Identification of P122rhogap (deleted in Liver Cancer-1) Serine-322 as a Substrate for Protein Kinase B and Rsk in Insulin-stimulated Cells

Protein kinase B (Akt) plays an essential role in the actions of insulin, cytokines and growth factors, although the substrates for PKB that are relevant to many of its actions require identification. In this study we report the identification of p122RhoGAP, a GTPase activating protein selective for RhoA and rodent homologue of the tumour suppressor gene Deleted in Liver Cancer (DLC1), as a novel insulin-stimulated phosphoprotein in primary rat adipocytes. We demonstrate that Ser-322 is phosphorylated upon insulin stimulation of intact cells and that this site is directly phosphorylated in vitro by PKB and RSK, members of the AGC-family of insulinstimulated protein kinases. Furthermore, expression of constitutively-active mutants of PKB or MAPK kinase (MEK) stimulate Ser-322 phosphorylation in intact cells, demonstrating that activation of the PKB or MEK pathway is sufficient for Ser-322 phosphorylation in vivo. Indeed, in primary adipocytes, insulinstimulated Ser-322 phosphorylation was almost exclusively by the PI3-kinase/PKB pathway, whereas in immortalised cells, insulinstimulated phosphorylation was predominantly by the MEK/ERK/RSK pathway, with the PI3kinase/PKB pathway playing a minor role. These results demonstrate that p122RhoGAP Ser-322 acts as an integrator of signal transduction in a manner dependent on the cellular context. INTRODUCTION Protein kinase B (PKB or Akt) is a protein serine/threonine kinase that plays a key role in intracellular signalling and cellular homeostasis. The kinase is activated by dual phosphorylation on Thr-308 by PDK1 (1), and on Ser-473 in a hydrophobic motif by a kinase that remains to be identified although recent evidence suggests that ATM, DNA-PK, ILK and/or mTOR/Rictor may be involved (2-5). Phosphorylation of these residues requires the production of PtdIns(3,4,5)P3 in response to the activation of Class I PI3-kinases (reviewed in (6,7)). Insulin, numerous growth factors, cytokines and other stimuli can activate PKB in this manner. Insulin utilises the PKB signalling pathway to regulate many intracellular events including the stimulation of glucose uptake (via the translocation GLUT4 to the plasma membrane), glycolysis and glycogen synthesis, and alterations in gene expression. Constitutively-active PKB mutants induce GLUT4 translocation in the absence of insulin, whereas dominant-negative PKB mutants and ablation of PKB by using siRNAs decrease insulin-stimulated glucose uptake (8,9), PKBβ knockout mice exhibit reduced insulinstimulated glucose uptake into muscle and adipose tissue (10) and deregulation of PKB activation by insulin has been reported to be associated with insulin resistance in type II diabetes (11). PKB also plays a central role in the regulation of many other cellular processes such as apoptosis and anoikis, neuronal by gest on Sptem er 1, 2017 hp://w w w .jb.org/ D ow nladed from Phosphorylation of p122RhoGAP 2 development and degeneration, and the cell cycle (see (7) for a recent review). Despite the central role of PKB in insulin action, many of the substrates that mediate the actions of PKB remain to be identified. Several groups, including our own, have used a commercially available PAS (Phospho Akt Substrate) antibody raised against the reported minimal consensus phosphorylation site found in almost all known PKB substrates (RXRXX[pS/pT]) (12,13) to purify and identify new PKB substrates. For example, this antibody has been used to identify AS160 (14), ATPcitrate lysase (15), PRAS40 (16), PIKfyve (17), YAP (18) and WNK1 (19) as PKB substrates. AS160 is a GTPase activating protein for Rabs 2A, 8A, 10 and 14, and has been reported to play a role in insulin-stimulated GLUT-4 translocation to the membrane (20,21). PIKfyve is a PtdIns(3)P 5-kinase which also appears to play a role in regulating the intracellular trafficking of GLUT4 (17). The role of PRAS40 remains unclear whereas YAP attenuates p73induced apoptosis (18) and WNK1 negative regulates insulin-stimulated mitogenesis (19). Using this approach in the current study, we identified by mass spectrometry an insulinstimulated phosphoprotein of apparent molecular weight 120 kDa in primary adipocytes as p122RhoGAP, a member of the extensive family of GTPase activating proteins for the Rho, Rac and Cdc42 family. We demonstrate that insulin stimulates p122RhoGAP phosphorylation on Ser-322 in both primary adipocytes and in an insulin-responsive cultured cell line (CHO.T cells) and that PKB can directly phosphorylate p122RhoGAP on Ser-322 in vitro. We also show that p122RhoGAP can be phosphorylated in vitro by RSK1, another member of the AGC-family of protein kinases, and that this also occurs in intact cells in an insulin-stimulated manner via the MEK/ERK signalling pathway. Thus Ser-322 phosphorylation of p122RhoGAP acts as an integrator of two distinct signal transduction pathways with the relative contribution of the PKB and MEK pathways in insulin-stimulated p122RhoGAP phosphorylation clearly depending on the cellular background involved. MATERIALS AND METHODS MaterialsMale Wistar rats (160-210 g) were fed ad libitum on a stock diet (CRM; Bioshore, Manea, Cambridgeshire, UK). Wortmannin and rapamycin were from Calbiochem (Bad Soden, Germany) and UO126 was from Promega Biosciences (San Luis Obispo, CA, USA). The anti-p122RhoGAP polyclonal antibody was as described previously (22). The anti-PKB substrate antibody (PAS), anti-phospho PKB (Thr-308), anti-phospho ERK (Thr-202/Tyr204) and anti-phospho p70S6K (Thr-389) were all from Cell Signaling Technology (Beverly, MA, USA). The anti-PKBα antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA), whereas the anti-green fluorescent protein (GFP) monoclonal antibody came from Roche Diagnostics Ltd (Lewes, East Sussex, UK). Unless otherwise stated, all other reagents were as described (23). PlasmidsThe constitutively-active myristoylated PKB (myrPKB) and constitutively-active p110 subunit of PI3 kinase (p110CAAX) were kindly provided by B. Hemmings (Friedlich Miescher Institute, Basal) and J. Downward (Cancer Research UK, London Research Institute, London), respectively, and were subcloned into the vector pcDNA3 (Invitrogen). The constitutively-active MEK-MANE construct was kindly provided by Stephen Hooper (Institute of Cancer Research, London, UK). The GFP-p122RhoGAP construct was as described before (24). Mutagenesis using the wild type GFPp122RhoGAP as a template, was undertaken with the Quikchange system (Stratagene, Amsterdam, Holland) following the manufacturer’s protocol: three mutants were created (i) p122RhoGAP[S320A], (ii) p122RhoGAP[S322A] and (iii) p122RhoGAP[S559A]. Preparation of rat epididymal adipocytesAdipocytes were isolated from the epididymal fat pads of Wistar rats as described previously (23). Cells were subsequently washed in Krebsbicarbonate-HEPES buffer (130 mM NaCl, 4.7 mM KCl, 1.5 mM MgSO4, 1.2 mM CaCl2, 2.5 mM NaH2PO4, 15.5 mM NaHCO3, 10 mM HEPES and 11 mM glucose, pH 7.4) with 1% bovine serum albumin added prior to the experiment. Transfection of primary adipocytes-Primary adipocytes were transfected as described previously (15) with a few adjustments. In short, freshly isolated cells were washed in intracellular Krebs-bicarbonate-HEPES buffer, by gest on Sptem er 1, 2017 hp://w w w .jb.org/ D ow nladed from Phosphorylation of p122RhoGAP 3 pH 7.4 (4 mM NaCl, 125 mM KCl, 1 mM EGTA, 1 mM MgCl2, 2.5 mM NaH2PO4, 15.5 mM NaHCO3, 10 mM HEPES and 11 mM glucose) with 1% bovine serum albumin. A 0.4cm electrode gap Gene Pulser cuvette (Biorad, Richmond, CA) was used to electroporate 500 μl of cells (30% cytocrit) in the presence of 15 μg of plasmid DNA. Electroporation was performed by administering 5 pulses at 500 V and a capacitance of 50 microFarads using a Gene Pulser transfection apparatus (Biorad). After electroporation, the cells were transferred to a 30 ml tube (Bibby-Sterilin Ltd., Staffs, UK) and incubated for 30 min at 37°C before replacing the medium with 4 ml of Dulbecco’s modified Eagle’s medium (containing 1% bovine serum albumin, 2 mM glutamine, 200 nM phenylisopropyladenosine, 100 μg gentamycin and 25 mM HEPES, pH 7.4). The cells were incubated for an additional 17 h at 37°C and 5% CO2 and subsequently washed into Krebs-bicarbonate-HEPES buffer without bovine serum albumin prior to the experiment. Incubation of transfected rat epididymal adipocytesCells were washed into Krebsbicarbonate-HEPES buffer, pH 7.4 without bovine serum albumin and left untreated or incubated with 100 nM wortmannin, 10 μM UO126 or 200 nM rapamycin for 30 min at 37°C prior to stimulation with 87 nM insulin for the times indicated. The reaction was terminated by extracting the cells 1:1 (packed cell volume/volume) in ice cold NP40 extraction buffer (50 mM Tris pH 7.5, containing 1% NP40, 1 mM EDTA, 120 mM NaCl, 50 mM NaF, 40 mM β-glycerophosphate, 1 mM benzamidine, 1 μM microcystin, 10 mM sodium orthovanadate and 1 μg/ml each of pepstatin, leupeptin and antipain). Cell extracts were centrifuged at 10,000 x g for 10 min at 4°C and the infranatant was taken for subsequent analysis. Culture, transfection and incubation of CHO.T cellsCHO.T cells (CHO cells stably expressing the human insulin receptor) were cultured in Ham's F12 medium containing 5% (v/v) foetal calf serum, 200 units/ml benzylpenicillin, 100 μg/ml streptomycin and 0.25 mg/ml G-418. CHO.T cells at 70% confluence were transfected in 60 mm dishes with 5 μg of each plasmid (GFP-tagged wild type RhoGAP, p110CAAX, myrPKB or MEK-MANE) using a charge ratio of 3 μl of Fugene 6 reagent (Roche Molecular Biochemicals) per μg of plasmid DNA, according to the manufacturer's instructions. After 5 h the cells were serum starved for 16 h, treated with insulin (87 nM) for the time period indicated in the figure legends and washed twice in ice-cold PBS before extraction by scraping into 500 μl of ice cold NP40 extraction buffer (50 mM Tris pH 7.5, containing 1% NP40, 1 mM EDTA, 120 mM NaCl, 50 mM NaF, 40 mM β-glycerophosphate, 1 mM benzamidine, 1 μM microcystin, 10 mM sodium orthovanadate and 1 μg/ml each of pepstatin, leupeptin and antipain). Cell lysates were centrifuged at 10,000 x g for 10 min at 4°C and the supernatant was taken for subsequent analysis. ImmunoprecipitationThe GFP-labelled p122RhoGAP was immunoprecipitated by rotating 250 μl of total cell extract with 5 μl of anti-GFP antibody and 10 μl protein GSepharose (50% w/v) at 4°C. The protein GSepharose beads were isolated by centrifugation and washed three times in NP40 extraction buffer. Subsequently, Laemmli sample buffer was added and proteins were separated by SDSPAGE for immunoblotting. ImmunoblottingProteins were separated by SDS-PAGE using 3-8% gradient gels (p122RhoGAP immunoprecipitates) or 7.5% gels (total lysates) and transferred to polyvinylidene difluoride membranes (Immobilon). The membranes were blocked in 10% (w/v) bovine serum albumin dissolved in Tris-buffered saline with 0.1% Tween (TBS-T; 20 mM Tris, pH 7.6, 137 mM NaCl, 0.1% Tween) and subsequently incubated with primary and secondary antibodies, which were diluted in TBS-T containing 5% (w/v) bovine serum albumin. Blots were washed at least 5 times for 5 min after each antibody incubation and developed using an Enhanced ChemiLuminescence detection system (AP Biotech, Amersham, U.K.). Primary antibodies were used at a concentration of 1 μg/ml. Horseradish peroxidase–conjugated secondary antibody (Amersham) was diluted 1:10,000 for all antibodies. Production of recombinant GST-p122RhoGAPp122RhoGAP was expressed as a recombinant fusion with GST in E. coli. Briefly, 2 litres of cells were grown to an optical density (OD600) of by gest on Sptem er 1, 2017 hp://w w w .jb.org/ D ow nladed from Phosphorylation of p122RhoGAP 4 0.8 and protein expression was induced over 2 h by addition of 0.5 mM isopropyl β-dthiogalactopyranoside (IPTG). The cells were harvested by centrifugation and resuspended in 40 ml of ice-cold lysis buffer (50 mM Tris, pH 7.5, 1% Triton X-100, 150 mM NaCl, 5 mM MgCl, 1 mM dithiothreitol, 0.2 mM PMSF), and the cells were then broken by sonication. Insoluble material was removed by centrifugation and GST-p122RhoGAP protein was purified through binding to glutathioneSepharose beads. Purified GST-p122RhoGAP protein was stored on beads in aliquots at -80 °C.