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Molecular Endocrinology, doi:10.1210/me.2007-0114
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Molecular Endocrinology 21 (9): 2282-2293
Copyright © 2007 by The Endocrine Society

RhoA/Rho Kinase Blocks Muscle Differentiation via Serine Phosphorylation of Insulin Receptor Substrate-1 and -2

Min Jin Lim, Kyu Jin Choi, Yan Ding, Jin Hwan Kim, Bum Shik Kim, Yun Hong Kim, Jinhwa Lee, Wonchae Choe, Insug Kang, Joohun Ha, Kyung-Sik Yoon and Sung Soo Kim

Department of Biochemistry and Molecular Biology (BK21 Project) (M.J.L., K.J.C., Y.D., J.H.K., W.C., I.K., J.H., K.-S.Y., S.S.K.), Medical Research Center for Bioreaction to Reactive Oxygen Species and Biomedical Science Institute (M.J.L., K.J.C., Y.D., J.H.K., W.C., I.K., J.H., K.-S.Y., S.S.K.), Department of Thoracic Surgery (B.S.K.), School of Medicine, Kyung Hee University, Seoul 130-701, Korea; Department of Anesthesiology and Pain Medicine (Y.H.K.), Kangbuk Samsung Hospital, Sungkyunkwan University School of Medicine, Seoul 110-746, Korea; and Department of Biotechnology (J.L.), Dongseo University, Busan 617-716, Republic of Korea

Address all correspondence and requests for reprints to: Sung Soo Kim, Department of Biochemistry and Molecular Biology, School of Medicine, Kyung Hee University, #1, Hoegi-dong, Dongdaemoon-gu, Seoul 130-701, Korea. E-mail: sgskim{at}khu.ac.kr.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Although the RhoA/Rho kinase (RhoA/ROK) pathway has been extensively investigated, its roles and downstream signaling pathways are still not well understood in myogenic processes. Therefore, we examined the effects of RhoA/ROK on myogenic processes and their signaling molecules using H9c2 and C2C12 cells. Increases in RhoA/ROK activities and serine phosphorylation levels of insulin receptor substrate (IRS)-1 (Ser307 and Ser636/639) and IRS-2 were found in proliferating myoblasts, whereas IRS-1/2 tyrosine phosphorylation and phosphatidylinositol (PI) 3-kinase activity increased during the differentiation process. ROK strongly bound to IRS-1/2 in proliferation medium but dissociated from them in differentiation medium (DM). ROK inactivation by a ROK inhibitor, Y27632, or a dominant-negative ROK, decreased IRS-1/2 serine phosphorylation with increases in IRS-1/2 tyrosine phosphorylation and PI 3-kinase activity, which led to muscle differentiation even in proliferation medium. Inhibition of ROK also enhanced differentiation in DM. ROK activation by a constitutive active ROK blocked muscle differentiation with the increased IRS-1/2 serine phosphorylation, followed by decreases in IRS-1/2 tyrosine phosphorylation and PI 3-kinase activity in DM. Interestingly, fibroblast growth factor-2 added to DM also blocked muscle differentiation through RhoA/ROK activation. Fibroblast growth factor-2 blockage of muscle differentiation was reversed by Y27632. Collectively, these results suggest that the RhoA/ROK pathway blocks muscle differentiation by phosphorylating IRS proteins at serine residues, resulting in the decreased IRS-1/2 tyrosine phosphorylation and PI 3-kinase activity. The absence of the inhibitory effects of RhoA/ROK in DM due to low concentrations of myogenic inhibitory growth factors seems to allow IRS-1/2 tyrosine phosphorylation, which stimulates muscle differentiation via transducing normal myogenic signaling.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
MUSCLE DIFFERENTIATION IS a multistep process that involves cell cycle arrest, muscle-specific protein synthesis, myoblast elongation, and fusion into multinucleated myotubes (1, 2). When myoblasts are subjected to conditions that induce differentiation, specific signaling pathways are activated and basic helix-loop-helix muscle regulatory transcription factors, such as MyoD, Myf5, myogenin, and MRF4, are subsequently induced (3, 4). Insulin and IGF-I and -II are the most characterized ligands for muscle differentiation (5, 6, 7, 8). These factors stimulate muscle differentiation via phosphorylation of insulin receptor substrates (IRS) at multiple tyrosine residues, and subsequent activation of phosphatidylinositol 3-kinase (PI 3-kinase) (6, 9), Akt/protein kinase B (10, 11, 12), Rac (13, 14), p70S6kinase (15), phospholipase C-ß1 and -{gamma}1 (16, 17), and mammalian target of rapamycin (mTOR) pathway (18). p38 MAPK is another well-defined myogenic signaling molecule (19, 20).

RhoA is one of the Rho subfamily members of small GTPases, including Rho, Rac1, and Cdc42 that are evolutionally conserved. Among the Rho subfamily, RhoA is the best characterized. Similar to all GTPases, RhoA acts as a molecular switch and cycles between two conformation states: an active state (GTP-bound form) and an inactive state (GDP-bound form). Many agonists, including platelet-derived growth factor, angiotensin II, thrombin, endothelin-1, etc., activate guanine nucleotide exchange factors, which substitute the Rho-GTP for Rho-GDP forms (21, 22).

Rho kinase (ROK) is a well-known downstream target of Rho that functions as a serine/threonine kinase. ROK is activated after interaction of the Rho-binding domain with the GTP-bound RhoA. ROK has diverse cellular functions, such as stress fiber formation, cell migration, and proliferation (23, 24, 25). Rho is also known to have several other effector proteins, including protein kinase N (PKN), rhophilin, rhotekin, and citron kinase (26, 27, 28). The cellular function of the RhoA/ROK pathway has been investigated using a pharmacological inhibitor of ROK, such as Y27632, which targets its ATP-dependent kinase domain. Alternatively, a genetic approach involving a dominant-negative ROK (ROK-RB) and a constitutive active ROK (CAT-ROK) has also been used because Y27632 can also inhibit protein kinase C-related kinase-2, PKN, and citron kinase at higher concentrations (29).

The role of RhoA/ROK in the muscle differentiation process has been extensively investigated. RhoA has been proposed to be a positive regulator of muscle differentiation (13, 30, 31). In contrast, there are reports that RhoA inhibits the muscle differentiation process. Expression level and activity of RhoA were down-regulated in both primary avian myoblasts and mouse satellite cells undergoing differentiation, and ectopic expression of ROK impaired differentiation (32, 33).

We studied the RhoA/ROK function in muscle differentiation using rat cardiac H9c2 and mouse C2C12 myoblast cell lines as our model. We also studied the mechanism by which RhoA/ROK affects muscle differentiation. In this study, the activated RhoA/ROK pathway in cells undergoing proliferation blocked muscle differentiation by increasing IRS-1/2 serine phosphorylation, which in turn decreased their tyrosine phosphorylation, followed by down-regulation of PI 3-kinase activity.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
RhoA and ROK Activities Are Down-Regulated during Myogenesis
Although several studies have examined the function of RhoA in myogenesis, the role of RhoA in muscle differentiation is still not well understood (13, 30, 31, 32, 33). To investigate RhoA activity during myogenesis in vitro, we measured RhoA activity in H9c2 myoblasts using a pull-down assay that captured only the active GTP-bound form of RhoA. The level of the GTP-bound RhoA was high in proliferation medium (PM). However, it decreased rapidly within 3 h of being shifted to differentiation medium (DM), then continued to decrease gradually for up to 6 d after being transferred to DM. Total RhoA levels did not change during muscle differentiation (Fig. 1Go, A and B).


Figure 1
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  		Fig. 1. RhoA Activity during Muscle Differentiation

A, GTP-bound RhoA pull-down assay. Cell lysates prepared in PM and DM at the indicated time periods were subjected to a pull-down assay for the GTP-bound form of RhoA or Western blot analysis with anti-RhoA and anti-RBD antibodies. Total RhoA was immunoblotted to ensure comparable amount of cell lysates. RBD (Rho-binding domain) indicates the same amount of GST-rhotekin RBD agarose beads for this assay. B, Densitometric analysis. Data are shown as percentage over PM condition ± SE and are representatives of three independent experiments.

 
To monitor the changes of ROK activity during muscle differentiation, we first measured ROK activity by Western blot analysis. As observed in GTP-bound RhoA, the phosphorylation level of MYPT1, a known specific substrate of ROK, was high in PM but was reduced within 3 h in DM. ROK expression was sustained for up to 6 d in DM (Fig. 2Go, A and B). To further study the ROK activity during muscle differentiation, MYPT1 fused to GST was used as a substrate for the kinase assay. Endogenous ROK was immunoprecipitated from H9c2 cell extracts that were prepared in PM, as well as at different time points in DM, and kinase activity assays were performed using these immunoprecipitates. The changes of ROK activity measured by these assays were similar to those observed by Western blot analysis (Fig. 2CGo).


Figure 2
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  		Fig. 2. ROK Activity during Muscle Differentiation

A, Western blot analysis. ROK activity was measured by testing phosphorylation levels of MYPT-1, a ROK substrate. Expression levels of total MYPT-1 and ROK were measured by Western blot analysis. B, Densitometric analysis. Data are expressed as percentage of PM condition for each experiment and are shown as the mean ± SE. C, ROK activity assay. Phosphorylation levels of recombinant GST-MYPT1 were detected by radioactivity counting. Data are shown as percentage over PM condition and represent the mean ± SE of three independent experiments done in triplicate dishes. Asterisks indicate P < 0.05, compared with PM.

 
Y27632 Stimulates Muscle Differentiation
To further assess the inhibitory mechanisms of muscle differentiation by ROK, we examined the effects of Y27632, a pharmacological inhibitor of ROK. After H9c2 and C2C12 myoblast cells were cultured in PM and DM for 4 d in the presence or absence of Y27632, morphological changes were observed and the expression levels of myogenic markers, myogenin and myosin heavy chain (MHC), were measured. As shown in Fig. 3Go, A and B, syncytia that included a number of nuclei were increased in the presence of Y27632, even in PM, in both H9c2 (Fig. 3AGo) and C2C12 cells (Fig. 3BGo). Expression levels of myogenin and MHC were also strongly induced after Y27632 treatment in PM (Fig. 3Go, C and D). Consistent with these results, treatment of myoblasts with Y27632 in DM for 4 d accelerated formation of myotubes (Fig. 3Go, A and B) and increased expression of myogenin or MHC, compared with cells that were not treated with Y27632 in DM for 4 d (Fig. 3Go, C and D).


Figure 3
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  		Fig. 3. Effects of ROK Inhibitor Y27632 on Muscle Differentiation

H9c2 and C2C12 cells were incubated in PM and DM for 4 d in the absence or presence of 10 µM Y27632. Then, differentiation was evaluated based on morphological changes (A and B) and expression levels of differentiation markers, myogenin and MHC, using Western blot analysis (C and D). Actin protein was used to standardize the amount of sample proteins for Western blot analysis.

 
IRS-1/2 Tyrosine Phosphorylation and PI 3-Kinase Activity Increase during Differentiation
It is well known that insulin and IGF-I/2 induce muscle differentiation through PI 3-kinase activation (5, 6, 7). To determine whether IRS-1/2 was phosphorylated at serine or tyrosine residues during myogenic differentiation, the IRS-1/2 immunoprecipitates were analyzed by Western blotting in PM and DM for 2, 4, and 6 d. As shown in Fig. 4AGo, the serine phosphorylation and expression levels of IRS-1/2 were high, but their tyrosine phosphorylation, association with p85 subunit, or PI 3-kinase activity was low in PM, compared with DM. However, these behaviors reversed with time in DM.


Figure 4
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  		Fig. 4. Changes in Phosphorylation States of IRS-1/2 and Myogenic Signaling Molecules during Muscle Differentiation

A, Immunoprecipitation (IP). Cell lysates at the indicated time periods were immunoprecipitated by anti-IRS-1/2 antibodies. The immunoprecipitates were analyzed by Western blot (IB) analysis using antibodies specific to IRS-1/2, phosphoserine, phosphotyrosine (PY20), and p85. PI 3-kinase activity on IRS-1/2 immunoprecipitates was measured as described in Materials and Methods. PIP indicates the phosphorylated lipid. B, Western blot analysis. Cell lysates were analyzed for IRS-1 phosphorylation levels at Ser307 and Ser636/639 and its expression levels. C and D, Normalization of samples to IRS-1/2 protein amounts. To normalize sample amounts to IRS-1/2, three times higher amounts of cell lysates in DM than those in PM were subjected to immunoprecipitation by anti-IRS-1/2 antibodies (C), or Western blot analysis (D). Actin levels represent the loaded sample amounts. All these experiments were repeated at least three times and typical data are presented.

 
Because phospho-specific antibodies against Ser307 and Ser636/639 of IRS-1 were commercially available, we also tested the phosphorylation state of those serine residues during myogenesis. In accordance with changes in serine phosphorylation status, the phosphorylation levels at Ser307 and Ser636/639 of IRS-1 were high in PM, but decreased during myogenesis in both H9c2 and C2C12 cells (Fig. 4BGo). Interestingly, protein and serine phosphorylation levels of IRS-1/2 concomitantly decreased in DM, although serine phosphorylation seemed to decrease more dramatically than protein levels of IRS-1/2 (Fig. 4Go, A and B). Therefore, we tried to use IRS-1/2 proteins instead of actin as a normalization control to clarify the results. For this purpose, we immunoprecipitated IRS-1/2 from three times more samples prepared in DM than those in PM to set equal amounts of IRS-1/2. Actin level clearly indicates that samples prepared in DM were used much more in quantity than samples prepared in PM (Fig. 4CGo). Even in these experiments, serine phosphorylation levels of IRS-1/2 significantly decreased in DM, indicating that decrease in serine phosphorylation of IRS-1/2 really occurs during muscle differentiation. Consistently, Western blotting analysis also showed that phosphorylation of IRS-1 at Ser307 and Ser636/639 residues profoundly decreased 3 and 6 d after incubation in DM, even when protein levels were normalized to IRS-1 (Fig. 4DGo).

Increased Binding of ROK to IRS-1 and IRS-2 in PM Diminishes in DM
ROK has been demonstrated to directly phosphorylate IRS-1 at serine residues (34, 35, 36). To investigate the interaction between ROK and IRS-1/2 during myogenesis, we examined whether ROK is coimmunoprecipitated with IRS-1/2. First, we performed Western blotting for IRS-1/2 after immunoprecipitation with anti-ROK antibody. Then, we also tried to coimmunoprecipitate ROK, using anti-IRS-1/2 antibodies. In both experiments, we got the same results, that is, ROK bound to IRS-1/2 in PM, but this binding diminished in the cell lysates incubated in DM for 4 d (Fig. 5Go, A and B). Protein levels of ROK and IRS-1/2 detected by Western blot again indicates that IRS-1/2, but not ROK, changes in protein amount during muscle differentiation. Finally, to further clarify the diminished interaction between ROK and IRS-1/2 during muscle differentiation, we coimmunoprecipitated ROK using anti-IRS-1/2 antibodies after adjusting sample amounts as done in Fig. 4Go, C and D. These experiments again showed the decreased binding of IRS-1/2 to ROK during muscle differentiation. Western blotting analysis showed that protein levels of IRS-1/2 were equally applied for these experiments (Fig. 5CGo).


Figure 5
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  		Fig. 5. Binding of ROK to IRS-1 and IRS-2

Cell lysates were prepared from H9c2 cells in PM or in DM for 4 d. The lysates were subjected to immunoprecipitation (IP) by anti-ROK (A) or anti-IRS-1/2 (B) antibodies and the immunoprecipitates were analyzed by Western blotting (IB) with antibodies specific to IRS-1/2 and ROK. Note that IRS-1/2, but not ROK, change in their quantity during muscle differentiation. Samples were normalized to IRS-1/2 protein amounts and then subjected to immunoprecipitation (C). Western blot analysis shows the equal amount of IRS-1/2 in PM and DM. Data are representative of three different experiments. IP, Immunoprecipitation; IB, immunoblotting.

 
ROK Increases IRS-1/2 Serine Phosphorylation but Decreases Tyrosine Phosphorylation and PI 3-Kinase Activity
As shown above, Y27632 induced muscle differentiation, even in PM, and the binding of ROK to IRS-1/2 in PM was no longer present in DM for 4 d. Therefore, we hypothesized that myogenic signaling molecules could be activated in PM if ROK is inactivated. To test this hypothesis, we first examined the changes of GTP-RhoA in the presence or absence of Y27632, both in PM and DM for 4 d. Next, we determined IRS-1/2 serine or tyrosine phosphorylation state and PI 3-kinase activity as well as recruitment of the p85 subunit to tyrosine-phosphorylated IRS-1/2.

Levels of GTP-RhoA were not changed by Y27632 treatment in PM and DM for 4 d (Fig. 6AGo). However, IRS-1/2 serine phosphorylation decreased and their tyrosine phosphorylation increased in the presence of Y27632 in PM, despite Y27632 not affecting expression levels of IRS-1/2 (Fig. 6Go, B and C). Their association with p85 and the subsequent PI 3-kinase activity also increased in PM containing Y27632, compared with PM alone. Additionally, when Y27632 was present in DM for 4 d, the ratio of tyrosine to serine phosphorylation in IRS-1/2 was enhanced, as were their association with p85 and PI 3-kinase activity, compared with DM alone (Fig. 6Go, B and C). Additionally, phosphorylation level of p70S6kinase, known as a downstream molecule of PI 3-kinase, increased similarly to PI 3-kinase activity without change in its protein level, when Y27632 was added to PM and DM for 4 d (Fig. 6DGo).


Figure 6
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  		Fig. 6. Effects of Y27632 on Myogenic Signaling Molecules

H9c2 cells were cultured with or without Y27632 for 4 d in PM and DM. A, GTP-bound RhoA pull-down assay. Cell lysates were subjected to a pull-down assay for the GTP-bound form of RhoA. Total RhoA was also immunoblotted to ensure comparable amount of cell lysates. RBD indicates the same amount of GST-rhotekin RBD agarose beads for this assay. B and C, Immunoprecipitation (IP). Immunoprecipitates by anti-IRS-1 (B) and anti-IRS-2 (C) antibodies were subjected to Western blot (IB) analysis and PI 3-kinase assays. PIP indicates the phosphorylated lipid analyzed by ascending chromatography. D, Western blot analysis of p70S6kinase. Phosphorylation and protein levels of p70S6kinase were measured by Western blotting analysis using anti-phospho-p70S6kinase (anti-Thr421/Ser424) and anti-p70S6kinase antibodies. All experiments were repeated at least three times and typical data are presented.

 
Because Y27632 also inhibited PKC-related kinase-2 and PKN at high concentrations, we tested the effects of ROK on muscle differentiation and myogenic signaling molecules by overexpressing ROK-RB and a CAT-ROK using adenoviral-mediated gene transfer techniques. LacZ was used as a negative control. Similar to Y27632, ROK-RB induced the differentiation process, even in PM, and accelerated differentiation in DM for 4 d. However, CAT-ROK blocked differentiation both in PM and DM (Fig. 7AGo). ROK-RB showed the same effects as Y27632 on IRS-1/2 serine or tyrosine phosphorylation as well as PI 3-kinase activity. In contrast, the serine or tyrosine phosphorylation states of IRS-1/2 and PI 3-kinase activity that were observed in DM for 4 d were almost completely reversed by overexpression of CAT-ROK (Fig. 7BGo).


Figure 7
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  		Fig. 7. Effects of ROK-RB and CAT-ROK on Muscle Differentiation

A, Muscle differentiation. H9c2 cells were infected with recombinant adenovirus expressing LacZ, ROK-RB (RB), or CAT-ROK (CAT), and then further incubated for 4 d in PM and DM. Muscle differentiation was evaluated by morphological changes (top) and expression levels of myogenin. Actin protein was used to standardize the amount of sample proteins for Western blot analysis (bottom). B, Changes in myogenic signaling molecules. Immunoprecipitation (IP) was performed using anti-IRS-1 (top) and anti-IRS-2 (bottom) antibodies, and the immunoprecipitates were analyzed by Western blot (IB) analysis using the indicated antibodies. PI 3-kinase assays were performed with anti-IRS-1 or anti-IRS-2 immunoprecipitates. PIP indicates the phosphorylated lipid analyzed by ascending chromatography. All experiments were repeated at least three times, and typical data are presented.

 
Fibroblast Growth Factor (FGF)-2 Activates ROK in Differentiation Medium
Previous studies have indicated that myogenic cell lines do not differentiate when exposed to FGFs or TGFß (37, 38). Because FGF-2 receptors (FGFR-1) were reported to be deficient in H9c2 cells (39), but present in C2C12 cells, we tested C2C12 cells instead of H9c2 cells. FGF-2 inhibited muscle differentiation and enhanced cell proliferation both in PM and DM (data not shown), consistent with previous reports (37, 38, 40).

FGF-2 in DM for 4 d increased the levels of GTP-bound RhoA and ROK activity in comparison with DM alone, similar to the results observed in PM. As expected, inhibition of ROK by Y27632 in FGF-2-treated cells in DM for 4 d significantly reduced FGF-2-induced ROK activity, but not the level of GTP-bound RhoA (Fig. 8Go, A and B). In the coimmunoprecipitation experiments with ROK antibody, the loss of ROK binding to IRS-1 and IRS-2 in DM was restored by FGF-2 treatment in DM for 4 d (Fig. 8CGo).


Figure 8
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  		Fig. 8. Effects of FGF-2 on Myogenic Signaling Molecules

C2C12 Cells were induced to differentiate to myotubes in DM for 4 d with or without 10 µM Y27632. FGF-2 at 10 ng/ml concentration was added 30 min after Y27632 treatment. A, GTP-bound RhoA pull-down assay. Cell lysates were prepared in PM and DM and subjected to a pull-down assay for the GTP-bound form of RhoA. Total RhoA was immunoblotted to ensure comparable amount of cell lysates. RBD indicates the same amount of GST-rhotekin RBD agarose beads for this assay. B, ROK activity. Data are shown as percentage over PM condition ± SE and are representatives of three independent experiments. *, P < 0.05 vs. PM; **, P < 0.05 vs. DM; #, P < 0.05 vs. DM + FGF. C, Immunoprecipitation (IP). Cell lysates cultured with or without FGF-2 in DM for 4 d were subjected to immunoprecipitation by anti-ROK antibody, followed by Western blot (IB) analysis using antibodies specific to IRS-1/2. ROK represents the same amount of sample loading. D, Changes in myogenic signaling molecules. After immunoprecipitation using anti-IRS-1 antibody, IRS-1 serine or tyrosine phosphorylation level, and association with p85 were measured by Western blot analysis. PI 3-kinase activity was determined as described in Materials and Methods. E, Normalization of sample amounts to IRS-1. After sample amounts were normalized to IRS-1 protein amount, serine phosphorylation levels of IRS-1 were measured by Western blot analysis using phospho-IRS-1 (Ser307) antibody. Actin bands represent the loaded sample amounts. All experiments were repeated at least three times and typical data are presented.

 
Additionally, FGF-2 in DM increased Ser307 phosphorylation, but decreased tyrosine phosphorylation of IRS-1, which was followed by the decreased association of p85 with IRS-1 and PI 3-kinase activity, as observed in PM. The effects of FGF-2 on these molecules were reversed by cotreatment of Y27632 and FGF-2 in DM for 4 d (Fig. 8DGo). Because IRS-1 protein level decreased in DM regardless of absence or presence of FGF-2 and Y27632, we normalized the protein amounts to IRS-1, instead of actin, and then tested how phosphorylation of IRS-1 at Ser307 is changed by FGF-2 or FGF-2 plus Y27632 treatment in DM. Results showed that FGF-2 increases Ser307 phosphorylation of IRS-1, and this increase is inhibited by cotreatment of FGF-2 and Y27632 in DM (Fig. 8EGo). The increased actin level in all DM conditions indicates the higher amount of sample loadings to normalize protein levels to IRS-1, as shown by IRS-1 protein level. All of these findings were also observed in IRS-2 (data not shown).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
This study represents the first evidence of a key role of RhoA/ROK signaling in blocking myoblast cell differentiation by phosphorylating serine residues of IRS-1/2. Although RhoA was previously proposed to be a positive regulator of muscle differentiation (13, 30, 31), there have also been many reports that the RhoA/ROK pathway blocks muscle differentiation (32, 33, 41). Consistent with these reports, we demonstrated that inhibition of ROK by Y27632 and ROK-RB induced muscle differentiation in H9c2 cells as well as C2C12 cells in proliferating condition. Additionally, CAT-ROK and FGF-2, one of inhibitory growth factors for muscle differentiation, blocked muscle differentiation through RhoA/ROK activation. Therefore, our results clearly indicate that the RhoA/ROK pathway negatively regulates myoblast differentiation.

Insulin and IGF-I/2 stimulate muscle differentiation by activating PI 3-kinase, the pivotal myogenic molecule, after recruitment of its p85 subunit to tyrosine phosphorylated IRS proteins (5, 6, 16). In PM, which contains 10% serum, concentrations of insulin and IGF-I/2 are ten times greater than those in DM (1% serum), but myoblast cells do not differentiate. A number of purified serum growth factors, including FGFs, TGFß, and platelet-derived growth factor-BB, have been found to be present in large quantities to block muscle differentiation in PM, even in the presence of higher concentrations of insulin and IGF-I/2. The decreased concentration of these inhibitory factors allows muscle differentiation in DM (42). There are several reports that ROK participates in phosphorylation of serine residues of IRS proteins (34, 35, 36), and serine phosphorylation of IRS proteins has been reported to have varying effects on signaling, including; 1) the negative feedback control that inhibits further tyrosine phosphorylation of IRS proteins (43), 2) desensitization of insulin signaling by stimulating subcellular redistribution of IRS-1 and sensitizing IRS-1 to the action of the proteasome (44, 45), 3) dissociation of IRS proteins from the insulin receptor, hindering tyrosine phosphorylation (46, 47), and 4) maintaining their tyrosine phosphorylated active conformation (48). Based on these reports, we speculated that the increased ROK activity blocks muscle differentiation by serine phosphorylation of IRS-1/2 followed by inhibition of their tyrosine phosphorylation despite the high concentrations of insulin and IGF-I/2 in PM. Consistent with our hypothesis, ROK strongly bound to IRS-1/2 in PM but dissociated from them in DM (Fig. 5Go). In addition, inhibition of the activated RhoA/ROK in PM by Y27632 (Fig. 6Go) and ROK-RB (Fig. 7Go) decreased IRS-1/2 serine phosphorylation but increased tyrosine phosphorylation, which led to an increase in PI 3-kinase and p70S6kinase activities, resulting in stimulation of the muscle differentiation process in both the H9c2 and C2C12 cells. Furthermore, activation of RhoA/ROK by CAT-ROK (Fig. 7Go) blocked muscle differentiation even in DM.

To analyze the effects of ROK on IRS-1/2 serine phosphorylation in a more defined condition, we added FGF-2 to DM to determine its affect on these signaling molecules. The results demonstrated that FGF-2, when added to DM, has the similar effects on IRS-1/2 serine and tyrosine phosphorylation and PI 3-kinase activity to PM (Fig. 8Go). Y27632 reversed the FGF-2 action on these molecules by inhibiting ROK. Because the serine/threonine kinase activity of ROK could directly phosphorylate IRS-1/2 as mentioned above, we concluded that ROK phosphorylates IRS-1/2 at serine residues in PM, and the inhibitory effects of ROK seem to disappear in DM because the inhibitory growth factors, including FGF-2, do not activate ROK due to their low concentrations.

IRS-1 contains greater than 100 potential serine phosphorylation sites (49) that are recognized by many kinases, including JNK (50), mTOR (18), PKCs (51), p70S6kinase (52), and ROK (34, 35, 36). It has been widely reported that insulin signaling via tyrosine phosphorylation of IRS-1 is negatively regulated by Ser307 phosphorylation of IRS-1 (50, 53, 54). Additionally, Ser307 and Ser636/639 phosphorylation by p70S6kinase (52) and Ser636/639 by the activated PI 3-kinase/Akt/mTOR pathway (18) resulted in negative regulation of insulin signaling. In our results, Ser307 and Ser636/639 of IRS-1 also concomitantly increased in PM but decreased in DM both in H9c2 and C2C12 cells. In addition, Y27632 suppressed serine phosphorylation at these sites. Therefore, phosphorylation at these serine residues in IRS-1 seems to play a key role in blocking muscle differentiation. Recently, Furukawa et al. (34) reported that phosphorylation on Ser632/635 residues in human IRS-1 (homologs to Ser636/639 in mouse IRS-1) positively regulates insulin action by facilitating tyrosine phosphorylation of IRS-1. The reasons for this discrepancy are not currently known. Zick (55) proposed that the dual role of these serine phosphorylations in IRS-1 serves either to enhance or terminate insulin signaling under certain conditions. We also hypothesize that the discrepancy may be due to stage-specific conditions because we used myoblast cells, whereas Furukawa et al. (34) used the fully differentiated myotube cells.

In summary, the RhoA/ROK signaling pathway is activated by myogenic inhibitory growth factors including FGF-2 in PM, and the activation of this signaling pathway blocks muscle differentiation via increased serine phosphorylation of IRS-1/2, followed by decreases in tyrosine phosphorylation of IRS-1/2, and PI 3-kinase and p70S6kinase activities. When this inhibitory signaling is turned off due to low concentrations in DM, insulin and IGF-I/2 may turn on and propagate the myogenic signaling pathway by phosphorylating IRS-1/2 at tyrosine residues, which ultimately leads to full muscle differentiation. Therefore, we propose that serine phosphorylation of IRS-1/2 by RhoA/ROK activation represents an on/off switch for muscle differentiation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Reagents
DMEM, DMEM-F12, donor calf serum, horse serum, and fetal bovine serum (FBS) were purchased from Invitrogen Life Technologies (Grand Island, NY). Y27632 and basic fibroblast growth factor (FGF-2) were purchased from Calbiochem (San Diego, CA). [{gamma}-32P]ATP was purchased from Amersham Biosciences (Little Chalfont, Buckinghamshire, UK). Protein A/G plus-agarose, antibodies specific to MHC, myogenin, actin, MYPT-1, IRS-1/2, phosphoserine, phosphotyrosine (PY20), p85 PI 3-kinase, RhoA, ROK, goat antimouse IgG-horseradish peroxidase (HRP), goat antirabbit IgG-HRP, and donkey antigoat IgG-HRP were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-phospho-MYPT1 (Thr696), myosin phosphatase target MYPT1 substrate (GST-MYPT1), and rhotekin rho-binding domain (Rhotekin RBD) agarose beads were purchased from Upstate Biotechnology (Lake Placid, NY). Antibodies specific to p-IRS-1 (Ser307 and Ser636/639), p-p70S6kinase (Thr421/Ser424), and p70S6kinase were purchased from Cell Signal Technology (Beverly, MA). The Ser312 and Ser632/S635 sites of IRS-1 for humans are homologs to Ser307 and Ser636/639 in mice (34).

Cell Culture
H9c2 rat cardiac and C2C12 mouse myoblasts were grown in 10-cm-diameter dishes in DMEM/F-12 containing 10% (vol/vol) donor calf serum and DMEM supplemented with 10% FBS (proliferation medium, PM), respectively. Cells were induced to differentiate by placing them in DMEM/F-12 containing 1% (vol/vol) horse serum or 1% (vol/vol) FBS (differentiation medium, DM). Full differentiation was achieved 6 d after induction of differentiation. Differentiation was evaluated based on the morphological appearance of cells and expression levels of the differentiation markers such as myogenin and MHC. Unless otherwise specified, the culture media were replaced with fresh media daily.

Immunoprecipitation
Cells were washed twice with cold PBS and lysed in cold lysis buffer [50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Nonidet P-40, 1% sodium deoxycholate, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM Na3VO4, 1 mM NaF]. Cell lysates were incubated with anti-IRS-1, anti-IRS-2, or anti-ROK antibody (2 µg) for 2 h at 4 C. Proteins A/G conjugated to agarose beads were added for 1 h. Beads were washed three times with washing buffer [50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Nonidet P-40, 1 mM PMSF, 1 mM Na3VO4, 1 mM NaF] and boiled for 5 min before electrophoresis.

GTP-Bound RhoA Pull-Down Assay
The activation of RhoA was assessed using a previously described pull-down assay (56). Briefly, cells were lysed with cold Rho binding lysis buffer [50 mM Tris-HCl (pH 7.2), 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 500 mM NaCl, 10 mM MgCl2, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 1 mM PMSF]. Next, cell lysates were clarified by centrifugation at 14,000 x g at 4 C for 15 min. The supernatants (500 µg) were incubated with 20 µg of GST-rhotekin RBD agarose (Upstate Biotechnology, Chicago, IL) for 45 min at 4 C. The beads were washed three times with pull-down buffer (Tris-HCl buffer containing 1% Triton X-100, 150 mM NaCl, 10 mM MgCl2, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 0.1 mM PMSF), and bound GTP-RhoA was detected by Western blotting with anti-RhoA antibody.

ROK Enzyme Activity
ROK was immunoprecipitated from whole cell lysates by incubating the precleared lysate proteins (200 µg) with anti-ROK antibody (2 µg) at 4 C and the immunoprecipitates then washed twice with lysis buffer and three times with kinase buffer [5 mM MgCl2, 5 mM 2-glycerophosphate, 50 mM Tris-HCl (pH 7.5), 0.1 mM EGTA, 0.1% 2-mercaptoethanol], and immediately used for the kinase assay. The beads were incubated for 10 min at 30 C with 0.6 mg/ml MYPT1 in the presence of [{gamma}-32P]ATP (200 µCi/ml) and 10 µM ATP in the kinase buffer. The aliquots (25 µl) were then added to phosphocellulose disks. The disks were washed for 10 min with 75 mM phosphoric acid and dried. The 32P radioactivity was then determined by adding 5 ml of scintillation fluid to vials containing the disks. Values were converted from counts to the percentage of control.

PI 3-Kinase Activity
PI 3-Kinase assays were performed as previously described (17). PI 3-kinase activity was assayed in the IRS-1 or 2 immunoprecipitates by conversion of PI to phosphatidylinositolphosphate (PIP). Cells were solubilized by incubation for 30 min in 1 ml of lysis buffer [20 mM Tris-HCl (pH 7.5), 137 mM NaCl, 1 mM MgCl2, 1 mM CaCl2, 100 µM Na3VO4, 2 mM EDTA, 1% Nonidet P-40, 10% glycerol, 5 µg/ml leupeptin and 1 mM PMSF]. Anti-IRS-1 or -2 antibody conjugated agarose (20 µl) was incubated with supernatants containing 500 µg of protein for 1 h at 4 C. After washing, the immunoprecipitates were resuspended in 100 µl of kinase assay buffer [20 mM Tris-HCl (pH 7.6), 75 mM NaCl, 10 mM MgCl2, 200 µg/ml PI, and 10 µCi [{gamma}-32P] ATP], and incubated for 20 min at room temperature with constant shaking. The reaction was stopped by addition of 100 µl of 1 N HCl and 200 µl of CHCl3-methanol (1:1). The samples were centrifuged, and the lower organic phase was harvested and applied to a silica gel thin-layer chromatography plate (Merck, Whitehouse Station, NJ) coated with 1% potassium oxalate. Thin-layer chromatography plates were developed in CHCl3-CH3OH-H2O-NH4OH (60:47:11.3:2), dried, and visualized by autoradiography.

Adenovirus-Mediated ROK Expression
Adenoviral vectors carrying CAT-ROK and ROK-RB were kindly provided by Dr. Sandro Rusconi (University of Fribourg, Switzerland). H9c2 cells were rinsed twice with serum-free medium and infected with the recombinant adenovirus CAT-ROK or ROK-RB at titer of a multiplicity of infection of 200 for 3 h followed by addition of 10% serum medium and further incubated for 24 h.

Western Blot Analysis
The immunoprecipitates or total lysates were separated by SDS-PAGE and transferred onto nitrocellulose membrane. After blocking with 1.5% BSA, the membrane was incubated with the indicated primary antibody followed by a secondary antibody. Samples were finally detected using enhanced chemiluminescence (Amersham Biosciences). For quantification, band intensity was measured by Bio-Rad imaging densitometric scan (Quantity One, software version 4.6.2).

Statistical Analysis
Results were expressed as mean ± SE. The difference between two mean values was analyzed using the Student’s t test. The difference was considered statistically significant when P < 0.05.


   ACKNOWLEDGMENTS
 
We thank Dr. Sandro Rusconi (University of Fribourg, Switzerland) for the generous gift of adenoviral vectors carrying CAT-ROK and ROK-RB.


   FOOTNOTES
 
This work was supported by grant from the Korea Science & Engineering Foundation (No. R13-2002-020-01001-0).

Disclosure Statement: The authors have nothing to disclose.

First Published Online June 19, 2007

Abbreviations: CAT-ROK, Constitutive active ROK; DM, differentiation medium; FBS, fetal bovine serum; FGF, fibroblast growth factor; HRP, horseradish peroxidase; IRS, insulin receptor substrate; MHC, myosin heavy chain; mTOR, mammalian target of rapamycin; PI, phosphatidylinositol; PIP, phosphatidylinositolphosphate; PKB, protein kinase B; PKC, protein kinase C; PKN, protein kinase N; PM, proliferation medium; PMSF, phenylmethylsulfonyl fluoride; PTB, phosphotyrosine binding; ROK, Rho kinase; ROK-RB, dominant-negative ROK.

Received for publication February 28, 2007. Accepted for publication June 8, 2007.


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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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