appl1 scaffolds tak1-mkk3-p38 mapk in adiponectin pathway

doi: 10.1152/ajpendo.00427.2010300:E103-E110, 2011. First published 26 October 2010;Am J Physiol Endocrinol Metab

Xiaoban Xin, Lijun Zhou, Caleb M. Reyes, Feng Liu and Lily Q. Dongpathwayactivation by scaffolding the TAK1-MKK3-p38 MAPK APPL1 mediates adiponectin-stimulated p38 MAPK

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APPL1 mediates adiponectin-stimulated p38 MAPK activation by scaffoldingthe TAK1-MKK3-p38 MAPK pathway

Xiaoban Xin,1 Lijun Zhou,2 Caleb M. Reyes,1 Feng Liu,2,3,4 and Lily Q. Dong1,2,4

Departments of 1Cellular and Structural Biology, 2Pharmacology, and 3Biochemistry, and 4The Barshop Center for Longevityand Aging Studies, University of Texas Health Science Center at San Antonio, San Antonio, Texas

Submitted 19 July 2010; accepted in final form 21 October 2010

Xin X, Zhou L, Reyes CM, Liu F, Dong LQ. APPL1 mediatesadiponectin-stimulated p38 MAPK activation by scaffolding theTAK1-MKK3-p38 MAPK pathway. Am J Physiol EndocrinolMetab 300: E103–E110, 2011. First published October 26, 2010;doi:10.1152/ajpendo.00427.2010.—The adaptor protein APPL1 me-diates the stimulatory effect of adiponectin on p38 mitogen-activatedprotein kinase (MAPK) signaling, yet the underlying mechanismremains unclear. Here we show that, in C2C12 cells, overexpression orsuppression of APPL1 enhanced or suppressed, respectively, adi-ponectin-stimulated p38 MAPK upstream kinase cascade, consistingof transforming growth factor-�-activated kinase 1 (TAK1) and mi-togen-activated protein kinase kinase 3 (MKK3). In vitro affinitybinding and coimmunoprecipitation experiments revealed that TAK1and MKK3 bind to different regions of APPL1, suggesting thatAPPL1 functions as a scaffolding protein to facilitate adiponectin-stimulated p38 MAPK activation. Interestingly, suppressing APPL1had no effect on TNF�-stimulated p38 MAPK phosphorylation inC2C12 myotubes, indicating that the stimulatory effect of APPL1 onp38 MAPK activation is selective. Taken together, our study demon-strated that the TAK1-MKK3 cascade mediates adiponectin signalingand uncovers a scaffolding role of APPL1 in regulating the TAK1-MKK3-p38 MAPK pathway, specifically in response to adiponectinstimulation.

APPL1; transforming growth factor-�-activated kinase 1; mitogen-activated protein kinase kinase 3; p38 mitogen-activated proteinkinase

ADIPONECTIN IS AN ADIPOSE-derived hormone that plays an im-portant role in the regulation of energy homeostasis (2, 10, 28).The binding of adiponectin to its membrane receptors, such asAdipoR1 and AdipoR2, leads to the activation of two majorsignal pathways in muscle cells, the AMP-activated proteinkinase (AMPK) and the p38 mitogen-activated protein kinase(MAPK) pathways (16, 27). Activation of these pathways hasbeen shown to be essential for adiponectin-induced glucoseuptake and fatty acid oxidation (16, 27, 28).

APPL1 (adaptor protein containing PH domain, PTB do-main and leucine zipper motif-1) is an adaptor protein contain-ing multiple protein-protein interaction domains and was orig-inally reported as an associating protein that interacts with thecatalytic subunit of phosphatidylinositol 3-kinase (p110) andAkt (17). Our laboratory has recently shown that APPL1mediates adiponectin signaling to activate both the AMPK andthe p38 MAPK signaling pathways (7, 16, 29). However,despite the finding that APPL1 mediates adiponectin-stimu-lated AMPK activation by promoting the cytosolic transloca-

tion of AMPK upstream kinase LKB1 (29), the molecularmechanism underlying APPL1-regulated p38 MAPK activa-tion remains elusive.

The p38 MAPK is a major kinase in the MAPK family andplays essential roles in regulating cell proliferation, inflamma-tion, and immune responses (19). Recent studies suggest thatp38 MAPK acts as an essential mediator in regulating adi-ponectin-induced glucose uptake and fatty acid oxidation inC2C12 myotubes (16, 27). However, the molecular mechanismunderlying adiponectin-stimulated p38 MAPK activation re-mains largely unknown. A kinase cascade consisting of trans-forming growth factor-�-activated kinase 1 (TAK1), mitogen-activated protein kinase kinase (MKK) 3, and MKK6 has beenreported to activate p38 MAPK in response to extracellularstimuli, including growth factors and inflammatory cytokines(18, 19). It was unclear whether adiponectin activates p38MAPK via a similar or a different mechanism.

In the present study, we showed that the TAK1-MKK3kinase pathway mediates adiponectin-stimulated p38 MAPKactivation in C2C12 myotubes. In addition, we demonstratedthat APPL1 serves as a docking platform for scaffolding theTAK1-MKK3-p38 MAPK cascade in response to adiponectinstimulation. Altering the cellular expression level of APPL1had no effect on TNF�-induced activation of the TAK1-MKK3-p38 MAPK kinase pathway, suggesting a selective roleof this adaptor protein in regulating adiponectin signaling.Taken together, our study reveals that the APPL1-TAK1-MKK3 cascade mediates the adiponectin signaling to stimulatep38 MAPK activity in muscle cells.

MATERIAL AND METHODS

Plasmids, adiponectin, and antibodies. The cDNAs encoding full-length and various truncation mutants of human APPL1 were gener-ated by PCR and subcloned into the mammalian expression vectorpcDNA3.1/Myc-His(�)A (Invitrogen) or the bacterial expressionvector pGEX-4T1 (Amersham Pharmacia Biotechnology). The re-combinant globular adiponectin was produced, as described previ-ously (16). The short hairpin RNAs for TAK1 (V2M_193230),MKK3 (V2M_218674), MKK6 (V2M_188205), and AMPK�2(RMM1766_96744125), as well as control vector pSM2c (RHS1704),were purchased from OpenBiosystem (Huntsville, AL). TNF� (cata-log no. T7539) was purchased from Sigma-Aldrich (St. Louis, MO).The antibody to APPL1 and AdipoR1 was generated as describedpreviously (16). All other antibodies were obtained from Cell Signal-ing Technologies (Danvers, MA).

Cell culture. C2C12 myoblasts [from the American Type CultureCollection (ATCC), Manassas, VA] were grown in DMEM (ATCC)supplemented with 10% fetal bovine serum and 1% penicillin-strep-tomycin. Differentiation of C2C12 myoblasts into myotubes wasinduced by growing the cells in low-serum differentiation medium(99% DMEM, 0.1% fetal bovine serum, 1% penicillin-streptomycin,and 100 nM insulin). The medium was changed daily, and multinu-

Address for reprint requests and other correspondence: L. Q. Dong, Dept. ofCellular and Structural Biology, Univ. of Texas Health Science Center at SanAntonio, 7703 Floyd Curl Drive, San Antonio, TX 78229 (e-mail:[email protected]).

Am J Physiol Endocrinol Metab 300: E103–E110, 2011.First published October 26, 2010; doi:10.1152/ajpendo.00427.2010.

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cleated myotubes were normally observed in 3–5 days. APPL1 scram-bled and RNA interference (RNAi) stable cell lines were generated asdescribed previously (16). Control and AMPK�2 RNAi stable celllines were generated by transfecting the cells with pSM2C or pTRIPZ/AMPK�2 RNAi construct and selected by puromycin, following themanufacturer’s protocol (OpenBiosystem, Huntsville, AL).

Immunoprecipitation and Western blot analysis. GST in vitroaffinity binding assays, coimmunoprecipitation, and Western blotexperiments were carried out as described previously (16).

RESULTS

Adiponectin activates p38 MAPK through the TAK1-MKK3kinase cascade. Three kinases, TAK1, MKK3, and MKK6, areknown as major upstream kinases, mediating p38 MAPKactivation induced by extracellular stimuli, including stress and

cytokines (5, 11, 19, 22). To determine whether these kinasesare involved in adiponectin-stimulated p38 MAPK activation,we suppressed their expression levels by RNAi in C2C12

myocytes (Fig. 1A). The adiponectin-induced activations ofp38 MAPK were notably reduced in cells in which the expres-sion levels of TAK1 or MKK3 are suppressed, but not in thecells in which MKK6 is suppressed (Fig. 1A), demonstratingthat the TAK1-MKK3, but not TAK1-MKK6, cascade is es-sential for mediating the stimulatory effect of adiponectin onp38 MAPK activation.

AMPK is not involved in adiponectin-stimulated p38 MAPKactivation. The AMPK has been suggested to be upstream ofp38 MAPK in the ischemic heart (12). Since adiponectinactivates both AMPK and p38 MAPK (16), we investigated

Fig. 1. Transforming growth factor-�-activatedkinase 1 (TAK1) and mitogen-activated proteinkinase kinase 3 (MKK3) are essential in mediat-ing adiponectin (Ad)-induced p38 mitogen-activated protein kinase (MAPK) activation.A: suppression of TAK1 or MKK3, but notMKK6, impaired Ad-induced p38 MAPK acti-vation. C2C12 myocytes with TAK1, MKK3, orMKK6 suppressed by transfecting short hairpinRNA were serum starved for 6 h and treated with1 �g/ml Ad for 10 min. The phosphorylated (P)(Thr180/Tyr182) and the protein levels of p38MAPK, as well as TAK1, MKK3, MKK6, andtubulin, were detected by Western blot analysiswith specific antibodies, as indicated. Graphicpresentation indicated the effect of suppressingTAK1, MKK3, or MKK6 on the Ad-stimulatedp38 MAPK activation shown in Western blotanalysis. Values are means � SE from threeindependent experiments. **P � 0.01. B: sup-pression of AMP-activated protein kinase(AMPK) expression does not affect Ad-stimu-lated p38 MAPK activation. The control orAMPK�2 RNA interference (RNAi) C2C12

myotubes were serum starved overnight andtreated with 1 �g/ml Ad for 10 min. The P-p38MAPK (Thr180/Tyr182) and the protein levels ofp38 MAPK, as well as AMPK�2, were detectedby Western blot analysis with specific antibodies,as indicated. Graphic presentation indicated theeffect of suppressing AMPK on the Ad-stimulated p38 MAPK activation shown inWestern blot analysis. Values are means � SEfrom three independent experiments. **P �0.01. C: Ad sequentially stimulates TAK1,MKK3, and p38 MAPK activities. C2C12 myo-tubes were serum starved overnight and treatedwith 1 �g/ml Ad for different times, as indicated.P-TAK1 (Thr184/187), P-p38 MAPK (Thr180/Tyr182), and their protein levels were detected byWestern blot analysis with specific antibodies, asindicated. MKK3 was immunoprecipitated (IP)with an antibody specific to MKK3, and P-MKK3 was detected by Western blot with anti-body to P-MKK3/6 (Ser189/207). Graphic presen-tation indicated the effect of Ad on activation ofTAK1, MKK3, and p38 MAPK shown in West-ern blot analysis. Values are means � SE fromthree independent experiments. The t-test wasperformed with the activity at each time pointcompared with the basal level. *P � 0.05 and**P � 0.01. Mock, mock control; Ctrl, control;ns, nonsignificant.

E104 APPL1 SCAFFOLDS TAK1-MKK3-p38 MAPK IN ADIPONECTIN PATHWAY

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whether AMPK is involved in the adiponectin-stimulated p38MAPK activation. To this end, we generated C2C12 stable celllines in which AMPK�2 was suppressed by RNAi, whichimpaired the intact activity of AMPK (12, 13, 21, 23, 24). Asshown in Fig. 1B, adiponectin-stimulated p38 MAPK activa-tion was not affected in the AMPK�2-suppressed cells. Thisobservation is consistent with a report that AMPK and p38MAPK pathways are dissociated in skeletal muscle (9).

Adiponectin sequentially activates TAK1, MKK3, and p38MAPK. We next examined whether adiponectin is able toactivate TAK1 and MKK3. Time course studies showed thatadiponectin stimulated TAK1 and MKK3 phosphorylation in atime-dependent manner, and the phosphorylation events oc-

curred sequentially, i.e., TAK1, the kinase at the top of thecascade, was phosphorylated first, whereas p38 MAPK, themost downstream component of the cascade, was phosphory-lated last (Fig. 1C, panels 1, 3, and 5). These results providedfurther evidence that TAK1 and MKK3 are the kinases in-volved in adiponectin-stimulated p38 MAPK activation.

APPL1 is essential for adiponectin-stimulated TAK1-MKK3-p38 MAPK activation. APPL1 has been shown as a keymediator conveying adiponectin signaling toward p38 MAPK(16). To determine whether APPL1 is required for adiponectin-stimulated TAK1-MKK3 activation, we examined the effect ofAPPL1 protein expression on TAK1 and MKK3 phosphoryla-tion in C2C12 myocytes. Overexpression of APPL1 signifi-

Fig. 2. The role of APPL1 in mediating Ad-stimulated activation of TAK1-MKK3-p38 MAPK pathway. A: overexpression of APPL1 enhanced Ad-stimulatedTAK1-MKK3-p38 MAPK cascade activation. C2C12 myocytes overexpressing pcDNA3.1/Myc-His(�)A vector (as a mock control) or pcDNA3.1/Myc-His(�)A/APPL1 were serum starved for 6 h and treated with 1 �g/ml Ad for different times, as indicated. P-TAK1 (Thr184/187) and P-p38 MAPK (Thr180/Tyr182)and their protein levels were detected by Western blot with specific antibodies, as indicated. MKK3 was IP with an antibody specific to MKK3, and P-MKK3 wasdetected by Western blot with antibody to P-MKK3/6 (Ser189/207). Graphic presentation indicated the effect of overexpression of APPL1 on Ad-stimulated activationof TAK1, MKK3, and p38 MAPK shown in the Western blot. Values are means � SE from three independent experiments. The t-test was performed by comparingthe activities between mock and overexpression group at each time point. *P � 0.05 and **P � 0.01. B: suppression of APPL1 expression impaired Ad-inducedTAK1-MKK3-p38 MAPK cascade activation. The scrambled control or APPL1 RNAi C2C12 myotubes were serum starved overnight and treated with 1 �g/mlAd for different times, as indicated. P-TAK1 (Thr184/187), P-p38 MAPK (Thr180/Tyr182), and their protein levels were detected by Western blot with specificantibodies, as indicated. MKK3 was IP with an antibody specific to MKK3, and P-MKK3 was detected by Western blot with antibody to P-MKK3/6 (Ser189/207).Graphic presentation indicated the effect of suppressing APPL1 expression on Ad-stimulated activation of TAK1, MKK3, and p38 MAPK shown in the Westernblot. The t-test was performed by comparing the activities between the scramble and APPL1-RNAi group at each time point. Values are means � SE from threeindependent experiments. *P � 0.05. au, Arbitrary units.

E105APPL1 SCAFFOLDS TAK1-MKK3-p38 MAPK IN ADIPONECTIN PATHWAY


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cantly enhanced basal TAK1, MKK3, and p38 MAPK phos-phorylation, but had little effect on adiponectin-stimulatedphosphorylation due to high basal phosphorylation of thesekinases (Fig. 2A, panels 1, 3, and 5, lane 5 vs. lane 4). Theeffect of APPL1 overexpression on the basal level of p38MAPK (Fig. 2A, panel 5, lane 4) is consistent with what ourlaboratory reported previously (16). In addition, we also found

that overexpression of APPL1 led to significant enhancementof basal activities of the kinases upstream of p38 MAPK (Fig.2A, panels 1 and 3, lane 4). A possible explanation could bethat a limited amount of endogenous APPL1 interacts with theadiponectin receptors, and the majority of the APPL1 is se-questrated away from the receptors by APPL2 under basalconditions (25). Overexpression of APPL1 disturbs the balance



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between APPL1 and APPL2 in cells, leading to excess amountof free APPL1 binding with the adiponectin receptors andsubsequently activating the TAK1-MKK3-p38 MAPK path-way downstream of the adiponectin receptors. Conversely,suppressing APPL1 expression by RNAi significantly impairedadiponectin-stimulated phosphorylation of TAK1, MKK3, andp38 MAPK (Fig. 2B). However, suppression of APPL1 expres-sion is not sufficient to completely inhibit adiponectin-stimu-lated TAK1-MKK3-p38 MAPK activation (Fig. 2B), suggest-ing that an APPL1-independent pathway may also contribute toadiponectin-stimulated p38 MAPK activation.

APPL1 plays a selective role in adiponectin and TNF�-stimulated p38 MAPK activation. TNF� has been shown tostimulate p38 MAPK activation in C2C12 cells (Ref. 6 and Fig. 3A,lanes 2–4). However, suppressing the expression levels ofAPPL1 had little effect on TNF�-induced TAK1, MKK3, orp38 MAPK activation in C2C12 myotubes (Fig. 3A, lanes 6–8vs. lanes 2–4), indicating that adiponectin and TNF� activatep38 MAPK through distinct mechanisms, and that the effect ofAPPL1 on p38 MAPK cascade is highly selective to adiponec-tin stimulation.

There are two major isoforms, � and �, of p38 MAPK inskeletal muscle cells (19), and the �-isoform of the kinase isselectively stimulated by TNF� (6). To determine whether thestimulatory effect of adiponectin is also selective, we examinedthe phosphorylation of the �- and �-isoforms of p38 MAPKimmunoprecipitated from C2C12 cells treated with adiponectinor TNF�. Consistent with previous report (6), TNF� specifi-cally activated the �-isoform, but not the �-isoform, of p38MAPK (Fig. 3B, lane 3, panels 1 vs. 3). On the other hand,adiponectin treatment stimulated the phosphorylation of bothisoforms (Fig. 3B, lane 2, panels 1 vs. 3). These data suggestthat the �-isoform selectively mediates adiponectin, but notTNF� signaling, in skeletal muscle cells.

We next investigated the effects of APPL1 on adiponectin-or TNF�-stimulated activation of the �- and/or �-isoforms ofp38 MAPK. As shown in Fig. 3C, suppression of APPL1expression led to a significant inhibition of adiponectin-stim-ulated activation of both isoforms (panels 1 and 3, lane 4 vs.lane 2). In contrast, suppression of APPL1 expression had noeffect on TNF�-induced activation of the �-isoform (Fig. 3D,panel 1, lane 4 vs. lane 2). Together, these data suggested thatAPPL1 selectively mediates adiponectin, but not TNF� signal,to activate both �- and �-isoforms of p38 MAPK.

APPL1 promotes p38 MAPK activation by scaffoldingTAK1, MKK3, and p38 MAPK. APPL1 consists of multipleprotein-protein interaction domains, suggesting that this pro-tein may function as an adaptor or a scaffold protein in avariety of pathways (7). Consistent with this hypothesis, GST-APPL1 fusion protein, but not GST itself, interacted withendogenous TAK1, MKK3, and MKK6 (Fig. 4A, panels 1–3,lane 2 vs. lane 1). In addition, GST-APPL1(BAR-PH), amutated form of APPL1 with a truncation in the COOH-terminal region (16), interacted with MKK3, but not TAK1 andMKK6 (Fig. 4A, panels 1–3, lane 3), suggesting that TAK1and MKK6 bind to the regions on APPL1 different fromMKK3. The BAR-PH truncation of APPL1, which is unable tobind to the adiponectin receptors, has been shown to act as adominant negative mutant that inhibits adiponectin-stimulatedp38 MAPK activation (16). The deficiency of APPL1(BAR-PH) mutant in the interaction with TAK1 could also contributeto its inhibitory role in adiponectin-induced p38 MAPK acti-vation.

By coimmunoprecipitation experiments, we found that en-dogenous APPL1 interacts with TAK1 and weakly associateswith AdipoR1, MKK3, and p38 MAPK in C2C12 myotubesunder the basal condition (Fig. 4B, panels 1–4, lane 3).Adiponectin treatment enhanced the interaction betweenAPPL1 and TAK1 (Fig. 4B, panel 2, lane 4 vs. lane 3) andinduced a binding of the APPL1-TAK1 complex with AdipoR1(Fig. 4B, panel 1, lanes 4 and 5 vs. lane 3), which, subse-quently, resulted in a sequential recruitment of MKK3 and p38MAPK onto AdipoR1-APPL1-TAK1 complex (Fig. 4B, panels3 and 4, lanes 5 and 6 vs. lane 3). Interestingly, the time courseof the interaction between APPL1 with TAK1, MKK3, and p38MAPK is correlated with adiponectin-stimulated activation ofthese kinases (Figs. 4B vs. 1C), suggesting the presence of adynamic mechanism by which adiponectin activates p38MAPK via promoting the interaction of APPL1 with all signalcomponents in the p38 MAPK pathway.

It was noted that, while endogenous p38 MAPK is coimmu-noprecipitated with APPL1 under the basal condition (Fig. 4B,panel 4, lane 3), we were unable to detect the interactionbetween p38 MAPK and APPL1 by in vitro binding assays(data not shown), implying that postmodification of APPL1could play an essential role in the interaction of APPL1 withp38 MAPK or p38 MAPK binds with APPL1 indirectly incells. Since the APPL1(BAR-PH) truncation mutant binds to

Fig. 3. APPL1 is not involved in mediating TNF�-induced TAK1-MKK3-p38 MAPK activation. A: the scrambled control or APPL1 RNAi C2C12 myotubes wereserum starved overnight and treated with TNF� (1 nM) for different times, as indicated. P-TAK1 (Thr184/187), P-p38 MAPK (Thr180/Tyr182), and their proteinlevels were detected by Western blot with specific antibodies, as indicated. MKK3 was IP with an antibody specific to MKK3, and P-MKK3 was detected byWestern blot analysis with antibody to P-MKK3/6 (Ser189/207). Graphic presentation indicated the effect of suppressing APPL1 expression on TNF�-stimulatedactivation of TAK1, MKK3, and p38 MAPK shown in the Western blot. Values are means � SE from three independent experiments. The t-test was performedby comparing the activities between scramble and RNAi groups at each time point. B: the effects of Ad and TNF� on the activation of �- and �-isoforms ofp38 MAPK in C2C12 myotubes. C2C12 myotubes were serum starved overnight and treated with either Ad (1 �g/ml, 10 min) or TNF� (1 nM, 10 min). The �-and �-isoforms of p38 MAPK were IP, and P-p38 MAPK (Thr180/Tyr182) was detected by Western blot analysis with antibodies, as indicated. Graphicpresentation indicated the effect of Ad or TNF� on activation of �- and �-isoforms of p38 MAPK shown in the Western blot. Values are means � SE from threeindependent experiments. *P � 0.05. C: suppression of APPL1 impaired the effect of Ad on activation of the �- and �-isoform of p38 MAPK. The scrambledcontrol or APPL1 RNAi C2C12 myotubes were serum starved overnight and treated with 1 �g/ml Ad for 10 min. The �- and �-isoforms of p38 MAPK wereIP, and the P-p38 MAPK (Thr180/Tyr182) was detected by Western blot analysis with specific antibodies, as indicated. Graphic presentation indicates the effectof suppression of APPL1 on Ad-stimulated activation of the �- and �-isoforms of p38 MAPK shown in the Western blot. Values are means � SE from threeindependent experiments. *P � 0.05. D: suppression of APPL1 had no effect of TNF� on activation of the �-isoform of p38 MAPK. The scrambled controlor APPL1 RNAi C2C12 myotubes were serum starved overnight and treated with TNF� (1 nM) for 10 min. The �-isoform of p38 MAPK was IP, and the P-p38MAPK (Thr180/Tyr182) was detected by Western blot analysis with specific antibodies, as indicated. Graphic presentation indicated the effect of suppression ofAPPL1 on TNF�-stimulated activation of �-isoform of p38 MAPK shown in the Western blot. Values are means � SE from three independent experiments.*P � 0.05.



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MKK3 but not TAK1 (Fig. 4A, panel 1 vs. panel 2, lane 3), wetested whether altering the expression levels of this mutantactivates MKK3. However, we found that overexpression ofthe APPL1(BAR-PH) truncation mutant was unable to activateMKK3 (data not shown), suggesting that an optimized bindingand/or topological orientation of all components in TAK1-MKK3-p38 MAPK cascade is necessary for adiponectin-in-duced activation of MKK3. This observation is also consistentwith our previous finding that overexpression of theAPPL1(BAR-PH) mutant does not activate p38 MAPK acti-vation in C2C12 myocytes (16).

DISCUSSION

Previous studies showed that adiponectin activates p38MAPK through AdipoR1- and APPL1-dependent mechanism(16, 27). However, the underlying mechanism remains un-known. In this study, we demonstrated that the TAK1-MKK3pathway mediates adiponectin-stimulated p38 MAPK activa-tion, and that APPL1 functions as a scaffolding protein to forma complex with TAK1, MKK3, and p38 MAPK in musclecells, leading to selective and effective activation of thiscascade in response to adiponectin (Fig. 5).

Our study has demonstrated that APPL1 scaffolds TAK1and MKK3 via different regions (Fig. 4A). While severalMKKs have been shown to activate p38 MAPK in response to

TNF� and UV stimulation (18, 19), we found that adiponectin-stimulated p38 MAPK activation selectively requires MKK3 inmuscle cells (Figs. 1A). Interestingly, we found that MKK6 canalso interact with APPL1 in vitro (Fig. 4A, panel 3), althoughit is not involved in adiponectin-stimulated p38 MAPK acti-vation (Fig. 1A). This result suggests that MKK6 may beinvolved in another signaling event that is also mediated byAPPL1. Recently, the TAK1-MKK6-p38MAPK axis was re-ported to be essential for the differentiation of C2C12 myocytesinduced by low serum (3). Interestingly, the APPL1-mediatedp38 MAPK activation is also involved in C2C12 myocytedifferentiation (1). Therefore, it is possible that the interactionbetween APPL1 and MKK6 plays a role in myogenesis.Further investigations will be needed to test this possibility.

In the present study, we have found that adiponectin stim-ulation leads to a multiprotein complex formation (APPL1,AdipoR1, TAK1, MKK3, and p38 MAPK) (Fig. 4B), resultingin chronological activation of the TAK1-MKK3-p38 MAPKkinase cascade (Figs. 1C and 5). Interestingly, once activated,the components in this cascade dissociate from APPL1 (Figs.4B and 5), followed by dephosphorylation of these kinases(Figs. 1C and 5). A possible explanation for these findings isthat the interaction with APPL1 ensures timely activation ofthis cascade and prevents dephosphorylation of these kinasesfrom the action of a protein phosphatase(s). Thus APPL1 acts

Fig. 4. APPL1 acts as a scaffold protein byinteracting with TAK1-MKK3-p38 MAPK.A: APPL1 interacts with TAK1, MKK3, andMKK6 in vitro. GST or GST-APPL1 (full-length, BAR-PH or CT truncations) fusion pro-tein was incubated with cell lysates of C2C12

myotubes. Endogenous TAK1, MKK3, andMKK6 associated with recombinant GST-APPL1 (full-length or truncations) and their pro-tein levels in the lysates were detected by West-ern blot analysis with specific antibodies, as in-dicated. Graphic presentation indicated thebinding affinity between APPL1 fusion proteinswith TAK1, MKK3, and MKK6 shown in theWestern blot. Values are means � SE from threeindependent experiments. *P � 0.05 and **P �0.01. B: the effect of Ad on the interactions ofAPPL1 with Ad receptor 1 (AdipoR1), TAK1,MKK3, and p38 MAPK in cells. C2C12 myo-tubes were serum starved overnight and treatedwith 1 �g/ml Ad for the indicated time. Endog-enous APPL1 was IP with the antibody specificto APPL1. Coimmunoprecipitated (Co-IP) Adi-poR1, TAK1, MKK3, and p38 MAPK, as wellas their protein levels in the cell lysates, weredetected by Western blot analysis with specificantibodies, as indicated. Graphic presentation in-dicated the effect of Ad on the interaction ofAPPL1 with AdipoR1, TAK1, MKK3, and p38MAPK shown in the Western blot. The t-test wasperformed by comparing the affinity with thebasal conditions. Values are means � SE fromthree independent experiments. *P � 0.05.



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as a docking platform to dynamically and efficiently regulatethe TAK1-MKK3-p38 MAPK kinase cascade in response toadiponectin stimulation (Fig. 5).

The data from the affinity-binding assay suggest that p38MAPK was unable to bind with GST-fused APPL1 under invitro conditions (data not shown), although endogenous p38MAPK was coimmunoprecipitated with APPL1 under basalconditions (Fig. 4B). One possible explanation is that post-translational modification on APPL1 may contribute to thebinding with p38 MAPK in cells. Alternatively, p38 MAPKmay bind to the NH2-terminus of APPL1, and the GST proteinfused to the NH2-terminus of APPL1 may interrupt this bind-ing. The other possibility is that MKK3 acts as a “carrier” tobring p38 MAPK onto the APPL1-MKK3 complex in responseto adiponectin stimulation. Together, our study indicates thatAPPL1 protein is essential for controlling adiponectin-inducedTAK1-MKK3-p38 MAPK cascade activation, which is ahighly dynamic process in cells.

It has been reported that AMPK functions as an upstreamkinase of p38 MAPK in regulating glucose uptake stimulatedby stretch and AICAR, however, it is still controversialwhether AMPK-stimulated p38 MAPK activation is a commonmechanism in skeletal muscle (4, 9, 12, 26). Suppression ofAMPK�2 expression significantly affected AMPK activity andimpaired the ischemia-induced p38 MAPK activation in isch-emic heart (12), suggesting a role of AMPK�2 in the activationof p38 MAPK. To test whether a similar mechanism is in-volved in adiponectin-induced p38 MAPK activation, we gen-erated a stable C2C12 cell line in which the expression levels ofthe AMPK�2 subunit are remarkably suppressed by RNAi(13). As shown in Fig. 1B, suppressing the expression of the�2-subunit of AMPK, a subunit essential for intact AMPKactivity (12, 13, 21, 23, 24), had no significant effect on thestimulatory role of adiponectin in p38 MAPK activation inC2C12 cells (Fig. 1B), suggesting that AMPK is dispensable foradiponectin-stimulated p38 MAPK activation in C2C12 myo-tubes. Thus the involvement of AMPK in p38 MAPK activa-tion may be a tissue- and pathway-specific event. Combinedwith the evidence that TAK1-MKK3 pathway transmits adi-ponectin signaling to p38 MAPK (Fig. 1A), our data demon-strate that the TAK1-MKK3 axis, but not AMPK, functions as

a major regulator, mediating the effect of adiponectin towardp38 MAPK.

The p38 MAPK is a vital enzyme in maintaining metabolichomeostasis (14), and its activation is essential for exercise-,insulin-, and adiponectin-induced glucose and lipid utilization(8, 14–16, 27). However, aberrant activation of p38 MAPKcould contribute to chronic inflammation-induced insulin re-sistance (6). Since both adiponectin and TNF� can activate p38MAPK (6, 16), while adiponectin acts as an anti-inflammatoryfactor by inhibiting the expression and activity of TNF� invivo (20), it implies that the activity of p38 MAPK is differ-ently regulated by adiponectin and TNF� in cells. Consistentwith this, our data showed that adiponectin activates both ofthe �-and the �-isoforms of p38 MAPK, whereas TNF� onlystimulates the �-isoform of this kinase in muscle cells (Fig.3B). In addition, we found that APPL1 specifically mediatesadiponectin- but not TNF�-induced p38 MAPK activation(Fig. 3).

In summary, we have demonstrated TAK1-MKK3 as theupstream kinases for mediating adiponectin signal to stim-ulate p38 MAPK activity in muscle cells. In addition, wehave shown that APPL1 acts as a scaffolding protein toorchestrate the activities of the TAK1-MKK3-p38 MAPK.Furthermore, we have demonstrated that the scaffoldingaction of APPL1 on TAK1-MKK3-p38 MAPK cascade isselective to adiponectin, but not TNF�. Identification of TAK1and MKK3 as new signal components in adiponectin pathwayand elucidation of the mechanism by which APPL1 scaffoldsTAK1-MKK3-p38 MAPK pathway should provide importantinformation for understanding the molecular mechanism ofadiponectin action and identification of novel targets of insulinresistance and associated metabolic diseases.

ACKNOWLEDGMENTS

We thank Derong Hu for excellent technical assistance.

GRANTS

This work was supported in part by National Institute of Diabetes andDigestive and Kidney Diseases grants (RO1 DK69930 to L. Q. Dong and R01DK76902 to F. Liu) and a Pre-doctoral Fellowship from the American HeartAssociation (10PRE3180019 to X. Xin).

Fig. 5. A model of APPL1-regulated TAK1-MKK3-p38 MAPK pathway in response toAd stimulation. C’, indicates the COOH ter-minus of AdipoR1. Under the basal condi-tion, APPL1 binds with inactive TAK1 andweakly associates with MKK3 and p38MAPK. On Ad stimulation, APPL1 interactswith AdipoR1, leading to activation ofTAK1 and, subsequently, recruitment ofMKK3 and p38 MAPK to form a complexconsisting of AdipoR1, APPL1, TAK1,MKK3, and p38 MAPK. Once MKK3 isactivated, TAK1 dissociates from APPL1,and the activity of TAK1 is rapidly down-regulated. This process occurs concurrentlywith the dissociation of APPL1 complexfrom AdipoR1 and, in turn, stimulates thedownstream events regulated by TAK1-MKK3-p38 MAPK pathway.



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DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

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