G.
      • Farese R.V.
      ). For in vitro assays, labeled cells were sonicated in Buffer I containing 25 mM HEPES (pH 7.4), 1 mM EDTA, 100 mM KCl, and 3 mM NaCl. After equilibration of homogenates for 20 min at room temperature to release GDP, 1 μM CaCl2, 5 mM MgCl2, 2.5% ethanol, and GTPγS were added, and incubation at 37°C was continued for 30 min. [32H]Phosphatidylethanol was isolated by TLC as described (
      • Standaert M.L.
      • Avignon A.
      • Yamada K.
      • Bandyopadhyay G.
      • Farese R.V.
      ).
      Glucose transport was assessed by measurement of [3H]2-deoxyglucose (2-DOG) uptake during a 1-min period, following treatment of adipocytes with vehicle (controls) or 10 nM insulin for 30 min as described (
      • Standaert M.L.
      • Avignon A.
      • Yamada K.
      • Bandyopadhyay G.
      • Farese R.V.
      ). Where indicated, cells were electroporated in the absence or the presence of C3 transferase and incubated for designated times prior to assessment of basal and insulin-stimulated 2-DOG uptake.

      DISCUSSION

      The present findings suggested that Rho and ARF may participate in the activation of PC-PLD by insulin in rat adipocytes. Both Rho and ARF translocated to plasma membranes sufficiently rapidly to contribute to the rapid activation of plasma membrane PC-PLD by insulin (see Ref.
      • Standaert M.L.
      • Avignon A.
      • Yamada K.
      • Bandyopadhyay G.
      • Farese R.V.
      ), and both G-proteins have been reported to activate PC-PLD (
      • Brown H.A.
      • Gutowski S.
      • Kahan R.A.
      • Sternweis P.C.
      ,
      • Terui T.
      • Kahn R.A.
      • Randazzo P.A.
      ,
      • Pertile P.
      • Liscovitch M.
      • Chalifa V.
      • Cantley L.C.
      ,
      • Siddiqi A.R.
      • Smith J.L.
      • Ross A.H.
      • Qiu R.G.
      • Symons M.
      • Exton J.H.
      ,
      • Cockcroft S.
      • Thomas G.M.
      • Fensome A.
      • Geny B.
      • Cunningham E.
      • Gout I.
      • Hiles I.
      • Totty N.F.
      • Truong O.
      • Hsuan J.J.
      ,
      • Bowman E.P.
      • Uhlinger D.J.
      • Lambeth J.D.
      ). In addition, GTPγS stimulated Rho and ARF translocation to the plasma membrane, as well as PLD activity, in adipocyte homogenates, and insulin increased GTP loading of Rho in intact cells. Moreover, insulin effects on PLD were inhibited by C3 transferase, which markedly depleted Rho.
      The present findings also suggested that PI 3-kinase activation, perhaps via polyphosphoinositides, may contribute to Rho translocation. Accordingly, both wortmannin and LY294002 inhibited the translocation of Rho in intact adipocytes, and PIP2 stimulated Rho translocation in vitro In addition, polyphosphoinositides may be required for PC-PLD activation (
      • Brown H.A.
      • Gutowski S.
      • Kahan R.A.
      • Sternweis P.C.
      ,
      • Terui T.
      • Kahn R.A.
      • Randazzo P.A.
      ,
      • Pertile P.
      • Liscovitch M.
      • Chalifa V.
      • Cantley L.C.
      ,
      • Siddiqi A.R.
      • Smith J.L.
      • Ross A.H.
      • Qiu R.G.
      • Symons M.
      • Exton J.H.
      ), and wortmannin inhibits PC-PLD activation in both insulin-treated rat adipocytes (
      • Standaert M.L.
      • Avignon A.
      • Yamada K.
      • Bandyopadhyay G.
      • Farese R.V.
      ) and f-Met-Leu-Phe-stimulated human neutrophils (
      • Reinhold S.L.
      • Prescott S.M.
      • Zimmerman G.A.
      • McIntyre T.M.
      ). It is therefore possible that insulin-induced activation of PI 3-kinase leads to production of polyphosphoinositides in the plasma membrane, followed by translocation of Rho, GTP loading, and PLD activation. Along these lines, PIP2 increases GDP dissociation from ARF (
      • Terui T.
      • Kahn R.A.
      • Randazzo P.A.
      ) and Cdc42Hs and Rho (see Ref.
      • Zheng Y.
      • Glaven J.A.
      • Wu W.J.
      • Cerione R.A.
      ); however, this dissociation does not necessarily result in GTP loading (
      • Zheng Y.
      • Glaven J.A.
      • Wu W.J.
      • Cerione R.A.
      ), and, as shown presently, GTP loading of Rho appeared to be due to a factor distinct from PI 3-kinase.
      Recently, there has been keen interest in mechanisms whereby small G-proteins activate PC-PLD on the one hand and, on the other hand, regulate vesicle formation and trafficking and in a variety of cytoskeletal events. Of Rho family members (Rho, Rac, and Cdc42Hs subtypes), Rho A is important in PC-PLD activation (
      • Brown H.A.
      • Gutowski S.
      • Kahan R.A.
      • Sternweis P.C.
      ,
      • Terui T.
      • Kahn R.A.
      • Randazzo P.A.
      ,
      • Pertile P.
      • Liscovitch M.
      • Chalifa V.
      • Cantley L.C.
      ,
      • Siddiqi A.R.
      • Smith J.L.
      • Ross A.H.
      • Qiu R.G.
      • Symons M.
      • Exton J.H.
      ,
      • Cockcroft S.
      • Thomas G.M.
      • Fensome A.
      • Geny B.
      • Cunningham E.
      • Gout I.
      • Hiles I.
      • Totty N.F.
      • Truong O.
      • Hsuan J.J.
      ) and formation of actin stress fibers and focal adhesions (
      • Ridley A.J.
      • Paterson H.F.
      • Johnston C.L.
      • Diekmann D.
      • Hall A.
      ). Rho has also been suggested to operate upstream of PI 3-kinase in human platelets (
      • Zhang J.
      • King W.G.
      • Dillon S.
      • Hall A.
      • Feig L.
      • Rittenhouse S.E.
      ,
      • Kumagai N.
      • Morii N.
      • Fujisawa K.
      • Nemoto Y.
      • Narumiya S.
      ), although in insulin-stimulated adipocytes, PI 3-kinase appears to be primarily activated by IRS-1 or other phosphotyrosine-containing proteins (
      • White M.F.
      • Kahn C.R.
      ). Rac1 is important in membrane ruffling (
      • Ridley A.J.
      • Paterson H.F.
      • Johnston C.L.
      • Diekmann D.
      • Hall A.
      ) and seems to operate downstream of PI 3-kinase in insulin-induced membrane ruffling (
      • Kotani K.
      • Hara K.
      • Kotani K.
      • Yonezawa K.
      • Kasuga M.
      ). Rac1 and Cdc42Hs (but not Rho A) activate a 62-65-kDa protein kinase that regulates stress-activated protein kinase/c-Jun NH2-terminal kinase (
      • Bagrodia S.
      • Taylor S.J.
      • Creasy C.L.
      • Chernoff J.
      • Cerione R.A.
      ,
      • Zhang S.
      • Han J.
      • Sells M.A.
      • Chernoff J.
      • Knaus U.G.
      • Ulevitch R.J.
      • Bokoch G.M.
      ,
      • Teo M.
      • Manser E.
      • Lim L.
      ,
      • Polverino A.
      • Frost J.
      • Yang P.
      • Hutchison M.
      • Neiman A.M.
      • Cobb M.H.
      • Marcus S.
      ). GTPγS-containing forms of Cdc42Hs and Rac1 also bind to and activate PI 3-kinase, and although this suggested that PI 3-kinase may be a downstream effector, it was surmised from other evidence that PI 3-kinase operates upstream of Cdc42Hs and Rac1 (
      • Tolias K.F.
      • Cantley L.C.
      • Carpenter C.L.
      ). Of further interest, PI 3-kinase and PC-PLD may control vesicle formation through the generation of membrane curvature-perturbing, acidic phospholipids, i.e. PI 3,4,5-(PO4)3, PI 3,4-(PO4)2, and PI 3-PO4, via PI 3-kinase action and phosphatidic acid via PLD action (
      • Liscovitch M.
      • Cantely L.C.
      ). Also, conventional (α, β, and γ) and novel (Δ, ε, η, and θ) PKCs may be activated by diacylglycerol derived through PC-PLD action, and these PKCs, along with atypical PKCs (ζ and λ) and protein kinase C-related kinase (PRK1 or PKN; see below), may be activated by D3-PO4 polyphosphoinositides (
      • Nakanishi H.
      • Brewer K.A.
      • Exton J.H.
      ,
      • Palmer R.H.
      • Dekker L.V.
      • Woscholski R.
      • LeGood J.A.
      • Gigg R.
      • Parker P.J.
      ) derived from PI 3-kinase action. Although not a member of the Rho family, ARF, like Rho, activates PC-PLD (
      • Brown H.A.
      • Gutowski S.
      • Kahan R.A.
      • Sternweis P.C.
      ,
      • Terui T.
      • Kahn R.A.
      • Randazzo P.A.
      ,
      • Pertile P.
      • Liscovitch M.
      • Chalifa V.
      • Cantley L.C.
      ,
      • Siddiqi A.R.
      • Smith J.L.
      • Ross A.H.
      • Qiu R.G.
      • Symons M.
      • Exton J.H.
      ,
      • Cockcroft S.
      • Thomas G.M.
      • Fensome A.
      • Geny B.
      • Cunningham E.
      • Gout I.
      • Hiles I.
      • Totty N.F.
      • Truong O.
      • Hsuan J.J.
      ), and, as suggested by the finding that polyphosphoinositides stimulate GTP/GDP exchange in ARF (
      • Terui T.
      • Kahn R.A.
      • Randazzo P.A.
      ), PI 3-kinase may function upstream of ARF, as well as Rho. Obviously, both Rho and ARF may operate through or in conjunction with a variety of lipid and protein kinases and other signaling factors in regulating vesicle trafficking and cytoskeletal events.
      Similar to our present observation of Rho and ARF translocation, f-Met-Leu-Phe stimulates ARF and Rho translocation in HL-60 cells (
      • Houle M.G.
      • Kahn R.A.
      • Naccache P.H.
      • Bourgoin S.
      ) and guanosine nucleotide exchange in Rho in lymphocytes (
      • Laudanna C.
      • Campbell J.J.
      • Butcher E.C.
      ). Thus, ARF and Rho may function in parallel or in tandem and co-ordinately activate PC-PLD and other processes in response to agonist treatment in various cell types.
      In addition to PLD activation, our observation that insulin-induced Rho translocation is sensitive to polyphosphoinositides and PI 3-kinase inhibitors is of interest, because: (a) GTP-Rho binds to an activation site in the NH2-terminal regulatory domain of a 120-kDa protein kinase, variously called PKN (
      • Amano M.
      • Mukai H.
      • Ono Y.
      • Chihara K.
      • Matsui T.
      • Hamajima Y.
      • Okawa K.
      • Iwamatsu A.
      • Kaibuchi K.
      ,
      • Watanabe G.
      • Saito Y.
      • Madaule P.
      • Ishizaki T.
      • Fujisawa K.
      • Morii N.
      • Mukai H.
      • Ono Y.
      • Kakizuka A.
      • Narumiya S.
      ) or PRK1 (
      • Palmer R.H.
      • Dekker L.V.
      • Woscholski R.
      • LeGood J.A.
      • Gigg R.
      • Parker P.J.
      ), and (b) PRK1 (
      • Palmer R.H.
      • Dekker L.V.
      • Woscholski R.
      • LeGood J.A.
      • Gigg R.
      • Parker P.J.
      ), like various PKCs (
      • Nakanishi H.
      • Brewer K.A.
      • Exton J.H.
      ,
      • Palmer R.H.
      • Dekker L.V.
      • Woscholski R.
      • LeGood J.A.
      • Gigg R.
      • Parker P.J.
      ), is activated by polyphosphoinositides. Thus, insulin-induced increases in polyphosphoinositides (
      • Farese R.V.
      • Larson R.E.
      • Sabir M.A.
      ,
      • Farese R.V.
      • Davis J.S.
      • Barnes D.E.
      • Standaert M.L.
      • Babischkin J.S.
      • Hock R.
      • Rosic N.K.
      • Pollet R.J.
      ,
      • Ruderman N.B.
      • Kapeller R.
      • White M.F.
      • Cantley L.C.
      ), which occur through the activation of PI 3-kinase (
      • Ruderman N.B.
      • Kapeller R.
      • White M.F.
      • Cantley L.C.
      ), may activate PKN (PRK1), both directly via polyphosphoinositides and indirectly through Rho. The co-activation of Rho and PKN (PRK1) by polyphosphoinositides may facilitate their co-localization and may also co-ordinate the activation of PKN (PRK1) with other PKCs that are activated by PI 3-kinase through its lipid products or PC-PLD. With respect to PKC, it should be noted that we have not observed significant activation of PC-PLD or translocation of Rho during phorbol ester treatment in rat adipocytes.
      Finally, it was of interest to find that insulin-stimulated glucose transport was inhibited in cells depleted of Rho by C3 transferase treatment. This apparent requirement for Rho, coupled with the fact that Rho is translocated and activated by insulin, suggests that Rho may have a role in insulin stimulation of glucose transport. Clearly, more studies are needed to test this possibility and further define the role of Rho.
      In summary, insulin provoked rapid increases in Rho and ARF translocation to the plasma membrane and GTP loading of Rho in rat adipocytes. In addition, wortmannin and LY294002 inhibited insulin effects on Rho translocation in intact adipocytes, but wortmannin did not inhibit GTP loading of Rho. Of further note, PIP2 and GTPγS stimulated Rho translocation in adipocyte homogenates, and C3 transferase inhibited PLD activation in intact adipocytes. Collectively, these findings suggest that insulin translocates Rho by a PI 3-kinase-dependent mechanism but stimulates GTP loading of Rho by a PI 3-kinase-independent mechanism, and both Rho and ARF may participate in the activation of PLD. Further studies will be required to define: (a) the precise mechanisms for activation and translocation of Rho and ARF and (b) the interrelated roles of PI 3-kinase and these small G-proteins in the activation of PC-PLD, various lipid-regulated protein kinases, vesicle trafficking, cytoskeletal events, and other cellular processes.

      REFERENCES

        • Standaert M.L.
        • Avignon A.
        • Yamada K.
        • Bandyopadhyay G.
        • Farese R.V.
        Biochem. J. 1996; 313: 1039-1046
        • Baldini P.M.
        • Zannetti A.
        • Donchenko V.
        • Dini L.
        • Luly P.
        Biochem. Biophys. Acta. 1992; 1170: 208-214
        • Donchenko V.
        • Zannetti A.
        • Baldini P.M.
        Biochem. Biophys. Acta. 1994; 1222: 492-500
        • Standaert M.L.
        • Musunuru K.
        • Yamada K.
        • Cooper D.R.
        • Farese R.V.
        Cell. Signalling. 1994; 6: 707-716
        • Hoffman J.M.
        • Standaert M.L.
        • Nair G.P.
        • Farese R.V.
        Biochemistry. 1991; 30: 3315-3322
        • White M.F.
        • Kahn C.R.
        J. Biol. Chem. 1994; 269: 1-4
        • Farese R.V.
        • Larson R.E.
        • Sabir M.A.
        J. Biol. Chem. 1982; 257: 4042-4045
        • Farese R.V.
        • Davis J.S.
        • Barnes D.E.
        • Standaert M.L.
        • Babischkin J.S.
        • Hock R.
        • Rosic N.K.
        • Pollet R.J.
        Biochem. J. 1985; 231: 269-278
        • Ruderman N.B.
        • Kapeller R.
        • White M.F.
        • Cantley L.C.
        Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 1411-1415
        • Brown H.A.
        • Gutowski S.
        • Kahan R.A.
        • Sternweis P.C.
        J. Biol. Chem. 1995; 270: 14935-14943
        • Terui T.
        • Kahn R.A.
        • Randazzo P.A.
        J. Biol. Chem. 1994; 269: 28130-28135
        • Pertile P.
        • Liscovitch M.
        • Chalifa V.
        • Cantley L.C.
        J. Biol. Chem. 1995; 270: 5130-5135
        • Siddiqi A.R.
        • Smith J.L.
        • Ross A.H.
        • Qiu R.G.
        • Symons M.
        • Exton J.H.
        J. Biol. Chem. 1995; 270: 8466-8473
        • Cockcroft S.
        • Thomas G.M.
        • Fensome A.
        • Geny B.
        • Cunningham E.
        • Gout I.
        • Hiles I.
        • Totty N.F.
        • Truong O.
        • Hsuan J.J.
        Science. 1994; 263: 523-526
        • Bowman E.P.
        • Uhlinger D.J.
        • Lambeth J.D.
        J. Biol. Chem. 1993; 268: 21509-21512
        • Cooper D.R.
        • Watson J.E.
        • Hernandez H.
        • Yu B.
        • Standaert M.L.
        • Ways D.K.
        • Arnold T.
        • Ishizuka T.
        • Farese R.V.
        Biochem. Biophys. Res. Commun. 1992; 188: 142-148
        • Aktories K.
        • Just I.
        Methods Enzymol. 1995; 256: 184-195
        • Kelly K.L.
        • Ruderman N.B.
        • Chen K.S.
        J. Biol. Chem. 1992; 267: 3423-3428
        • Malcolm K.C.
        • Elliott C.M.
        • Exton J.H.
        J. Biol. Chem. 1996; 271: 13135-13139
        • Reinhold S.L.
        • Prescott S.M.
        • Zimmerman G.A.
        • McIntyre T.M.
        FASEB J. 1990; 4: 208-214
        • Amano M.
        • Mukai H.
        • Ono Y.
        • Chihara K.
        • Matsui T.
        • Hamajima Y.
        • Okawa K.
        • Iwamatsu A.
        • Kaibuchi K.
        Science. 1996; 271: 648-650
        • Zheng Y.
        • Glaven J.A.
        • Wu W.J.
        • Cerione R.A.
        J. Biol. Chem. 1996; 271: 23815-23819
        • Ridley A.J.
        • Paterson H.F.
        • Johnston C.L.
        • Diekmann D.
        • Hall A.
        Cell. 1992; 70: 401-410
        • Zhang J.
        • King W.G.
        • Dillon S.
        • Hall A.
        • Feig L.
        • Rittenhouse S.E.
        J. Biol. Chem. 1993; 268: 22251-22254
        • Kumagai N.
        • Morii N.
        • Fujisawa K.
        • Nemoto Y.
        • Narumiya S.
        J. Biol. Chem. 1993; 268: 24535-24538
        • Kotani K.
        • Hara K.
        • Kotani K.
        • Yonezawa K.
        • Kasuga M.
        Biochem. Biophys. Res. Commun. 1995; 208: 985-990
        • Bagrodia S.
        • Taylor S.J.
        • Creasy C.L.
        • Chernoff J.
        • Cerione R.A.
        J. Biol. Chem. 1995; 270: 22731-22737
        • Zhang S.
        • Han J.
        • Sells M.A.
        • Chernoff J.
        • Knaus U.G.
        • Ulevitch R.J.
        • Bokoch G.M.
        J. Biol. Chem. 1995; 270: 23934-23936
        • Teo M.
        • Manser E.
        • Lim L.
        J. Biol. Chem. 1995; 270: 26690-26697
        • Polverino A.
        • Frost J.
        • Yang P.
        • Hutchison M.
        • Neiman A.M.
        • Cobb M.H.
        • Marcus S.
        J. Biol. Chem. 1995; 270: 26067-26070
        • Tolias K.F.
        • Cantley L.C.
        • Carpenter C.L.
        J. Biol. Chem. 1995; 270: 17656-17659
        • Liscovitch M.
        • Cantely L.C.
        Cell. 1995; 81: 659-662
        • Nakanishi H.
        • Brewer K.A.
        • Exton J.H.
        J. Biol. Chem. 1993; 268: 13-16
        • Palmer R.H.
        • Dekker L.V.
        • Woscholski R.
        • LeGood J.A.
        • Gigg R.
        • Parker P.J.
        J. Biol. Chem. 1995; 270: 22412-22416
        • Houle M.G.
        • Kahn R.A.
        • Naccache P.H.
        • Bourgoin S.
        J. Biol. Chem. 1995; 270: 22795-22800
        • Laudanna C.
        • Campbell J.J.
        • Butcher E.C.
        Science. 1996; 271: 981-983
        • Watanabe G.
        • Saito Y.
        • Madaule P.
        • Ishizaki T.
        • Fujisawa K.
        • Morii N.
        • Mukai H.
        • Ono Y.
        • Kakizuka A.
        • Narumiya S.
        Science. 1996; 271: 645-648