serine/threoninekinase40(stk40)functionsasanovel ... · either one impaired the formation of...

Serine/Threonine Kinase 40 (Stk40) Functions as a NovelRegulator of Skeletal Muscle Differentiation*□S

Received for publication, February 4, 2016, and in revised form, November 8, 2016 Published, JBC Papers in Press, November 29, 2016, DOI 10.1074/jbc.M116.719849

Ke He‡1, Jing Hu‡1, Hongyao Yu‡, Lina Wang‡, Fan Tang‡, Junjie Gu‡, Laixiang Ge‡, Hongye Wang§, Sheng Li§,Ping Hu§, and Ying Jin‡¶2

From the ‡Laboratory of Molecular Developmental Biology, Shanghai Jiao Tong University School of Medicine, 280 SouthChongqing Road, Shanghai 200025, China, the ¶Key Laboratory of Stem Cell Biology, Chinese Academy of Sciences Center forExcellence in Molecular Cell Science, Institute of Health Sciences, Shanghai Institute for Biological Sciences, Chinese Academy ofSciences, 320 Yueyang Road, Shanghai 200031, China, and the §Institute of Biochemistry and Cell Biology, Shanghai Institute forBiological Sciences, Chinese Academy of Sciences, 320 Yueyang Road, Shanghai 200032, China

Edited by John M. Denu

Skeletal muscle differentiation is a precisely coordinated pro-cess, and the molecular mechanism regulating the processremains incompletely understood. Here we report the identifi-cation of serine/threonine kinase 40 (Stk40) as a novel positiveregulator of skeletal myoblast differentiation in culture and fetalskeletal muscle formation in vivo. We show that the expressionlevel of Stk40 increases during skeletal muscle differentiation.Down-regulation and overexpression of Stk40 significantlydecreases and increases myogenic differentiation of C2C12myoblasts, respectively. In vivo, the number of myofibers andexpression levels of myogenic markers are reduced in the fetalmuscle of Stk40 knockout mice, indicating impaired fetal skele-tal muscle formation. Mechanistically, Stk40 controls the pro-tein level of histone deacetylase 5 (HDAC5) to maintain tran-scriptional activities of myocyte enhancer factor 2 (MEF2), afamily of transcription factor important for skeletal myogenesis.Silencing of HDAC5 expression rescues the reduced myogenicgene expression caused by Stk40 deficiency. Together, our studyreveals that Stk40 is required for fetal skeletal muscle develop-ment and provides molecular insights into the control of theHDAC5-MEF2 axis in skeletal myogenesis.

Skeletal muscle differentiation occurs in both normal muscledevelopment and muscle regeneration in the postnatal period.Skeletal muscle development in the mouse initiates fromembryonic day 8.5/9 (E8.5/9)3 to birth (�19 days), followed byfurther maturation for about 2–3 weeks after birth (1, 2). Satel-lite cells are responsible for the regeneration of adult skeletal

muscle and undergo activation, proliferation, and terminal dif-ferentiation after injury (3). Terminal differentiation of musclecells during muscle development and regeneration consists ofseveral processes, including cell cycle exit of mononucleatedmyoblasts, myogenic gene expression, and fusion of myocytesto multinucleated myotubes (4, 5).

The major transcription factors modulating skeletal myo-genesis are myogenic regulatory factors (MRFs), a family shar-ing a common basic helix-loop-helix domain. The members ofthe family, including MyoD, Myogenin, Myf5, and MRF4, canform heterodimers with E proteins to bind the E box sequencepresent in the regulatory region of skeletal muscle-specificgenes (6 – 8). Notably, MyoD can convert non-muscle cells,such as mesenchymal stem cells of the C3H10T1/2 line, intomyotubes (9). Another important family of transcription fac-tors regulating skeletal myogenesis is myocyte enhancer factor2 (MEF2), which works as the coactivator of the MRF family toactivate myogenic gene expression (10 –13). The family ofMEF2 contains four members, MEF2A, B, C, and D, in verte-brates and has a common DNA-binding MCM1, agamous, defi-ciens, and serum-response factor (MADS)/MEF2 domain,forming homo- and heterodimers with coactivators and core-pressors as well as its own family members (11). Muscle-spe-cific knockout of Mef2c causes some defects in skeletal muscledevelopment, including myofiber disarray and sarcomere dis-organization (14). Mef2a-null mice show delayed muscle regen-eration (15). Conditional triple knockout of Mef2a, c, and d insatellite cells impairs muscle regeneration (16). Interestingly,Mef2c is the direct target of the MRF and MEF2 families. Hence,MEF2C regulates its own expression during skeletal muscledevelopment (17), consistent with the autoregulatory activity ofDrosophila MEF2 (18).

Numerous coactivators and corepressors of MEF2 have beenreported. Class IIa histone deacetylases (HDACs), includingHDAC4, 5, 7, and 9, control muscle gene expression, acting ascorepressors of MEF2. Among these, cellular localization andprotein levels of HDAC5 are known to influence its repressiveeffect on the transcriptional activity of MEF2. HDAC5 shuttlesbetween the nucleus and cytoplasm, depending on its phosphor-ylation at the conserved serine residues. Calcium/calmodulin-dependent protein kinase phosphorylates HDAC5 at Ser-259

* This study was supported by grants from the Ministry of Science and Tech-nology of China (2016YFA0100100 and 2013CB966801), the National Nat-ural Science Foundation (31301015 and 91419309), and the Chinese Acad-emy of Sciences (XDB19020100). The authors declare that they have noconflicts of interest with the contents of this article.

□S This article contains supplemental Table 1.1 Both authors contributed equally to this work.2 To whom correspondence should be addressed: Laboratory of Molecular

Developmental Biology, Shanghai Jiao Tong University School of Medi-cine, 280 South Chongqing Rd., Shanghai 200025, China. E-mail: [email protected]. Tel.: 86-21-54923342.

3 The abbreviations used are: E, embryonic day; MEF, myocyte enhancer fac-tor; HDAC, histone deacetylase; MyHC, myosin heavy chain; P, postnatalday; MCK, muscle creatine kinase; TA, tibialis anterior; RT-qPCR, quantita-tive RT-PCR.

crossmarkTHE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 292, NO. 1, pp. 351–360, January 6, 2017

© 2017 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

JANUARY 6, 2017 • VOLUME 292 • NUMBER 1 JOURNAL OF BIOLOGICAL CHEMISTRY 351

by guest on August 6, 2020

http://ww

w.jbc.org/

Dow

nloaded from

mailto:[email protected]

mailto:[email protected]

http://crossmark.crossref.org/dialog/?doi=10.1074/jbc.M116.719849&domain=pdf&date_stamp=2016-11-29

http://www.jbc.org/

and Ser-498, resulting in the nuclear export of HDAC5 and, inturn, relieving its repression on MEF2 (19 –22). Moreover,HDAC5 can be ubiquitinated and degraded by the proteasomepathway in the nucleus of C2C12 cells. MEF2 activationdecreases when HDAC5 protein levels increase because of theblock of proteasomes (23), indicating that the nuclear proteinlevel of HDAC5 negatively controls MEF2 transcriptional activ-ity. However, the regulatory mechanism for the control of theHDAC5 level is not clearly understood.

Stk40, a putative serine/threonine kinase, can activate theErk/MAPK pathway to induce mouse embryonic stem cell dif-ferentiation into the extraembryonic endoderm (24). Stk40knockout mice suffer from immature lung development andneonatal lethality at birth (25). Besides, Stk40 represses adipo-genesis through controlling the translation of CCAAT/en-hancer binding proteins (C/EBP) proteins (26). Thus, the func-tion of Stk40 is multifarious. Here we find that the expression ofStk40 is positively related to MEF2 transcriptional activities butinversely correlated to the levels of HDAC5. Concomitantly,Stk40 is required for skeletal myogenic differentiation both invitro and in vivo. Therefore, our study sheds light on the regu-latory mechanism for the HDAC5 protein level, as well as for

the MEF2 transcriptional activity and myogenesis. In addition,the findings uncover an important function of Stk40 in skeletalmuscle development.

Results

Stk40 Expression Levels Increase during Skeletal Muscle Dif-ferentiation and Regeneration—To learn about the involvementof Stk40 in skeletal myogenesis, we began with an examinationof Stk40 expression patterns in both in vivo and in vitro mod-els of skeletal muscle differentiation. First, we used the C2C12myoblast line, a well established in vitro model for studyingskeletal muscle differentiation (27). Efficient myogenic differ-entiation of C2C12 myoblasts was demonstrated by the induc-tion of myogenic transcription factors, including Myogenin andMEF2C, as well as their downstream target myosin heavy chain(MyHC) (Fig. 1A). Simultaneously, protein expression of Stk40significantly increased after the induction of differentiation(Fig. 1, A and B). However, the mRNA level of Stk40 increasedslightly (Fig. 1C). Second, we examined the expression of Stk40in developing skeletal muscle in vivo. In this regard, hind limbmuscles at the fetal (E16.5 and E18.5), perinatal (postnatal day2, P2) and adult (postnatal week 8, P8 week) stages were isolated

FIGURE 1. Stk40 expression levels increase during skeletal muscle differentiation in vitro and in vivo. A, protein levels of Stk40, MyHC, Myogenin, andMEF2C at the indicated time points of C2C12 cell differentiation (Diff) were analyzed by Western blotting. �-Tubulin was used as a loading control. The meangray values of Stk40 analyzed by ImageJ are listed below the Stk40 blot. B, -fold changes of the mean gray values of the protein levels of Stk40 on differentiationdays 0 and 2. The mean gray values were analyzed by ImageJ. Data were normalized to the level of �-tubulin. Error bars represent S.D; Student’s t test; ***, p �0.001. C, the mRNA level of Stk40 at the indicated time points of C2C12 cell differentiation was detected by RT-qPCR assays. Data were normalized to the levelof GAPDH. Error bars represent S.D. D, hind limb muscles were isolated from E16.5, E18.5, P2, and P8 week embryos. Protein levels of Stk40, Myogenin, andMEF2C in the hind limb muscle at the indicated time point of muscle development were analyzed by Western blotting. GAPDH was used as a loading control.E, Protein levels of Stk40 in different tissues and organs isolated from mouse embryos at E18.5 were analyzed by Western blotting. GAPDH was used as a loadingcontrol. F, protein levels of Stk40, Myogenin, and MEF2C in TA muscle treated with CTX for 0, 3, 5, 9, and 14 days, respectively, were analyzed by Westernblotting. GAPDH was used as a loading control.

Stk40 Regulates Skeletal Myogenesis

352 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 292 • NUMBER 1 • JANUARY 6, 2017


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

from C57BL/6 mice. Similar to Myogenin and MEF2C, thesteady-state levels of Stk40 proteins were highest in the musclefrom E16.5 embryos and gradually declined afterward (Fig. 1D).By P8 week, little Stk40 could be detected in the muscle. Nev-ertheless, Stk40 was not specifically expressed in the muscle atfetal stage. It was ubiquitously expressed in various tissues andorgans at E18.5, including skeletal muscle tissues such as thetongue, diaphragm, and hind limb muscle as well as the kidney,lung, heart, thymus, and so on (Fig. 1E). In addition, the myo-genic process also takes place during muscle regeneration afterinjury. Cardiotoxin (CTX) treatment induces the muscle

degeneration and regeneration program (28). Interestingly,expression of Stk40 was also induced during this process, in amanner similar to that of MEF2C (Fig. 1F). These results indi-cate that the expression of Stk40 is developmentally regulatedin the skeletal muscle and that it is potentially involved in skel-etal muscle differentiation.

Stk40 Positively Regulates Skeletal Myogenic Differentiation—To study the function of Stk40 in myogenesis, we knockeddown Stk40 in C2C12 myoblasts by specific shRNA deliveredvia retroviral plasmids. Two independent shRNA sequenceswere used (Stk40 shRNA-1 and shRNA-2), and expression of

FIGURE 2. Stk40 positively regulates the myogenic differentiation of C2C12 myoblasts. A, knockdown of Stk40 by two different shRNAs, respectively, viaretroviral delivery in C2C12 myoblasts. Bright-field photos were taken on differentiation day 4. Scale bars � 50 �m. Ctrli, control shRNA; Stk40i, Stk40 shRNA. B,immunostaining of MyHC (green) in control and Stk40-deficient C2C12 cells on differentiation day 4. DAPI was used to stain the nuclei (blue). Scale bars � 50 �m.C, quantification of the ratio of MyHC-positive nuclei to total nuclei in control and Stk40-deficient C2C12 cells on differentiation day 4. Error bars represent S.D.;Student’s t test; *, p � 0.05. D, transcript levels of Stk40, MyHC, Myogenin, and MEF2C in control and Stk40-deficient C2C12 cells at the indicated time points ofdifferentiation were detected by RT-qPCR assays. Data were normalized to the level of GAPDH. Error bars represent S.D.; Student’s t test; *, p � 0.05; **, p � 0.01;***, p � 0.001. E, protein levels of Stk40, MEF2C, Myogenin, MyHC, and MyoD in control and Stk40-deficient C2C12 cells at the indicated time points ofdifferentiation (Diff) were analyzed by Western blotting. �-Tubulin was used as a loading control. F, overexpression of Stk40-GFP fusion proteins by retroviraldelivery in C2C12 myoblasts. Shown is immunostaining of MyHC (red) in Stk40-GFP� and Stk40-GFP� C2C12 cells on differentiation day 3. GFP-positive andGFP-negative groups were sorted by a flow cytometer. DAPI was used to stain the nuclei (blue). Scale bars � 20 �m. G, quantification of the ratio of MyHC-positive nuclei to total nuclei in Stk40-GFP� and Stk40-GFP� C2C12 cells on differentiation day 3. Error bars represent S.D.; Student’s t test; *, p � 0.05. H, proteinlevels of Stk40-GFP, MEF2C, Myogenin, and MyHC in Stk40-GFP� and Stk40-GFP� C2C12 cells on differentiation day 3 were analyzed by Western blotting.�-Tubulin was used as a loading control.




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

either one impaired the formation of multinucleated myo-tubes (Fig. 2, A and B) with a concomitant decrease in thepercentage of MyHC-positive cells and expression levels ofmyogenic markers during the C2C12 myogenic differentia-tion process (Fig. 2, C–E). The findings suggest that Stk40might play an important role in the normal myogenicprocess.

We next ectopically expressed Stk40-GFP fusion proteins inC2C12 cells through retroviral transduction. Ectopicallyexpressed Stk40-GFP protein distributed mainly in the nucleus(Fig. 2F). Overexpression of Stk40 enhanced the myogenesis ofC2C12 cells moderately, as shown by increases in the expres-sion level of myogenic markers and the percentage of MyHC-positive cells (Fig. 2, F–H). Thus, Stk40 could enhance myo-genic differentiation of C2C12 myoblasts.

To determine whether Stk40 could have the same function inanother independent cell model, we knocked down Stk40 dur-ing MyoD-mediated myogenesis in C3H10T1/2, a mesenchy-mal stem cell line widely utilized for the study of skeletal muscledifferentiation (13, 29). Stk40-deficient C3H10T1/2 cells dis-played attenuated myogenesis, as shown by a significant reduc-tion in the percentage of MyHC-positive cells compared withcontrol cells on differentiation day 2 (Fig. 3, A and B). More-over, the protein levels of myogenic markers such as MyHC,MEF2C and Myogenin were lower in Stk40-deficient cells thanin control cells (Fig. 3C). Therefore, our results reveal thatStk40 has a pro-myogenesis role in different myogenic cellmodels.

Stk40 Controls Myogenesis through a Cell Cycle- and CellSurvival-independent Mechanism—Having shown that Stk40deficiency led to attenuated myogenesis, we explored whetherStk40 could control the cell cycle or cell survival during myo-genesis. To address this question, we compared the percentageof cells in the S phase between control and Stk40-deficientC2C12 cells, as the cell cycle exit occurs at the very beginning ofmyoblast differentiation (4). As shown in Fig. 4, A and B, therewas no significant difference in the percentage of the S phasecells between control and Stk40-deficient cells, although a sub-stantial reduction in the percentage of cells in the S phase wasobserved on differentiation day 1 for both control and Stk40-deficient cells. Moreover, mRNA levels of the cyclin-dependentkinase inhibitor p21 were comparable between the two groups(Fig. 4C). Furthermore, protein levels of cleaved Caspase-3, amarker of cell apoptosis, were similar in control and Stk40-deficient cells, although they increased significantly on day 1 ofmyogenic differentiation of C2C12 myoblasts for cells of bothtypes (Fig. 4D). Therefore, the impaired myogenesis observedin Stk40-deficient C2C12 cells was not caused by an altered cellcycle or cell survival.

Stk40 Modulates the Level of HDAC5 Proteins during Myo-genic Differentiation of C2C12 Myoblasts—To search for themolecular mechanism by which Stk40 regulates skeletal myo-genesis, we investigated the regulatory role of Stk40 in the tran-scriptional activity of important factors modulating myogen-esis. Interestingly, overexpression of Stk40 enhanced theluciferase activity of the MEF2-responsive gene reporter (3 �MEF2) (Fig. 5A), which contained three MEF2 binding sitesupstream of a c-fos minimal promoter, suggesting that Stk40might play a role in the control of MEF2 transcriptionalactivities.

To know how Stk40 positively modulated MEF2 activity, weexamined the expression levels of HDAC5, as it is known thatHDAC5 represses MEF2 activity (19). Compared with controlC2C12 cells, Stk40-deficient cells had a higher level of HDAC5proteins during myogenic differentiation (Fig. 5B). Asexpected, Stk40-deficient cells had reduced levels of the MEF2downstream target genes MEF2C and MyHC (Figs. 2E and 5B),in line with the previous finding that higher levels of HDAC5correspond to lower transcriptional activity of MEF2 (30). Incontrast, overexpression of Stk40 reduced the protein levels ofHDAC5, accompanied by enhanced protein levels of MEF2Cand MyHC on day 2 of differentiation (Fig. 5C), further indicat-ing a negative regulatory role of Stk40 for HDAC5 protein levelsduring myogenesis.

HDAC5 carries out its suppressive effect on MEF2 transcrip-tional activities in the nucleus (22, 30). Moreover, HDAC5 wasreported to be phosphorylated and undergo nuclear exportduring myogenic differentiation (22). Therefore, we examinedwhether Stk40 could modulate HDAC5 protein levels in thenucleus during myogenic differentiation of C2C12 cells. Twoindependent approaches were applied: one was cytoplasmicand nuclear protein fractionation and another was immunoflu-orescence staining. First, the efficient isolation of cytoplasmicand nuclear proteins was verified by the correct distribution ofcytoplasmic GAPDH and nuclear H3 proteins, respectively. Inaddition, Stk40 proteins were mainly detected in the nuclear

FIGURE 3. Knockdown of Stk40 attenuates MyoD-mediated myogenic dif-ferentiation of C3H10T1/2 cells. A, immunostaining of MyHC (red) in controland Stk40-deficient MyoD-expressed C3H10T1/2 cells on differentiation day2. DAPI was used to stain the nuclei (blue). Scale bars � 100 �m. Ctrli, controlshRNA; Stk40i, Stk40 shRNA. B, quantification of the percentages of MyHC-positive cells to total cells in control and Stk40-deficient MyoD-expressedC3H10T1/2 cells on differentiation day 2. Error bars represent S.D.; Student’s ttest; **, p � 0.01. C, protein levels of MyHC, MEF2C, Stk40, and Myogenin incontrol and Stk40-deficient MyoD-expressed C3H10T1/2 cells on differentia-tion day 2 were analyzed by Western blotting. �-Tubulin was used as a load-ing control.




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

extract, consistent with its nuclear location observed by confo-cal microscopy (Fig. 2F). We found that Stk40 knockdownincreased, whereas Stk40 overexpression decreased, HDAC5proteins in the nuclei, respectively (Fig. 5, D and E). Second,immunofluorescence staining showed that HDAC5 proteinsmainly located in the cytoplasm on differentiation day 4 in con-trol C2C12 cells. However, Stk40-deficient cells had evidentlymore HDAC5 proteins located in the nuclei than controlC2C12 cells (Fig. 5F). The results from both approaches indi-cate that Stk40 negatively modulates HDAC5 protein levels inthe nuclei. It is worth mentioning that the level of Ser-259 phos-phorylated HDAC5 proteins, mostly located in the cytoplasm,was also higher in Stk40-deficient cells (Fig. 5D), suggesting thatStk40 might control the steady-state levels of HDAC5 proteinsregardless of their subcellular localization.

HDAC5 Is an Important Factor for Stk40-controlled Myo-genesis—To validate the involvement of HDAC5 in Stk40-reg-ulated myogenesis, we examined the expression patternof HDAC5 during C2C12 differentiation. Its protein leveldecreased along with myogenic differentiation (Fig. 6A), similarto the HDAC4 expression pattern reported previously (31).Functionally, overexpression of HDAC5 blocked myogenesisand attenuated the expression of MEF2C and MyHC on differ-entiation day 2 (Fig. 6, B and C), resembling the phenotypeobserved in Stk40-deficient cells. Importantly, knockdown ofHDAC5 reverted the reduction in MyHC protein levels causedby Stk40 deficiency (Fig. 6D). Therefore, HDAC5 repressesskeletal myogenesis and functions as an important player inStk40-controlled myogenic differentiation. As a member ofclass IIa HDACs, HDAC4 has been reported to inhibit skeletalmyogenesis by repressing MEF2 activity similarly as HDAC5(22, 30, 31). We examined whether HDAC4 was also involved inStk40-controlled skeletal myogenesis. HDAC4 displayed a sim-ilar expression pattern as HDAC5 during the myogenic differ-entiation of C2C12 cells (Fig. 6, A and E). Also, Stk40-deficient

cells had a higher level of HDAC4 proteins during myogenicdifferentiation (Fig. 6F). Therefore, it seems that Stk40 regu-lates the protein levels of both HDAC4 and HDAC5, whichmight both be involved in Stk40-controlled myogenesis.

Stk40 Is Required for Normal Fetal Skeletal Muscle De-velopment—Stk40�/� mice died at birth, which prevented usfrom studying its role in adult myogenesis (25). To investigatethe physiological function of Stk40 in skeletal myogenesis invivo, we examined whether there existed some defects in fetalskeletal muscle development at E18.5. Histological immuno-staining showed that the hind limb muscle tissue of Stk40�/�

mice was smaller and had fewer myofibers at E18.5, as indicatedby the reduced number of Laminin-positive cells, comparedwith that of wild-type mice (Fig. 7, A and B). The ratio of num-bers of Laminin-positive cells to body weight was significantlydecreased in Stk40�/� mice, which suggested a specific role ofStk40 in the control of myofiber number in development. How-ever, we did not detect any remarkable changes in the musclepattern (Fig. 7A). The size of each myofiber was comparable inStk40�/� and Stk40�/� mice (Fig. 7C), suggesting that thesmaller muscle tissue mainly resulted from the fewer number ofmyofibers but not the smaller size of myofibers. In addition, weexamined the expression level of muscle-specific markers in thehind limb muscle at E18.5. As shown in Fig. 7D, the level ofthe neonatal MyHC isoform was significantly decreased inStk40�/� mice, which was the major MyHC isoform in the fetalmuscle (32). Also, downstream targets of MEF2 such as �-skel-etal actin (Acta1), muscle creatine kinase (MCK), and desminwere all down-regulated in Stk40�/� muscle. These results sup-port the notion that Stk40 is required for normal fetal skeletalmuscle development.

Discussion

In this study, we show that Stk40 can positively modulateskeletal muscle differentiation. Stk40 expression levels in-

FIGURE 4. Knockdown of Stk40 does not alter the cell cycle process and cell apoptosis during the differentiation of C2C12 cells. A, percentages of the cellnumber of C2C12 cells in the different phases of the cell cycle on differentiation days 0 (D0) and 1 (D1). Error bars represent S.D.; Student’s t test; *, p � 0.05. Ctrli,control shRNA; Stk40i, Stk40 shRNA. B, percentages of the cell number of the control and Stk40-deficient C2C12 cells in the different phases of the cell cycle ondifferentiation days 0 and 1. Error bars represent S.D. C, mRNA levels of p21 in control and Stk40-deficient C2C12 cells on differentiation days 0 and 2 weredetected by RT-qPCR assays. Data were normalized to the level of GAPDH. Error bars represent S.D. D, protein levels of cleaved Caspase-3 and Stk40 in controland Stk40-deficient C2C12 cells at the indicated time points of differentiation (Diff) were analyzed by Western blot. �-Tubulin was used as a loading control.




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

creased during skeletal muscle differentiation, whereas theywere down-regulated in myotubes, implying that Stk40 has apotential role in the differentiation process rather than in themaintenance or the survival of mature myotubes. Moreover,the loss-of-function and gain-of-function studies conducted inC2C12 cells suggest that Stk40 is essential for skeletal myogen-esis in vitro. Stk40 had higher expression levels in developingfetal muscle and muscle tissues in regeneration after injurycompared with normal adult muscle. The hind limb muscletissue from Stk40�/� embryos at E18.5 was smaller than thatfrom wild-type embryos because of the reduced number ofmyofibers, providing in vivo evidence for the essential role ofStk40 in normal skeletal muscle differentiation. We did notinvestigate the role of Stk40 in muscle regeneration because ofthe lack of viable adult Stk40�/� mice (25). Nevertheless, the

increased expression level of Stk40 during muscle regeneration,together with the function of Stk40 in myogenesis of C2C12myoblasts derived from satellite cells and in fetal muscle devel-opment, implies that Stk40 might play a role in muscle regen-eration as well. Identification of Stk40 as a new regulator forskeletal muscle differentiation is important for understandinghow mammalian myogenesis is controlled at the molecularlevel.

Skeletal muscle development and regeneration are impor-tant biological processes and regulated by multiple transcrip-tion factors (2, 33). However, how the transcriptional activitiesof these factors are regulated is poorly elucidated. We showedthat Stk40 could control the transcriptional activity of MEF2, afamily of transcription factors required for the activation ofmyogenic gene expression. First, Stk40 enhanced MEF2-spe-

FIGURE 5. Stk40 enhances MEF2 transcriptional activity and negatively modulates the protein level of HDAC5. A, C2C12 cells were transfected with the3 � MEF2 reporter plasmid and pCMV-MEF2C together with the pMXS-Stk40 or pMXS-GFP plasmid as a control. Luciferase activity was analyzed with the dualRenilla/firefly luciferase system. Error bars represent S.D.; Student’s t test; *, p � 0.05. B, protein levels of HDAC5, MyHC, Stk40, and MEF2C in control andStk40-deficient C2C12 cells on differentiation day 2 were analyzed by Western blotting. �-Tubulin was used as a loading control. Ctrli, control shRNA; Stk40i,Stk40 shRNA. C, protein levels of HDAC5, MyHC, Stk40-GFP, and MEF2C in Stk40-GFP� and Stk40-GFP� C2C12 cells on differentiation (Diff) days 0 and 2 wereanalyzed by Western blotting. �-Tubulin was used as a loading control. D, protein levels of Stk40, HDAC5, p-HDAC5(S259), and MEF2C in the cytoplasm (C) andnucleus (N) of control and Stk40-deficient C2C12 cells on differentiation day 2 were analyzed by Western blotting. GAPDH and H3 were used as loading controlsfor the cytoplasmic and nuclear proteins, respectively. E, protein levels of Stk40, HDAC5, and MEF2C in the cytoplasm and nucleus of Stk40-GFP� andStk40-GFP� C2C12 cells on differentiation day 2 were analyzed by Western blotting. GAPDH and H3 were used as loading controls for the cytoplasmic andnuclear proteins, respectively. F, immunostaining of HDAC5 (red) in control and Stk40-deficient C2C12 cells on differentiation day 4. DAPI was used to stain thenuclei (blue). Scale bars � 10 �m.




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

cific reporter activities; second, Stk40 deficiency led to aremarkable reduction in protein levels of the MEF2 down-stream targets MEF2C and MyHC. Thus, Stk40 is required forthe appropriate activity of MEF2. Interestingly, further studyshowed that Stk40-deficient C2C12 cells had higher levels ofHDAC5 proteins in both whole-cell lysate and nuclear lysateduring myogenic differentiation. Consistent with the previousreport that nuclear HDAC5 proteins repressed MEF2 tran-scriptional activity, we found that overexpression of HDAC5disrupted myogenesis substantially and attenuated the expres-sion of MEF2C and MyHC during C2C12 cell differentiation.We propose that Stk40 positively regulates MEF2 activitiesthrough controlling the protein level of HDAC5 in the nucleus.Indeed, we found that down-regulation of Stk40 in C2C12 cellsgave rise to higher protein levels of HDAC5, which, in turn,repressed the MEF2 activity and resulted in down-regulation ofMEF2 target genes as well as attenuated myogenesis. Support-ing this proposal, silencing of HDAC5 partially rescued the phe-notype induced by Stk40 deficiency. Our data indicate thatStk40 modulates myogenesis, at least partially, through con-trolling the HDAC5-MEF2 axis.

Currently, we are not clear about how Stk40 regulates theprotein level of HDAC5 at a posttranscriptional level, as it didnot affect the transcript level of HDAC5 (data not shown). Sev-eral studies demonstrate that class IIa HDACs are posttran-scriptionally controlled (34 –36). HDAC5 is posttranscription-ally regulated by miR-2861 during osteoblast differentiation(37). For myogenic differentiation, miR-1, miR-206, andmiR-29 target HDAC4 to promote myogenesis (31, 38). How-ever, whether HDAC5 is regulated by microRNA in myogenesishas not yet been elucidated. Another reported posttranscrip-tional regulation for HDAC5 is ubiquitination. ExogenousHDAC5 proteins can be ubiquitinated and undergo degrada-tion in C2C12 cells (23). In addition, previous studies showedthat protein kinases such as calcium/calmodulin-dependentprotein kinase could promote skeletal myogenesis and MEF2transcriptional activity via phosphorylating HDAC5 to induceHDAC5 nuclear export (20, 30). As Stk40 is a putative proteinkinase and could control HDAC5 protein levels in the nucleus,we hypothesized that Stk40 might regulate the myogenesis ofC2C12 myoblasts and MEF2 activities through enhancingnuclear export of HDAC5 proteins. Unexpectedly, the phos-

FIGURE 6. The elevated HDAC5 protein level is responsible for the attenuated myogenesis in Stk40-deficient C2C12 myoblasts. A, protein levels ofHDAC5 in C2C12 cells at the indicated time points of differentiation (Diff) were analyzed by Western blotting. �-Tubulin was used as a loading control. B,bright-field photos of control C2C12 cells expressing GFP or C2C12 cells expressing HDAC5 were taken on differentiation day 3. Scale bars � 100 �m. C, proteinlevels of HDAC5, MyHC, and MEF2C in GFP- or HDAC5-expressed C2C12 cells on differentiation day 2 were analyzed by Western blotting. �-Tubulin was usedas a loading control. D, protein levels of HDAC5, MyHC, and Stk40 in Ctri, Stk40i, and Stk40i/HDAC5i C2C12 cells were analyzed by Western blotting. �-Tubulinwas used as a loading control. The mean gray values of MyHC analyzed by ImageJ are listed below. Ctrli, control shRNA; Stk40i, Stk40 shRNA; HDAC5i, HDAC5shRNA. E, protein levels of HDAC4 in C2C12 cells at the indicated time points of differentiation were analyzed by Western blotting. �-Tubulin was used as aloading control. F, protein levels of HDAC4, HDAC5, Stk40, and MEF2C in control and Stk40-deficient C2C12 cells on differentiation day 2 were analyzed byWestern blotting. �-Tubulin was used as a loading control.




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

phorylation level of HDAC5 was higher in Stk40-deficient cellsthan in control cells (Fig. 5D), which might be caused by theincreased whole-cell protein level of HDAC5. The resultexcludes the possibility that Stk40 controls the levels of nuclearHDAC5 by modulating HDAC5 nuclear export. Further inves-tigations are needed to understand the molecular mechanismsunderpinning the regulatory function of Stk40 for HDAC5proteins.

Class IIa HDAC members are involved in many physiologicalprocesses, such as the formation of slow-twitch myofibers, reg-ulation of cardiac hypertrophy, fibrosis, and pathologicalremodeling as well as cardiovascular growth and function (23,39, 40). They also share mechanisms in the control of develop-ment, such as skeletal myogenesis. Our study raises the possi-bility of involvement of Stk40 in these processes and providesnew clues to explore HDAC inhibitors for therapeutic applica-tion of the related diseases.

Experimental Procedures

Animals and Muscle Regeneration—The muscle tissue wasobtained from C57BL/6 mice. All animals were raised under theconditions described previously and handled according to theguidelines approved by the Shanghai Jiao Tong UniversitySchool of Medicine (26). The genotype of Stk40�/� andStk40�/� mice was determined as described previously (26).For muscle injury, 100 �l of CTX (10 �M) was injected into thetibialis anterior (TA) muscle of 12-week-old C57BL/6 mice.The injected TA muscle was isolated at the indicated time pointafter treatment with CTX.

Cell Culture and Differentiation—C2C12 cells were culturedin DMEM supplemented with 20% FBS, 1% penicillin/strepto-mycin, and 1% L-glutamine. C3H10T1/2 cells were cultured in

DMEM supplemented with 10% FBS, 1% penicillin/streptomy-cin, and 1% L-glutamine. For myogenic differentiation, afterconfluence, the medium of C2C12 or C3H10T1/2 cells waschanged to DMEM supplemented with 2% horse serum, 1%penicillin/streptomycin and 1% L-glutamine.

Virus Package and Transduction—For Stk40 knockdownassays, shRNAs specific to Stk40 were cloned into the pSIRENplasmid. Plasmids for HDAC5 knockdown assays were pur-chased from the GIPZ Lentiviral Mouse shRNA Library (GEDharmacon). The shRNA interference sequence for HDAC5was 5�-CCGGGAAGGCTCTACAGAA-3�. The shRNA inter-ference sequences for Stk40 were 5�-GGACCCATCGGATA-ACTAT-3� and 5�-TGCATACCGAGTACTCTCT-3�. TheshRNA interference sequence for control was 5�-GTGCGCT-GCTGGTGCCAAC-3�. For overexpression assays, Stk40-GFPcDNA and HDAC5 cDNA were cloned into the pMXS plasmid.Retroviral and lentiviral packaging and transduction were per-formed as described previously (25).

RNA Extraction and RT-qPCR—Whole-cell RNA was pre-pared using TRIzol (Invitrogen). 2 �g of total RNA was used toperform reverse transcription by a Fastquant reverse kit (Tian-gen). Quantitative PCR was performed on the ABI 7900 usingFastStart Universal SYBR Green Master (Roche). Primers usedfor RT-qPCR are provided in supplemental Table 1.

Immunoblotting—Total protein extract from C2C12 cells ormuscle tissue was prepared with lysis buffer consisting of 2 mM

EDTA, 0.5% Nonidet P-40, 50 mM Tris (pH 7.5), 150 mM NaCl,and 10% glycerol and quantified with the BCA kit (Pierce).Nuclear and cytoplasmic extraction reagents (Thermo) wereused to isolate the nuclear and cytoplasmic proteins fromC2C12 cells according to the recommendations of the manu-

FIGURE 7. Stk40 deficiency impairs fetal muscle development in mice. A, immunostaining of laminin (red) and MyHC (green) in the cross-section of Stk40-WTand Stk40-KO hind limb muscle at E18.5. DAPI was used to stain the nuclei (blue). The white frame marks the soleus muscle. Scale bars � 100 �m. B, quantificationfor the ratio of Laminin-positive cells of Stk40-WT and Stk40-KO soleus muscle to body weight (BW). Error bars represent S.D.; Student’s t test; *, p � 0.05. C,immunostaining of laminin (red) and MyHC (green) in the cross-section of Stk40-WT and Stk40-KO hind limb muscle at E18.5. DAPI was used to stain nuclei (blue).Scale bars � 20 �m. D, transcript levels of MyHC, Acta1, MCK, Desmin, neo-MyHC, and emb-MyHC in Stk40-WT and Stk40-KO hind limb muscle at E18.5 weredetected by RT-qPCR assays (n � 5). Data were normalized to the level of S26. Error bars represent S.D.; Student’s t test; *, p � 0.05; **, p � 0.01; ***, p � 0.001.




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

facturer. The protein samples were further examined by West-ern blotting, which was conducted as described previously (26).Antibodies used for immunoblotting in this study are providedin supplemental Table 1.

Flow Cytometric Analysis—C12C12 cells were infected with aretrovirus containing the Stk40-GFP cDNA sequence. After 2days of infection, the Stk40-GFP� and Stk40-GFP� groupswere sorted by a flow cytometer (BD Aria II).

Immunostaining—C2C12 cells cultured in 4-well plates orfrozen sections of the mouse hind limb muscle at E18.5 werefixed with 4% paraformaldehyde for 10 min, permeabilized with0.2% Triton X-100 for 5 min, and blocked with 3% BSA for 30min at room temperature. Incubation with primary antibodiesagainst MyHC (MF-20) and HDAC5 for C2C12 cells as well asMyHC and Laminin for frozen sections, respectively, wasconducted in 3% BSA at 4 °C overnight. After washing, cellsor sections were incubated with FITC-conjugated or CY3-conjugated secondary antibodies (Jackson ImmunoResearchLaboratories) in 3% BSA for 1 h. Images were captured usinga confocal microscope (TCS SP5, Leica Microsystems, Wet-zlar, Germany).

MEF2 Luciferase Reporter Assays—The 3 � MEF2-lucreporter plasmid was provided by Ron Prywes (Addgene plas-mid 32967). C2C12 cells were transfected with the 3 � MEF2reporter plasmid and pCMV-MEF2C together with the pMXS-Stk40 or pMXS-GFP plasmid using X-tremeGENE HP DNAtransfection reagent (Roche). Cell lysate was prepared aftertransfection for 2 days. A Dual-Luciferase reporter assay system(Promega) was used to detect the firefly and Renilla luciferaseactivity in the samples.

Statistical Analyses—Data were presented as the mean S.D. from at least three independent experiments or samples.Statistical significance was analyzed by two-tailed Student’s ttest and is shown as follows: *, p � 0.05; **, p � 0.01; ***, p �0.001.

Author Contributions—Y. J. and K. H. designed the study and ana-lyzed the results. K. H., J. H., H. Y., L. W., J. G., F. T., S. L., H. W., andL. G. performed the experiments. Y. J. and K. H. wrote themanuscript.

Acknowledgments—We thank Dr. Guang Ning for providing cell lines(C3H10T1/2) and Dr. Xin Fu for technical support.

References1. Biressi, S., Molinaro, M., and Cossu, G. (2007) Cellular heterogeneity dur-

ing vertebrate skeletal muscle development. Dev. Biol. 308, 281–2932. Tajbakhsh, S. (2009) Skeletal muscle stem cells in developmental versus

regenerative myogenesis. J. Int. Med. 266, 372–3893. Yin, H., Price, F., and Rudnicki, M. A. (2013) Satellite cells and the muscle

stem cell niche. Physiol. Rev. 93, 23– 674. Walsh, K., and Perlman, H. (1997) Cell cycle exit upon myogenic differ-

entiation. Curr. Opin. Genet. Dev. 7, 597– 6025. Bailey, P., Holowacz, T., and Lassar, A. B. (2001) The origin of skeletal

muscle stem cells in the embryo and the adult. Curr. Opin. Cell Biol. 13,679 – 689

6. Rudnicki, M. A., and Jaenisch, R. (1995) The MyoD family of transcriptionfactors and skeletal myogenesis. BioEssays 17, 203–209

7. Weintraub, H., Davis, R., Tapscott, S., Thayer, M., Krause, M., Benezra, R.,Blackwell, T. K., Turner, D., Rupp, R., and Hollenberg, S. (1991) The myoD

gene family: nodal point during specification of the muscle cell lineage.Science 251, 761–766

8. Massari, M. E., and Murre, C. (2000) Helix-loop-helix proteins: regulatorsof transcription in eucaryotic organisms. Mol. Cell Biol. 20, 429 – 440

9. Davis, R. L., Weintraub, H., and Lassar, A. B. (1987) Expression of a singletransfected cDNA converts fibroblasts to myoblasts. Cell 51, 987–1000

10. Braun, T., and Gautel, M. (2011) Transcriptional mechanisms regulatingskeletal muscle differentiation, growth and homeostasis. Nat. Rev. Mol.Cell Biol. 12, 349 –361

11. Black, B. L., and Olson, E. N. (1998) Transcriptional control of muscledevelopment by myocyte enhancer factor-2 (MEF2) proteins. Annu. Rev.Cell Dev. Biol. 14, 167–196

12. Bentzinger, C. F., Wang, Y. X., and Rudnicki, M. A. (2012) Building mus-cle: molecular regulation of myogenesis. Cold Spring Harb. Perspect. Biol.4, a008342

13. Molkentin, J. D., Black, B. L., Martin, J. F., and Olson, E. N. (1995) Coop-erative activation of muscle gene expression by MEF2 and myogenicbHLH proteins. Cell 83, 1125–1136

14. Potthoff, M. J., Arnold, M. A., McAnally, J., Richardson, J. A., Bassel-Duby,R., and Olson, E. N. (2007) Regulation of skeletal muscle sarcomereintegrity and postnatal muscle function by Mef2c. Mol. Cell Biol. 27,8143– 8151

15. Snyder, C. M., Rice, A. L., Estrella, N. L., Held, A., Kandarian, S. C., andNaya, F. J. (2013) MEF2A regulates the Gtl2-Dio3 microRNA mega-clus-ter to modulate WNT signaling in skeletal muscle regeneration. Develop-ment 140, 31– 42

16. Liu, N., Nelson, B. R., Bezprozvannaya, S., Shelton, J. M., Richardson, J. A.,Bassel-Duby, R., and Olson, E. N. (2014) Requirement of MEF2A, C, and Dfor skeletal muscle regeneration. Proc. Natl. Acad. Sci. U.S.A. 111,4109 – 4114

17. Wang, D. Z., Valdez, M. R., McAnally, J., Richardson, J., and Olson, E. N.(2001) The Mef2c gene is a direct transcriptional target of myogenicbHLH and MEF2 proteins during skeletal muscle development. Develop-ment 128, 4623– 4633

18. Cripps, R. M., Lovato, T. L., and Olson, E. N. (2004) Positive autoregula-tion of the Myocyte enhancer factor-2 myogenic control gene during so-matic muscle development in Drosophila. Developmental Biology 267,536 –547

19. McKinsey, T. A., Zhang, C. L., and Olson, E. N. (2001) Control of muscledevelopment by dueling HATs and HDACs. Curr. Opin. Genet. Dev. 11,497–504

20. McKinsey, T. A., Zhang, C. L., and Olson, E. N. (2000) Activation of themyocyte enhancer factor-2 transcription factor by calcium/calmodulin-dependent protein kinase-stimulated binding of 14 –3-3 to histonedeacetylase 5. Proc. Natl. Acad. Sci. U.S.A. 97, 14400 –14405

21. McKinsey, T. A., Zhang, C. L., and Olson, E. N. (2002) MEF2: a calcium-dependent regulator of cell division, differentiation and death. TrendsBiochem. Sci. 27, 40 – 47

22. McKinsey, T. A., Zhang, C. L., Lu, J., and Olson, E. N. (2000) Signal-de-pendent nuclear export of a histone deacetylase regulates muscle differ-entiation. Nature 408, 106 –111

23. Potthoff, M. J., Wu, H., Arnold, M. A., Shelton, J. M., Backs, J., McAnally,J., Richardson, J. A., Bassel-Duby, R., and Olson, E. N. (2007) Histonedeacetylase degradation and MEF2 activation promote the formation ofslow-twitch myofibers. J. Clin. Invest. 117, 2459 –2467

24. Li, L., Sun, L., Gao, F., Jiang, J., Yang, Y., Li, C., Gu, J., Wei, Z., Yang, A., Lu,R., Ma, Y., Tang, F., Kwon, S. W., Zhao, Y., Li, J., and Jin, Y. (2010) Stk40links the pluripotency factor Oct4 to the Erk/MAPK pathway and controlsextraembryonic endoderm differentiation. Proc. Natl. Acad. Sci. U.S.A.107, 1402–1407

25. Yu, H., He, K., Li, L., Sun, L., Tang, F., Li, R., Ning, W., and Jin, Y. (2013)Deletion of STK40 protein in mice causes respiratory failure and death atbirth. J. Biol. Chem. 288, 5342–5352

26. Yu, H., He, K., Wang, L., Hu, J., Gu, J., Zhou, C., Lu, R., and Jin, Y. (2015)Stk40 represses adipogenesis through translational control of CCAAT/enhancer-binding proteins. J. Cell Sci. 128, 2881–2890

27. Yaffe, D., and Saxel, O. (1977) Serial passaging and differentiation of myo-genic cells isolated from dystrophic mouse muscle. Nature 270, 725–727




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

28. Chargé, S. B., and Rudnicki, M. A. (2004) Cellular and molecular regula-tion of muscle regeneration. Physiol. Rev. 84, 209 –238

29. Jeong, H., Bae, S., An, S. Y., Byun, M. R., Hwang, J. H., Yaffe, M. B., Hong,J. H., and Hwang, E. S. (2010) TAZ as a novel enhancer of MyoD-mediatedmyogenic differentiation. FASEB J. 24, 3310 –3320

30. Lu, J., McKinsey, T. A., Zhang, C. L., and Olson, E. N. (2000) Regulation ofskeletal myogenesis by association of the MEF2 transcription factor withclass II histone deacetylases. Mol. Cell 6, 233–244

31. Chen, J. F., Mandel, E. M., Thomson, J. M., Wu, Q., Callis, T. E., Ham-mond, S. M., Conlon, F. L., and Wang, D. Z. (2006) The role of mi-croRNA-1 and microRNA-133 in skeletal muscle proliferation and differ-entiation. Nat. Genet. 38, 228 –233

32. Lyons, G. E., Ontell, M., Cox, R., Sassoon, D., and Buckingham, M. (1990)The expression of myosin genes in developing skeletal muscle in themouse embryo. J. Cell Biol. 111, 1465–1476

33. Schienda, J., Engleka, K. A., Jun, S., Hansen, M. S., Epstein, J. A., Tabin,C. J., Kunkel, L. M., and Kardon, G. (2006) Somitic origin of limb musclesatellite and side population cells. Proc. Natl. Acad. Sci. U.S.A. 103,945–950

34. Weeks, K. L., and Avkiran, M. (2015) Roles and post-translational regula-tion of cardiac class IIa histone deacetylase isoforms. J. Physiol. 593,1785–1797

35. Wang, Z., Qin, G., and Zhao, T. C. (2014) HDAC4: mechanism of regula-tion and biological functions. Epigenomics 6, 139 –150

36. Swierczynski, S., Klieser, E., Illig, R., Alinger-Scharinger, B., Kiesslich, T.,and Neureiter, D. (2015) Histone deacetylation meets miRNA: epigeneticsand post-transcriptional regulation in cancer and chronic diseases. ExpertOpin. Biol. Ther. 15, 651– 664

37. Li, H., Xie, H., Liu, W., Hu, R., Huang, B., Tan, Y. F., Xu, K., Sheng, Z. F.,Zhou, H. D., Wu, X. P., and Luo, X. H. (2009) A novel microRNA targetingHDAC5 regulates osteoblast differentiation in mice and contributes toprimary osteoporosis in humans. J. Clin. Invest. 119, 3666 –3677

38. Winbanks, C. E., Wang, B., Beyer, C., Koh, P., White, L., Kantharidis, P.,and Gregorevic, P. (2011) TGF- regulates miR-206 and miR-29 to controlmyogenic differentiation through regulation of HDAC4. J. Biol. Chem.286, 13805–13814

39. Haberland, M., Montgomery, R. L., and Olson, E. N. (2009) The manyroles of histone deacetylases in development and physiology: implicationsfor disease and therapy. Nat. Rev. Genet. 10, 32– 42

40. Chang, S., McKinsey, T. A., Zhang, C. L., Richardson, J. A., Hill, J. A., andOlson, E. N. (2004) Histone deacetylases 5 and 9 govern responsiveness ofthe heart to a subset of stress signals and play redundant roles in heartdevelopment. Mol. Cell Biol. 24, 8467– 8476




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

Wang, Sheng Li, Ping Hu and Ying JinKe He, Jing Hu, Hongyao Yu, Lina Wang, Fan Tang, Junjie Gu, Laixiang Ge, Hongye

Muscle DifferentiationSerine/Threonine Kinase 40 (Stk40) Functions as a Novel Regulator of Skeletal

doi: 10.1074/jbc.M116.719849 originally published online November 29, 20162017, 292:351-360.J. Biol. Chem.

10.1074/jbc.M116.719849Access the most updated version of this article at doi:

Alerts:

When a correction for this article is posted•

When this article is cited•

to choose from all of JBC's e-mail alertsClick here

Supplemental material:

http://www.jbc.org/content/suppl/2016/11/29/M116.719849.DC1

http://www.jbc.org/content/292/1/351.full.html#ref-list-1

This article cites 40 references, 15 of which can be accessed free at


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/lookup/doi/10.1074/jbc.M116.719849

http://www.jbc.org/cgi/alerts?alertType=citedby&addAlert=cited_by&cited_by_criteria_resid=jbc;292/1/351&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/292/1/351

http://www.jbc.org/cgi/alerts?alertType=correction&addAlert=correction&correction_criteria_value=292/1/351&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/292/1/351

http://www.jbc.org/cgi/alerts/etoc

http://www.jbc.org/content/suppl/2016/11/29/M116.719849.DC1

http://www.jbc.org/content/292/1/351.full.html#ref-list-1

http://www.jbc.org/

serine/threoninekinase40(stk40)functionsasanovel ... · either one impaired the formation of...

Documents