2606 Diabetes Volume 65, September 2016

Pragya Sharma,1 Edward B. Arias,1 and Gregory D. Cartee1,2,3

Protein 1-a Regulates AS160 Ser588 and Thr642 Dephosphorylation in Skeletal Muscle

Diabetes 2016;65:2606–2617 | DOI: 10.2337/db15-0867

Akt substrate of 160 kDa (AS160) on insulin resistance is crucial for whole-body insulin re- Thr642 and Ser588 by Akt is essential for insulin’s full ef- sistance and type 2 diabetes (1). Muscle insulin resistance fect on glucose transport. However, protein phosphory- is secondary, in large part, to defective GLUT4 transloca- lation is determined by the balance of actions by tion and glucose transport (2). Insulin’s stimulation of glu- fi and , and the speci c phosphatase(s) cose transport is triggered by a complex insulin-signaling controlling AS160 dephosphorylation is (are) unknown. pathway that begins with insulin’s binding to its receptor, Accordingly, we assessed roles of highly expressed leading to receptor autophosphorylation and activation of skeletal muscle serine/threonine phosphatases (PP1, receptor tyrosine (2). The insulin receptor kinase PP2A, PP2B, and PP2C) on AS160 dephosphorylation. Preliminary screening of candidate phosphatases used phosphorylates insulin receptor substrate (IRS) proteins an AS160 dephosphorylation assay. Lysates from insu- on multiple tyrosine residues, resulting in IRS protein en- lin-stimulated skeletal muscle were treated with phar- gagement with phosphatidylinositol (PI) 3-kinase (PI3K), macological phosphatase inhibitors and assessed for that in turn, phosphorylates PI 4,5-bisphosphate to create AS160 Ser588 and Thr642 dephosphorylation. AS160 de- 3,4,5-trisphosphate (PIP3). The serine/threonine kinase phosphorylation on both phosphorylation sites was un- Akt is recruited to bind PIP3 and become activated second- altered by PP2B or PP2C inhibitors. Okadaic acid (low 308

SIGNAL TRANSDUCTION ary to phosphorylation on Thr via phosphoinositide- dose inhibits PP2A; high dose inhibits PP1) delayed dependent kinase-1 (PDK1) and Ser473 via mTORC2. Akt AS160 Ser588 (both doses) and Thr642 (high dose only) phosphorylates many protein substrates, several of which dephosphorylation concomitant with greater Akt phos- have been implicated in insulin’s regulation of GLUT4 traf- phorylation (both doses). AS160 was coimmunoprecipi- fic to the cell surface membranes, including a Rab-GTPase tated with PP1-a but not with PP1-b, PP1-g1, or PP2A. activating protein known as Akt substrate of 160 kDa Recombinant inhibitor-2 protein (a selective PP1 inhibi- (AS160; also known as TBC1D4) (3–5). Akt can phosphor- tor) delayed AS160 dephosphorylation on both phos- ylate several residues on AS160. Mutation of serine or phorylation sites without altering Akt phosphorylation. threonine to alanine to prevent phosphorylation of Ser588 Furthermore, knockdown of PP1-a but not PP1-b or 642 PP1-g1 by small interfering RNA caused greater or Thr resulted in attenuation of insulin-stimulated AS160 Ser588 and Thr642 phosphorylation concomitant GLUT4 translocation, and mutation of several other Akt with unaltered Akt phosphorylation. Together, these phosphomotifs did not produce any further effects on results identified PP1-a as a regulator of AS160 Thr642 GLUT4 localization (6). Fully understanding the regulation and Ser588 dephosphorylation in skeletal muscle. of AS160 phosphorylation is essential given the crucial role that it plays in regulating insulin-stimulated glucose uptake by skeletal muscle. Skeletal muscle accounts for the largest portion of insulin- The reversible serine/threonine phosphorylation of pro- mediated whole-body glucose disposal, and skeletal muscle teins is balanced by the opposing actions of kinases and

1Muscle Biology Laboratory, School of Kinesiology, University of Michigan, Ann Received 24 June 2015 and accepted 23 May 2016. Arbor, MI © 2016 by the American Diabetes Association. Readers may use this article as 2 Department of Molecular and Integrative Physiology, University of Michigan, Ann long as the work is properly cited, the use is educational and not for profit, and Arbor, MI the work is not altered. More information is available at http://diabetesjournals 3 Institute of Gerontology, University of Michigan, Ann Arbor, MI .org/site/license. Corresponding author: Gregory D. Cartee, [email protected]. diabetes.diabetesjournals.org Sharma, Arias, and Cartee 2607 phosphatases, but for most proteins, there has been an (#ABS54), anti–PP1-b (#07–1217), anti–PP1-g1 (#07-1218), overwhelming bias to focus on serine/threonine kinases, anti–a-tubulin (#04-1117), and normal rabbit IgG polyclonal with strikingly fewer studies assessing the role of serine/ antibody control (#12-370) were purchased from EMD Milli- threonine phosphatases (7). Serine/threonine protein phos- pore. Anti-PP2Aa (#610556) was from BD Bioscience (San phatases regulate diverse aspects of growth, development, Jose, CA). PP1 Inh-2 antibody (#AF4719) was from R&D and metabolism, but relatively few protein serine/threonine Biosystems (Minneapolis, MN). Anti–PP1-a (#sc-443), anti- phosphatases control the specific dephosphorylation of a GADD34 (#sc-8327), anti-goat IgG horseradish peroxidase muchgreaternumberofphosphoprotein substrates (8). conjugate (#sc-2020), and anti-mouse IgG horseradish perox- With specific regard to AS160, many studies have analyzed idase conjugate (#sc-2060), were from Santa Cruz Biotech- the role of Akt in the insulin-stimulated phosphorylation of nology (Santa Cruz, CA). Anti-phostensin (#MB1057) was AS160 (9–13), but essentially nothing is known about the fromBioWorldTechnology,Inc.(St.LouisPark,MN).siRNA serine/threonine (s) regulating AS160 against rat PP1 catalytic subunits a (#L-100270-02-0020), dephosphorylation. b (#L-100263-02-0020), or g1 (#L-096319-02-0020) Protein phosphatase 1 (PP1), PP2A, PP2B, and PP2C and RNA interference-negative control (#D-001810-10-20) are among the most abundant serine/threonine protein were purchased as SMARTpools from Dharmacon (Lafayette, phosphatases expressed by skeletal muscle (14), and we CO). Protein G magnetic beads (#10004D), RNAiMAX trans- hypothesized that AS160 dephosphorylation on Thr642 fection reagent (#13778-150), and DMEM (#11995) were and Ser588 would be regulated by one or more of these from Life Technologies (Grand Island, NY). . We evaluated the hypothesis using multiple ap- proaches, including assessment of Animal Treatment Procedures for animal care were approved by the Univer- 1. the effects of several pharmacologic serine/threonine sity of Michigan Committee on Use and Care of Animals. – protein phosphatase inhibitors on AS160 Ser588 and Male Wistar rats (aged 8 10 weeks) were from Harlan Thr642 dephosphorylation; (Indianapolis, IN). Lean (Fa/Fa) and obese (fa/fa) male – 2. the physical association of AS160 with serine/threo- Zucker rats (aged 7 8 weeks) were from Charles River nine protein phosphatases; Laboratories (Wilmington, MA). Animals were provided 3. the influence of a selective inhibitor of PP1, known as with rodent chow (Lab Diet No. 5001; PMI Nutrition In- inhibitor 2 (Inh-2) (15), on AS160 Ser588 and Thr642 ternational, Brentwood, MO) ad libitum until 1700 h the phosphorylation; and night before the experiment, when food was removed. 4. the consequences of knockdown of serine/threonine The next day at 1000 h to 1200 h, rats were anesthetized protein phosphatases by small interfering (si)RNA si- (intraperitoneal injection of sodium pentobarbital), and lencing on AS160 Ser588 and Thr642 phosphorylation. both epitrochlearis muscles were isolated and treated as described below. These experiments identified PP1-a as a serine/threonine Muscle Incubation protein phosphatase that regulates AS160 Ser588 and Thr642 Isolated epitrochlearis muscles were incubated in glass dephosphorylation in skeletal muscle. vials containing Krebs-Henseleit buffer, 0.1% BSA, RESEARCH DESIGN AND METHODS 2 mmol/L sodium pyruvate, 6 mmol/L mannitol, without (basal) or with insulin at 0.6 nmol/L (for coimmunopre- Materials cipitation [Co-IP] assays described below) or 30 nmol/L ThereagentsandapparatusforSDS-PAGEandnonfatdry (for dephosphorylation assays described below) for milk (#170-6404XTU) were from Bio-Rad (Hercules, CA). 30 min in a heated, shaking water bath at 35°C with MemCode Reversible Protein Stain (#24580) and bicincho- continuous gassing (95% O2 and 5% CO2). Immediately ninic acid (#23227) protein assay kits and tissue protein after the incubation, muscles were blotted, rapidly extraction reagent (T-PER; #78510) were from Thermo Fisher trimmed of connective tissue, and freeze-clamped with (Waltham, MA). Luminata Forte Western HRP Substrate – liquid N2 cooled aluminum tongs. Frozen muscles were (#WBLUF0100) was from EMD Millipore (Billerica, MA). stored at 280°C until subsequent homogenization and Sanguinarine chloride (#ALX-350-076) was from Enzo Life analysis. Sciences (Farmingdale, NY). FK-506 (#3631) was purchased from Tocris (Bristol, U.K.). Okadaic acid (OA; #459620) was L6 Cell Culture and Treatment purchased from Merck Millipore (Billerica, MA). Recombinant L6 myoblasts were purchased from the American Type protein phosphatase Inh-2 (#P0755) was from New England Culture Collection (Manassas, VA). L6 cells were cultured Biolabs (Ipswich, MA). Anti-phosphorylated (p)AktThr308 in DMEM, supplemented with 10% (vol/vol) FBS, 1% (#9275), anti-pAktSer473 (#9271), anti-Akt (#4691), anti- penicillin, and 100 mg/mL streptomycin in a humidified Thr642 Ser588 pAS160 (#8881), anti-pAS160 (#8730), anti– atmosphere with 5% CO2 at 37°C. Cells were washed twice PP1-a (#2582), anti-spinophilin (#14136), and anti-rabbit with 13 PBS, and starved for 5 h in serum-free DMEM IgG horseradish peroxidase conjugate (#7074) were from medium before 20 min of incubation without insulin (basal) Technology (Danvers, MA). Anti-AS160 or with insulin (100 nmol/L). 2608 PP1-a Regulates AS160 Dephosphorylation Diabetes Volume 65, September 2016

Muscle and Cell Lysate Preparation added. The lysates were then incubated at 37°C, with ali- Unless otherwise noted, epitrochlearis muscles were quots (20 mL) withdrawn at 30, 60, and 120 min. Upon homogenized (TissueLyser II homogenizer; Qiagen Inc., withdrawal, each aliquot was rapidly mixed with an equal Valencia, CA) using ice-cold T-PER buffer supplemented volume of 23 SDS loading buffer and heated to 95°C for with 2.5 mmol/L sodium pyrophosphate, 1 mmol/L sodium 6min.SDS-PAGEandimmunoblotting were used to assess vanadate, 1 mmol/L b-glycerophosphate, 1 mg/mL leupep- the phosphorylation levels of AS160Ser588 and AS160Thr642 tin, 1 mg/mL pepstatin, 1 mg/mL aprotinin, and 1 mmol/L in these aliquots. When Inh-2 protein was used, dephos- phenylmethyl sulfonyl fluoride (PMSF). Homogenates were phorylation assays were performed as described above for then transferred to microcentrifuge tubes and rotated (1 h, chemical inhibitors, except that epitrochlearis lysates were 4°C) before being centrifuged (15,000g,20min,4°C).L6 incubated with or without recombinant Inh-2 (rInh-2) pro- cells were scraped in T-PER that was supplemented as de- tein (50 mg/mL lysate) rather than a chemical inhibitor. scribed above, incubated (4°C, 20 min), and then centri- Dephosphorylation assays inleanZucker(LZ)andobese fuged (15,000g, 20 min, 4°C). Total protein in supernatants Zucker (OZ) rats were performed as described above, except from muscle or cell lysates was measured by the bicincho- lysates were incubated without any phosphatase inhibitors. ninic acid method. Co-IP Epitrochlearis muscles and L6 cells used for Co-IP were IP was performed in lysates prepared from epitrochlearis processed as described above using Co-IP buffer (50 mmol/L muscles (300 mg total protein) and L6 cells (400 mgtotal HEPES [pH 7.5], 150 mmol/L NaCl, 1% IGEPAL, 10% glycerol) protein) using AS160 antibody or normal rabbit IgG at 4°C supplemented with 2.5 mmol/L sodium pyrophosphate, overnight. The next morning, the protein-antibody com- 1 mmol/L sodium vanadate, 1 mmol/L b-glycerophosphate, plex was incubated with 50 mL magnetic protein G beads 1 mg/mL leupeptin, 1 mg/mL pepstatin, 1 mg/mL aprotinin, for 2 h at 4°C with gentle rotation. The antibody-protein- and 1 mmol/L PMSF. beads complex was washed three times with Co-IP buffer. Epitrochlearis muscles used for the dephosphorylation The protein in the complex was then eluted with 30 mLof assay described below were homogenized for 2 min in cold 23 SDS loading buffer and boiled before running on a T-PER buffer supplemented with 1 mg/mL leupeptin, polyacrylamide gel. Proteins were transferred to polyviny- 1 mg/mL pepstatin, 1 mg/mL aprotinin, and 1 mmol/L lidene fluoride membranes, and AS160-associated proteins PMSF, but without phosphatase inhibitors (2.5 mmol/L so- were immunoblotted using antibodies against PP1-a,PP1-b, dium pyrophosphate, 1 mmol/L sodium vanadate, 1 mmol/L PP1-g1, and PP2A. The association of AS160 with PP1-a b-glycerophosphate). Lysates were centrifuged (15,000g, was also assessed by IP using anti–PP1-a, followed by im- 2 min, 4°C), and the resultant supernatants were immedi- munoblotting with anti-AS160. In addition, the association ately used for the dephosphorylation assay described below. of AS160 with several PP1-a regulatory subunits (20–22) AS160 Dephosphorylation Assay was evaluated by immunoprecipitation using anti-AS160, Preliminary insights about the regulation of AS160 followed by immunoblotting with antibodies against spino- dephosphorylation were provided by testing the ability of philin (also known as PPP1R9B), GADD34 (also known as several chemical phosphatase inhibitors, with differing PPP1R15A), phostensin (also known as PPP1R18), and Inh-2 specificity for inhibiting selected protein phosphatases, to (also known as PPP1R2). delay the rate of AS160 dephosphorylation in lysates siRNA Silencing prepared from isolated epitrochlearis muscles. The AS160 L6 cells were transfected with 200 nmol/L control dephosphorylation assay was a modification of a method scrambled siRNA, siPP1-a, siPP1-b, or siPP1-g1 using previously used to identify the serine/threonine phospha- RNAiMAX transfection reagent from Invitrogen using tase that dephosphorylates calcium-binding protein 4 (16) the manufacturer’s instructions, with minor modifica- The muscles used for this AS160 dephosphorylation assay tions. Briefly, cells were seeded in 60-mm plates at 50% were first incubated with 30 nmol/L insulin to ensure confluence in DMEM supplemented with 10% FBS. On initially high levels of AS160 phosphorylation. Freeze- day 2, myoblasts were transfected with 200 nmol/L of clamped muscles were rapidly homogenized in ice-cold siRNA in reduced serum Opti-MEM media without anti- buffer in the absence of protein phosphatase inhibitors. biotics. The media changed 48 h later to DMEM supple- An initial aliquot (20 mL) was rapidly withdrawn from mented with 10% FBS. At 72 h of transfection, the cells each muscle lysate and immediately mixed with an equal were treated without insulin (basal) or with 100 nmol/L volume of 23 SDS loading buffer and heated (95°C, insulin in serum-free medium for 20 min before being 6 min). This initial aliquot was denoted as the 0-min time harvested. The extent of PP1-a, PP1-b, or PP1-g1 knock- point for the AS160 dephosphorylation assay. To the down was assessed by immunoblot. remaining lysate, a chemical protein phosphatase inhibitor (5 nmol/L OA for PP2A inhibition and 1,000 nmol/L OA for Immunoblotting PP1 inhibition (17); 10 mmol/L sanguinarine for PP2C in- Equal amounts of protein from each sample were loaded on hibition (18); or 100 ng/mL FK-506 for PP2B inhibition a Tris glycine acrylamide gel and transferred to polyviny- (19)) or an equal volume of vehicle (DMSO) was rapidly lidene fluoride membrane. The membranes were incubated diabetes.diabetesjournals.org Sharma, Arias, and Cartee 2609

Figure 1—Effects of the PP2B inhibitor FK-506 (100 ng/mL) and the PP2C inhibitor sanguinarine (10 mmol/L) on AS160 phosphorylation in epitrochlearis muscles. The time course for AS160 dephosphorylation was assessed using isolated muscles that were incubated with insulin (30 nmol/L) before being rapidly homogenized. To determine initial AS160 phosphorylation, an aliquot from each lysate was immediately combined with 23 SDS buffer to stop dephosphorylation (denoted as 0 min). Aliquots from the remaining lysate were incubated for 30, 60, or 120 min at 37°C in the presence of vehicle, FK-506, denoted as PP2B (I), or the sanguinarine, denoted as PP2C (I). After each incubation time, lysates were combined with 23 SDS buffer before undergoing immunoblotting: pAS160Thr642 (A); AS160Ser588 (B); representative immunoblots of pAS160Thr642, pAS160Ser588, and total AS160 (C); pAktThr308 (D); pAktSer473 (E); and representative immunoblots of pAktThr308, pAktSer473, and total Akt (F). Values are expressed as mean 6 SEM (n = 4 muscle lysates per treatment). Data were analyzed using one-way ANOVA.

with appropriate primary and secondary antibodies. Im- was identified using Tukey post hoc analysis. A P value munoreactive proteins were detected using Luminata Forte of #0.05 was considered statistically significant. Western HRP Substrate and quantified by densitometry RESULTS (Alpha Innotech, San Leandro, CA). Neither FK-506 nor Sanguinarine Delay AS160 Statistical Analysis Dephosphorylation Statistical analyses were performed using Prism 4.0 Neither the PP2B inhibitor FK-506 nor the PP2C inhibitor software (GraphPad Software, La Jolla, CA). Data are sanguinarine differed from the vehicle for pAS160Thr642 expressed as means 6 SEM. Differences between two (Fig. 1A and C)orpAS160Ser588 (Fig. 1B and C)in groups were evaluated using a two-tailed t test. Differ- epitrochlearis muscle lysates. Similarly, neither inhib- ences between more than two groups were evaluated us- itor differed from vehicle for pAktThr308 (Fig. 1D ing one-way ANOVA. The source of significant variance and F)orpAktSer473 (Fig. 1E and F). These results 2610 PP1-a Regulates AS160 Dephosphorylation Diabetes Volume 65, September 2016

Figure 2—Effects of OA (5 nmol/L for PP2A inhibition or 1,000 nmol/L for PP1 inhibition) on AS160 dephosphorylation in epitrochlearis muscles. The procedure described for Fig. 1 was used except that OA was the phosphatase inhibitor used rather than FK-506 or sanguinarine: pAS160Thr642 (A); AS160Ser588 (B); representative immunoblots of pAS160Thr642, pAS160Ser588, and total AS160 (C); pAktThr308 (D); pAktSer473 (E); and representative immunoblots of pAktThr308, pAktSer473, and total Akt (F). Values are expressed as mean 6 SEM (n = 4 muscle lysates per treatment). Data were analyzed using one-way ANOVA. Tukey post hoc analysis was performed to identify the source of significant variance. *P < 0.05, 1,000 nmol/L OA exceeds both 0 and 5 nmol/L OA at the corresponding time; #P < 0.05, 1,000 nmol/L OA exceeds 0 nmol/L OA at the corresponding time; cP < 0.05, 5 nmol/L exceeds 0 nmol/L OA at the corresponding time.

provide no evidence that PP2B or PP2C regulate AS160 the 1,000 nmol/L values were significantly (P , 0.05) dephosphorylation. greater than vehicle at 30, 60, and 120 min (Fig. 2B and C). For phosphorylation of AktThr308, there were no Dose-Dependent Effects of OA on AS160 significant differences between the 5 nmol/L OA values Dephosphorylation and vehicle at 30, 60, and 120 min, but the 1,000 nmol/L There were no differences for the phosphorylation of values significantly (P , 0.05) exceeded both vehicle and AS160Thr642 of epitrochlearis muscle lysates treated with 5 nmol/L OA values at 30, 60, and 120 min (Fig. 2D and vehicle compared with 5 nmol/L OA, a dose sufficient to F). For phosphorylation of AktSer473, the values with inhibit PP2A but not PP1 (23) at 30, 60, and 120 min. In 5 nmol/L OA were significantly (P , 0.05) greater than contrast, for lysates treated with 1,000 nmol/L OA, a dose vehicle at 30 min, and the 1,000 nmol/L OA values sig- sufficient to inhibit PP1 (23), the phosphorylation of nificantly (P , 0.05) exceeded vehicle and 5 nmol/L val- AS160Thr642 was significantly (P , 0.05) greater than ues at 30, 60, and 120 min (Fig. 2E and F). Taken the vehicle at 30, 60, and 120 min and significantly together, these results suggest that AS160 dephosphory- (P , 0.05) greater than the 5 nmol/L OA values at lation is influenced by OA in a dose-dependent and site- 30 and 60 min (Fig. 2A and C). For phosphorylation of selective manner. Only the higher OA dose delayed AS160Ser588, the values with 5 nmol/L OA significantly dephosphorylation of AS160Thr642, which would be con- (P , 0.05) exceeded the vehicle at 30 and 120 min, and sistent with an effect related to inhibition of PP1. In diabetes.diabetesjournals.org Sharma, Arias, and Cartee 2611

Figure 3—Co-IP of PP1-a, PP1-b, PP1-g1, and PP2A with AS160 in epitrochlearis muscles. Anti-AS160 antibody was used to precipitate AS160 in lysates prepared from isolated muscles that had been incubated with or without insulin, and IgG was used as a control for nonspecific Co-IP. Immune complexes were immunoblotted for AS160, PP1-a, PP1-b, PP1-g1, and PP2A. Representative immunoblots (IBs) (A) and data (B) for the Co-IP of PP1-a with AS160. Representative IBs (C) and data (D) for the Co-IP of PP1-b with AS160. Representative IBs (E) and data (F) for the Co-IP of PP1-g1 with AS160. Representative IBs (G) and data (H) for the Co-IP of PP2A with AS160. Values are expressed as mean 6 SEM (n =3–4 per treatment). Data were analyzed using a two-tailed t test. *P < 0.05, IP using anti- AS160 exceeds IP using IgG control for lysates from muscles incubated without insulin; cP < 0.05, IP using anti-AS160 exceeds IP using IgG control for lysates from muscles incubated with insulin.

contrast, both OA doses delayed dephosphorylation of confirmed by IP using anti–PP1-a, followed by immuno- AS160Ser588, suggesting possible roles of PP2A and/or PP1. blotting with anti-AS160, compared with normal IgG con- trol (data not shown). In contrast, there was no evidence Selective AS160 Co-IP With PP1-a but Not PP1-b, that AS160 had any specific association with PP-b1(Fig.3C PP1-g1, or PP2A and D), PP1-g1(Fig.3E and F), or PP2A (Fig. 3G and H). Because the OA results were not definitive, we used Similar results were obtained for L6 cells in which specific additional approaches to probe the relationship of AS160 interaction was detected for AS160 with PP1-a but not for with PP1 and PP2A in skeletal muscle. Specific interaction AS160 with PP1-b,PP1-g1, or PP2A (data not shown). The between PP1-a and AS160 in epitrochlearis was indicated selective association of AS160 with PP1-a supports the by the significantly (P , 0.05) greater amount of PP1-a idea that PP1-a may regulate AS160 dephosphorylation. that Co-IP with AS160 compared with normal IgG con- The association of AS160 with several PP1-a regulatory trol regardless of insulin concentration (Fig. 3A and B). subunits (spinophilin, GADD34, phostensin, and Inh-2) The specific association between AS160 and PP1-a was was evaluated in epitrochlearis lysates immunoprecipitated 2612 PP1-a Regulates AS160 Dephosphorylation Diabetes Volume 65, September 2016

Figure 4—Effects of rInh-2 on AS160 dephosphorylation in epitrochlearis muscles. The procedure described for Fig. 1 was used except that the specific PP1 inhibitor protein rInh-2 was assessed rather than pharmacologic inhibitors: pAS160Thr642 (A); AS160Ser588 (B); representative immunoblots of pAS160Thr642, pAS160Ser588, and total AS160 (C); pAktThr308 (D); pAktSer473 (E); and representative immu- noblots of pAktThr308, pAktSer473, and total Akt (F). Values are expressed as mean 6 SEM (n = 4 muscle lysates per treatment). Data were analyzed using a two-tailed t test. *P < 0.05, plus rInh-2 exceeds minus rInh-2.

using anti-AS160, followed by immunoblotting with anti- phosphorylation is consistent with PP1 being a modulator bodies against the respective regulatory subunits. How- of AS160 dephosphorylation. ever, this analysis did not detect evidence of selective association of these proteins with AS160 (data not shown). Silencing PP1-a but Not Other PP1 Isoforms Leads to Greater AS160 Phosphorylation rInh-2 Protein Delays AS160 Dephosphorylation Because PP1-a was selectively associated with AS160 based Inh-2 was studied because it is a selective biological on Co-IP, it was also critical to perform a functional assess- inhibitor of PP1 (15). Epitrochlearis lysates incubated ment of the roles of PP1 isoforms on AS160’sphosphory- with rInh-2 protein compared with control lysates had lation status in intact muscle cells. L6 cells transfected with greater (P , 0.05) phosphorylation of AS160Thr642, at siPP1-a had a significant (P , 0.05) reduction in PP1-a 15, 30, 45, and 60 min (Fig. 4A and C). Furthermore, protein abundance compared with cells transfected with rInh-2 treatment versus controls produced greater (P , scrambled control siRNA (Fig. 5A). The specificity of the 0.05) phosphorylation of AS160Ser588 at 15, 30, and PP1-a knockdown in the siPP1-a–transfected cells was evi- 45 min (Fig. 4B and C). Greater AS160 phosphorylation denced by the lack of changes in PP1-b or PP1-g1protein was not accompanied by any significant effects of rInh-2 levels (Fig. 5B). Lower PP1-a abundance led to significantly on the phosphorylation of AktThr308 (Fig. 4D and F)or (P , 0.05) greater phosphorylation of AS160Thr642 (Fig. 5C) AktSer473 (Fig. 4E and F). The delayed AS160 dephosphor- and AS160Ser588 (Fig. 5D) in the insulin-stimulated siPP1-a– ylation on both Thr642 and Ser588 with unaltered Akt transfected cells compared with insulin-stimulated control diabetes.diabetesjournals.org Sharma, Arias, and Cartee 2613

Figure 5—Effects of silencing the PP1-a on AS160 phosphorylation in L6 myocytes. Cells were transfected with siPP1-a or scrambled (Scr) siRNA. At 72 h after transfection, cells were incubated without or with insulin (ins; 100 nmol/L) for 20 min. Protein abundance and phosphorylation were assessed by immunoblotting. PP1-a (Α); representative immunoblots of PP1-a, PP1-b, PP1-g1, and a-tubulin (B); pAS160Thr642 (C); pAS160Ser588 (D); representative immunoblots of AS160Thr642, pAS160Ser588, and AS160 (E); pAktThr308 (F); pAktSer473 (G); and representative immunoblots of pAktThr308, pAktSer473, and Akt (H). Values are expressed as mean 6 SEM (n =5–6 experiments). Data were analyzed using a two-tailed t test. #P < 0.05, siPP1-a is less than Scr for cells incubated without insulin. cP < 0.05, siPP1-a is less than Scr for cells incubated with insulin. *P < 0.05, siPP1-a exceeds Scr for cells incubated with insulin.

cells. Greater AS160 phosphorylation was not attributable AS160 Dephosphorylation in Muscles From LZ to any effect of siPP1-a transfection on the phosphorylation versus OZ Rats Thr642 of AktThr308 (Fig. 5F)orAktSer473 (Fig. 5G). The relative phosphorylation of AS160 was signifi- Transfection of L6 cells with siPP1-b produced a ro- cantly (P , 0.05) lower for epitrochlearis muscle lysates bust and significant (P , 0.05) reduction in PP1-b protein from OZ versus LZ rats at 5 min (Fig. 8A and C). The Ser588 abundance compared with cells transfected with scrambled relative phosphorylation of AS160 was significantly control siRNA (Fig. 6A). The specificity of the PP1-b knock- (P , 0.05) lower for OZ versus LZ at 30 and 40 min (Fig. down was confirmed by the absence of changes in PP1-a or 8B and C). In contrast to AS160 phosphorylation, there PP1-g1abundance(Fig.6B). The lower PP1-b content had no were no significant differences between LZ and OZ rats Thr308 Ser473 detectable effects on the phosphorylation of AS160Thr642 (Fig. for phosphorylation of Akt (Fig. 8D and F)orAkt 6C), AS160Ser588 (Fig. 6D), AktThr308 (Fig. 6F), or AktSer473 (Fig. (Fig. 8E and F). 6G) in siPP1-b–transfected cells compared with control cells. Epitrochlearis muscle lysates from LZ versus OZ did Transfection of L6 cells with siPP1-g1 caused a sub- not differ for protein abundance of AS160 (P = 0.42), stantial and significant (P , 0.05) reduction in PP1-g1 PP1-a (P = 0.78), or Inh-2 (P = 0.27) (data not shown). protein abundance compared with cells transfected with Neither did epitrochlearis muscle lysates from LZ versus the scrambled control siRNA (Fig. 7A). The specificity of OZ differ with regard to the amount of AS160-associated the PP1-g1 knockdown was demonstrated by unchanged PP1-a (P = 0.40) determined by Co-IP (data not shown). PP1-a or PP1-b levels (Fig. 7B). The lower PP1-g1 abun- dance resulted in no changes in the phosphorylation of DISCUSSION AS160Thr642 (Fig. 7C), AS160Ser588 (Fig. 7D), AktThr308 Protein phosphorylation status reflects the balance of (Fig. 7F), or AktSer473 (Fig. 7G)insiPP1-g1–transfected cells phosphorylation by kinases and dephosphorylation by versus control cells. phosphatases. However, there is an extreme disparity in 2614 PP1-a Regulates AS160 Dephosphorylation Diabetes Volume 65, September 2016

Figure 6—Effects of silencing PP1-b on AS160 phosphorylation in L6 myocytes. L6 cells were transfected with siPP1b or scrambled (Scr) siRNA and treated with insulin (ins) as described for Fig. 5. Protein abundance and phosphorylation were assessed by immunoblotting. PP1-b (Α); representative immunoblots of PP1-b, PP1-a, PP1-g1, and a-tubulin (B); pAS160Thr642 (C); pAS160Ser588 (D); representative immunoblots of pAS160Thr642, pAS160Ser588, and total AS160 (E); pAktThr308 (F); pAktSer473 (G); and representative immunoblots of pAktThr308, pAktSer473, and total Akt (H). Values are expressed as mean 6 SEM (n = 4 experiments). Data were analyzed using a two-tailed t test. #P < 0.05, siPP1-b is less than Scr for cells incubated without insulin; cP < 0.05, siPP1-b is less than Scr for cells incubated with insulin.

the level of knowledge about the roles of specific kinases on Thr642. This result is consistent with the possibility compared with phosphatases in the regulation of protein that PP1 regulated AS160 dephosphorylation on this phosphorylation. Although AS160 phosphorylation is a site, but a caveat is that this OA dose also produced key determinant of insulin-stimulated glucose transport, greater Akt phosphorylation. Both OA doses delayed prior research had not identified the serine/threonine AS160 dephosphorylation on Ser588 concomitant with in- protein phosphatases that regulate AS160 dephosphory- creased Akt phosphorylation. Earlier research has indi- lation. The current study tackled this important problem cated that OA can attenuate Akt dephosphorylation using a series of different experimental approaches (24–26). The OA results did not conclusively isolate the leading to the discovery that PP1-a modulates AS160 de- possible roles of PP1 or PP2A for controlling AS160 de- phosphorylation on Ser588 and Thr642 in skeletal muscle. phosphorylation, so we subsequently used additional ex- PP1, PP2A, PP2B, and PP2C are four of the most highly perimental approaches to gain more specific insights. expressed serine/threonine phosphatases in skeletal mus- For our first alternative approach, we used Inh-2 to cle (14). We initially tested AS160 dephosphorylation in specifically assess the role of PP1 in regulating AS160 muscle using several pharmacologic inhibitors that are dephosphorylation. The results from the rInh-2 experi- commonly used to block each of these phosphatases. ment were simpler to interpret because Inh-2 selectively This first screening step provided no evidence that inhibits PP1 without inhibiting PP2A (27) and because PP2B or PP2C inhibitors delayed AS160 dephosphoryla- Inh-2 caused greater phosphorylation on both AS160 sites tion on either AS160 phosphorylation site. OA was tested in the absence of altered Akt phosphorylation. These ob- at two doses because it has differing potency for inhibit- servations implicated PP1 in the regulation of AS160 de- ing PP2A (requiring a lower dose) compared with PP1 phosphorylation on both Ser588 and Thr642. However, (requiring a higher dose) (23). The higher OA dose but because skeletal muscle expresses three PP1 isoforms not the lower OA dose delayed AS160 dephosphorylation (PP1-a, PP1-b, and PP1-g1) and Inh-2 can bind and diabetes.diabetesjournals.org Sharma, Arias, and Cartee 2615

Figure 7—Effects of silencing PP1-g1 on AS160 phosphorylation in L6 myocytes. Cells were transfected with siPP1-g1 or scrambled (Scr) siRNA and treated with insulin (ins) as described for Fig. 5. Knockdown was confirmed by measuring the protein abundance of PP1-g1. Protein abundance and phosphorylation were assessed by immunoblotting. PP1g1(A); representative immunoblots of PP1-g1, PP1-a, PP1-b, and a-tubulin (B); pAS160Thr642 (C); pAS160Ser588 (D); representative immunoblots of pAS160Thr642, pAS160Ser588, and AS160 (E); pAktThr308 (F); pAktSer473 (G); and representative immunoblots of pAktThr308, pAktSer473, and Akt (H). Values are expressed as mean 6 SEM (n = 4 experiments). Data were analyzed using a two-tailed t test. #P < 0.05, siPP1-g1 is less than Scr for cells treated without insulin; cP < 0.05, siPP1-g1 is less than Scr for cells treated with insulin.

inhibit each of these PP1 isoforms (28), it was necessary abundance of the other PP1 isoforms. In contrast to the to next address the possibility of PP1 isoform selectivity. significant effects of PP1-a knockdown, neither PP1-b nor We used two distinct approaches to address the role of PP1-g1 knockdown altered AS160 phosphorylation. different PP1 isoforms. We first performed Co-IP analysis It seems possible that altered serine/threonine dephos- to evaluate the physical association between AS160 and phorylation of AS160 may contribute to attenuated AS160 candidate phosphatases (29). We found that AS160 was phosphorylation that has been reported in insulin-resistant selectively associated with PP1-a and not PP1-b or PP1- muscles. For example, OZ rats compared with LZ rats are g1 in both rat skeletal muscle and L6 myocytes. There was characterized by reductions in insulin-stimulated AS160 also no evidence for specific interaction between AS160 phosphorylation and glucose uptake (30–33). The current and PP2A in rat skeletal muscle or L6 cells. study included the first evaluation of the potential role of The Co-IP analysis demonstrated that PP1-a and AS160 accelerated AS160 dephosphorylation in insulin-resistant are binding partners, but this approach cannot establish if muscles. Results from the AS160 dephosphorylation assay there is a functional relationship between the two proteins. revealed modestly faster dephosphorylation of AS160 on Accordingly, we next turned to siRNA silencing to test for Ser588 and Thr642 in muscle lysates from insulin-resistant PP1 isoform–specific effects. The results of this experiment OZ rats versus LZ controls. These differences were not were invaluable because they clearly revealed that a selective explained by disparities in the abundance of AS160, PP1-a, reduction in PP1-a protein abundance produced greater or Inh-2 protein or in the association between PP1-a and AS160 phosphorylation on Ser588 and Thr642. Importantly, AS160. However, these results do not eliminate the possibil- Akt phosphorylation was unaltered by PP1-a knockdown, so ity of a role for PP1-a in the dysregulation of AS160 phos- the greater AS160 phosphorylation was not an indirect con- phorylationininsulin-resistantmuscle. sequence of greater Akt phosphorylation. Furthermore, PP1-a In conclusion, AS160 plays a pivotal role in insulin’s knockdown was specific, because there were no changes in the regulation of glucose transport in skeletal muscle. Many 2616 PP1-a Regulates AS160 Dephosphorylation Diabetes Volume 65, September 2016

Figure 8—Comparison of AS160 dephosphorylation in epitrochlearis muscle lysates from LZ vs. OZ rats. Isolated muscles from LZ and OZ rats were incubated with insulin (30 nmol/L) for 30 min. The dephosphorylation assay was as described for Fig. 1, except that lysates were incubated at 37°C for 5, 10, 20, 30, and 40 min without the addition of any phosphatase inhibitors. The initial phosphorylation levels for AS160 and Akt were determined for each sample and denoted as 0 min, with a value equal to 100%. The relative phosphorylation values for AS160 and Akt were plotted at each time point: pAS160Thr642 (A); pAS160Ser588 (B); representative immunoblots of pAS160Thr642, pAS160Ser588, and total AS160 (C); pAktThr308 (D); pAktSer473 (E); and representative immunoblots of pAktThr308, pAktSer473, and total Akt (F). Values are expressed as mean 6 SEM (n = 7 animals for each group). Data were analyzed using a two-tailed t test. *P < 0.05 indicates lean exceeds obese at the corresponding times.

previousstudieshavefocusedontheroleofAktintheinsulin- Duality of Interest. No potential conflicts of interest relevant to this article stimulated phosphorylation of AS160. In contrast, the current were reported. study was the first to focus on identifying the serine/threonine Author Contributions. P.S. performed the experiments, analyzed the phosphatases that control the dephosphorylation of AS160. data, designed the experiments, discussed the manuscript, developed the hypothesis, and wrote the manuscript. E.B.A. performed the experiments, We discovered that PP1-a regulates AS160 dephosphoryla- 588 642 analyzed the data, and discussed the manuscript. G.D.C. designed the tion on Ser and Thr , two key sites that control insulin- experiments, coordinated and directed the project, developed the hypothesis, stimulated glucose transport. This knowledge represents an discussed the manuscript, and wrote the manuscript. G.D.C. is the guarantor of essential building block for fully understanding the processes this work and, as such, had full access to all the data in the study and takes that control this key insulin signaling protein that is a crucial responsibility for the integrity of the data and the accuracy of the data analysis. regulator of insulin-stimulated glucose transport. References 1. DeFronzo RA, Jacot E, Jequier E, Maeder E, Wahren J, Felber JP. The effect Acknowledgments. The authors acknowledge Naveen Sharma, PhD, of insulin on the disposal of intravenous glucose. Results from indirect calo- currently of Central Michigan University, for his role in the initial development of rimetry and hepatic and femoral venous catheterization. Diabetes 1981;30:1000– the dephosphorylation assay. 1007 Funding. This research was supported by a grant from the National Institutes 2. Karlsson HK, Zierath JR. Insulin signaling and glucose transport in insulin of Health (R01-DK-071771). resistant human skeletal muscle. Cell Biochem Biophys 2007;48:103–113 diabetes.diabetesjournals.org Sharma, Arias, and Cartee 2617

3. Cartee GD. Roles of TBC1D1 and TBC1D4 in insulin- and exercise-stimulated activity and IgE activation-induced cytokine transcripts, but not exocytosis, in glucose transport of skeletal muscle. Diabetologia 2015;58:19–30 mouse mast cells. J Immunol 1995;154:1846–1851 4. Sakamoto K, Holman GD. Emerging role for AS160/TBC1D4 and TBC1D1 in 20. Esteves SL, Domingues SC, da Cruz e Silva OA, Fardilha M, da Cruz e Silva the regulation of GLUT4 traffic. Am J Physiol Endocrinol Metab 2008;295:E29–E37 EF. Protein phosphatase 1a interacting proteins in the human brain. OMICS 5. Cartee GD, Funai K. Exercise and insulin: convergence or divergence at 2012;16:3–17 AS160 and TBC1D1? Exerc Sport Sci Rev 2009;37:188–195 21. Kao SC, Chen CY, Wang SL, et al. Identification of phostensin, a PP1 F-actin 6. Sano H, Kane S, Sano E, et al. Insulin-stimulated phosphorylation of a Rab cytoskeleton targeting subunit. Biochem Biophys Res Commun 2007;356:594– GTPase-activating protein regulates GLUT4 translocation. J Biol Chem 2003;278: 598 14599–14602 22. Heroes E, Lesage B, Görnemann J, Beullens M, Van Meervelt L, Bollen M. 7. Brautigan DL. Protein Ser/Thr phosphatases–the ugly ducklings of cell The PP1 binding code: a molecular-lego strategy that governs specificity. FEBS signalling. FEBS J 2013;280:324–345 J 2013;280:584–595 8. Shi Y. Serine/threonine phosphatases: mechanism through structure. Cell 23. Bialojan C, Takai A. Inhibitory effect of a marine-sponge toxin, okadaic acid, 2009;139:468–484 on protein phosphatases. Specificity and kinetics. Biochem J 1988;256:283–290 9. Gonzalez E, McGraw TE. Insulin-modulated Akt subcellular localization 24. Thayyullathil F, Chathoth S, Shahin A, et al. Protein phosphatase determines Akt isoform-specific signaling. Proc Natl Acad Sci U S A 2009;106: 1-dependent dephosphorylation of Akt is the prime signaling event in sphingo- 7004–7009 sine-induced apoptosis in Jurkat cells. J Cell Biochem 2011;112:1138–1153 10. Sharma N, Arias EB, Sequea DA, Cartee GD. Preventing the calorie re- 25. Liu W, Akhand AA, Takeda K, et al. Protein phosphatase 2A-linked and striction-induced increase in insulin-stimulated Akt2 phosphorylation eliminates -unlinked caspase-dependent pathways for downregulation of Akt kinase trig- calorie restriction’s effect on glucose uptake in skeletal muscle. Biochim Biophys gered by 4-hydroxynonenal. Cell Death Differ 2003;10:772–781 Acta 2012;1822:1735–1740 26. Borgatti P, Martelli AM, Tabellini G, Bellacosa A, Capitani S, Neri LM. 11. Ng Y, Ramm G, Lopez JA, James DE. Rapid activation of Akt2 is sufficient to Threonine 308 phosphorylated form of Akt translocates to the nucleus of PC12 stimulate GLUT4 translocation in 3T3-L1 adipocytes. Cell Metab 2008;7:348–356 cells under nerve growth factor stimulation and associates with the nuclear 12. Kramer HF, Witczak CA, Fujii N, et al. Distinct signals regulate AS160 matrix protein nucleolin. J Cell Physiol 2003;196:79–88 phosphorylation in response to insulin, AICAR, and contraction in mouse skeletal 27. Hurley TD, Yang J, Zhang L, et al. Structural basis for regulation of protein muscle. Diabetes 2006;55:2067–2076 phosphatase 1 by inhibitor-2. J Biol Chem 2007;282:28874–28883 13. Ikubo M, Wada T, Fukui K, et al. Impact of lipid phosphatases SHIP2 and 28. Alessi DR, Street AJ, Cohen P, Cohen PT. Inhibitor-2 functions like a PTEN on the time- and Akt-isoform-specific amelioration of TNF-alpha-induced chaperone to fold three expressed isoforms of mammalian protein phosphatase-1 insulin resistance in 3T3-L1 adipocytes. Am J Physiol Endocrinol Metab 2009; into a conformation with the specificity and regulatory properties of the native 296:E157–E164 . Eur J Biochem 1993;213:1055–1066 14. Ingebritsen TS, Blair J, Guy P, Witters L, Hardie DG. The protein phos- 29. Virshup DM, Shenolikar S. From promiscuity to precision: protein phos- phatases involved in cellular regulation. 3. Fatty acid synthesis, cholesterol phatases get a makeover. Mol Cell 2009;33:537–545 synthesis and glycolysis/gluconeogenesis. Eur J Biochem 1983;132:275–281 30. Thyfault JP, Cree MG, Zheng D, et al. Contraction of insulin-resistant muscle 15. Eto M, Brautigan DL. Endogenous inhibitor proteins that connect Ser/Thr normalizes insulin action in association with increased mitochondrial activity and kinases and phosphatases in cell signaling. IUBMB Life 2012;64:732–739 fatty acid catabolism. Am J Physiol Cell Physiol 2007;292:C729–C739 16. Haeseleer F, Sokal I, Gregory FD, Lee A. Protein phosphatase 2A dephos- 31. Benton CR, Holloway GP, Han XX, et al. Increased levels of peroxisome phorylates CaBP4 and regulates CaBP4 function. Invest Ophthalmol Vis Sci 2013; proliferator-activated receptor gamma, coactivator 1 alpha (PGC-1alpha) improve 54:1214–1226 lipid utilisation, insulin signalling and glucose transport in skeletal muscle of lean 17. Cohen P, Holmes CF, Tsukitani Y. Okadaic acid: a new probe for the study of and insulin-resistant obese Zucker rats. Diabetologia 2010;53:2008–2019 cellular regulation. Trends Biochem Sci 1990;15:98–102 32. Mackrell JG, Cartee GD. A novel method to measure glucose uptake and 18. Aburai N, Yoshida M, Ohnishi M, Kimura K. Sanguinarine as a potent and myosin heavy chain isoform expression of single fibers from rat skeletal muscle. specific inhibitor of protein phosphatase 2C in vitro and induces apoptosis via Diabetes 2012;61:995–1003 phosphorylation of p38 in HL60 cells. Biosci Biotechnol Biochem 2010;74:548–552 33. Etgen GJ Jr, Wilson CM, Jensen J, Cushman SW, Ivy JL. Glucose transport 19. Fruman DA, Bierer BE, Benes JE, Burakoff SJ, Austen KF, Katz HR. The and cell surface GLUT-4 protein in skeletal muscle of the obese Zucker rat. Am complex of FK506-binding protein 12 and FK506 inhibits phosphatase J Physiol 1996;271:E294–E301