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Originally published In Press as doi:10.1074/jbc.M907412199 on April 11, 2000

J. Biol. Chem., Vol. 275, Issue 25, 18767-18776, June 23, 2000
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Stabilization of MyoD by Direct Binding to p57Kip2*

Emmanuel G. ReynaudDagger§, Marie Pierre Leibovitch§, Lionel A. J. Tintignac, Karine Pelpel, Martine Guillier, and Serge A. Leibovitch||

From the Laboratoire de Génétique Oncologique UMR 1599, Centre National de la Recherche Scientifique, Institut Gustave Roussy, 39, rue Camille Desmoulins, 94805 Villejuif, France

Received for publication, September 10, 1999, and in revised form, February 22, 2000

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Recent data have demonstrated the role of Cdk1- and Cdk2-dependent phosphorylation of MyoDSer200 in the regulation of MyoD activity and protein turnover. In the present study, we show that in presence of p57Kip2, MyoDAla200, a MyoD mutant that cannot be phosphorylated by cyclin-Cdk complexes, displayed activity 2-5-fold higher than of MyoDAla200 alone in transactivation of muscle-specific genes myosin heavy chain, creatine kinase, and myosin light chain 1. Furthermore, p57Kip2 increases the levels of MyoDAla200 in cotransfected cells. This result implies that p57Kip2 may regulate MyoD through a process distinct from its function as a cyclin-dependent kinase inhibitors. We report that overexpression of p57Kip2 increased the half-life of MyoDAla200. This increased half-life of MyoD involves a physical interaction between MyoD and p57Kip2 but not with p16Ink4a, as shown by cross-immunoprecipitation not only on overexpressed proteins from transfected cells, but also on endogenous MyoD and p57Kip2 from C2C12 myogenic cells. Mutational and functional analyses of the two proteins show that the NH2 domain of p57Kip2 associates with basic region in the basic helix-loop-helix domain of MyoD. Competition/association assays and site-directed mutagenesis of the NH2 terminus of p57Kip2 identified the intermediate alpha -helix domain, located between the Cdk and the cyclin binding sites, as essential for MyoD interaction. These data show that the alpha -helix domain of p57Kip2, which is conserved in the Cip/Kip proteins, is implicated in protein-protein interaction and confers a specific regulatory mechanism, outside of their Cdk-inhibitory activity, by which the p57Kip2 family members positively act on myogenic differentiation.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

A broad range of cellular proteins have been identified that together form a central signaling pathway to govern cell cycle progression. The retinoblastoma tumor-suppressor protein pRb inhibits cell proliferation by repressing a subset of genes that are controlled by the E2F family of transcription factors and which are involved in progression from the G1 to the S phase of the cell cycle (1). Upstream of pRB is the basic cell cycle machinery consisting of various cyclin-dependent kinases (Cdks)1 that regulate pRB and its related proteins through phosphorylation. Cyclin-dependent kinases are themselves regulated by a number of regulators including the cyclin-dependent kinase inhibitors (Ckis) (2). Ckis induce cell cycle arrest in response to anti-proliferative signals, including contact inhibition and serum deprivation (3), transforming growth factor-beta (4) and myogenic (5), myeloid (6), and neuronal differentiation (7). Ckis can be divided in two families (2, 8). The Ink4 family includes p16Ink4a, p15Ink4b, and p18Ink4c, which specifically bind and inhibit Cdk4 and Cdk6; and p19ARF, which has a tumor suppression function dependent upon the p53 (9). p21Cip1, p27Kip1, and p57Kip2, members of the other family of inhibitors, the Cip/Kip family, have the ability to inhibit all G1/S phase Cdks complexes (10-12). Although p21Cip1 expression during development correlates with terminally differentiating tissues, mice lacking p21Cip1 have normal development (13). p27Kip1-deficient mice have a grossly normal development but display several phenotypes that seem to be linked to cell proliferation (14-16). p57Kip2 is also a tightly binding inhibitor of cyclin D-Cdk4-Cdk6 complexes and a negative regulator of cell proliferation (17, 18). The expression pattern of p57 mRNA in various adult human tissues suggests that its distribution is more restricted than that of p21Cip1 and p27Kip1 (17, 18). The p57Kip2 gene is located in 11p15.5, a region suggested to be involved in sporadic cancers and the Beckwith-Wiedmann syndrome (17). Specific loss of the maternal allele has been observed in several types of childhood tumors including Wilm's tumors and rhabdomyosarcoma, suggesting a genomic imprinting, which has been confirmed both in mice and human. Generation of null mice for p57Kip2 has revealed a phenotype close to the syndrome observed in human. Mice died upon birth and showed macroglossia, omphalocele, gigantism, various levels of limb shortening, and an important modification in the skeletal muscle distribution, suggesting that p57Kip2 has an important role during development (19, 20). The lack of p21Cip1 or p27Kip1 functions does not lead to gross developmental defects, suggesting the existence of compensatory mechanisms during development. Such a redundant mechanism has been recently shown; mice lacking both p21Cip1 and p57Kip2 display severe defects in skeletal muscle development (and other tissues including lung). These two Ckis cooperate as terminal effectors of signaling pathways that impinge on cell cycle control and differentiation and control muscle differentiation at the myogenin step (21).

To undergo differentiation, myogenic cells have to exit the cell cycle through the G1 checkpoint. Myogenic differentiation is under the control of a family of muscle-specific transcription factors (MRFs), which includes MyoD (22), myogenin (23, 24), Myf5 (25), and MRF4 (26), also known as herculin (27) or Myf6 (28). These proteins share a central basic helix-loop-helix (bHLH) domain that is involved in DNA binding and protein-protein interactions (29). This 70-amino acid region accounts for their ability to form heterodimers with the E protein bHLH factors (30, 31), to bind as heterodimers to an E-box DNA consensus sequence (CANNTG) (29), to transactivate muscle genes, and to efficiently convert non-muscle cells to a myogenic lineage (32). MyoD is expressed in proliferating myoblasts prior to terminal differentiation (33). A number of molecular mechanisms have been proposed to explain the functional inactivation of MyoD in proliferating myoblasts and the coupling of muscle differentiation with the cell cycle arrest (34). These include inhibitory phosphorylation of the myogenic bHLH proteins (35-37), inhibition of the myogenic bHLH function via the Id family of dominant-negative helix-loop-helix factors (38), and either direct or indirect inhibition by the cyclin D-dependent kinases (39, 40). It has been previously shown that overexpression of cyclin D-Cdk complexes inhibited myogenic transcriptional activation mediated by MyoD (41) and that cell cycle arrest correlates with the induction of p21Cip1 by MyoD (42, 43). The role of Cdks in inhibiting muscle differentiation has been substantiated by the observation that forced expression of p21Cip1 and/or p16Ink4a in mitogen-stimulated myoblasts facilitates muscle differentiation in the absence of mitogen deprivation suggesting that an active cyclin-Cdk complex suppresses MyoD function in proliferating myoblasts (40). It has been recently proposed that Cdk phosphorylation of MyoD can target this protein for rapid degradation (44). Indeed, recent data show that direct phosphorylation of MyoDSer200 by Cdk1 or Cdk2 plays a crucial role in modulating MyoD half-life and myogenic activity (45). In contrast to Cdk1 and Cdk2, cyclin D1-Cdk4 complexes fail to phosphorylate MyoD (46). The cyclin-Cdk-mediated inhibition of myogenesis by cyclin D1 involves nuclear translocation of Cdk4 by cyclin D1 and the subsequent formation of a MyoD-Cdk4 complex that specifically inhibits the transactivation functions of MyoD in absence of Cdk4 kinase activity (46). Interestingly, degradation of MyoD by the ubiquitin-proteasome pathway is inhibited by the specific DNA sequence to which MyoD binds independently of its phosphorylation state. Formation of a proteolysis-resistant complex seems to be dependent on dimerization and DNA binding of MyoD proteins (47).

Here we show that half-life of MyoDAla200, which is not phosphorylatable in vivo by the cyclin-Cdk complexes, is augmented in the presence of p57Kip2, suggesting that a mechanism distinct from phosphorylation-dependent degradation stabilizes MyoD. We show that MyoD but not its partner E12 physically interacts with p57Kip2. Mutational and functional analyses of the two proteins demonstrate that the NH2 domain of p57Kip2 associates with the basic domain of MyoD. Moreover, competition/association and site-directed mutagenesis of the NH2-terminal domain of p57Kip2 define the intermediate alpha -helix structure, located between the cyclin and the Cdk binding sites, as the important structural element involved in this interaction. These results strongly suggest that p57Kip2 may function in myogenic differentiation via two distinct mechanisms; one is an inhibitor of active cyclin-Cdk complexes, which control the G1 phase of cell cycle and the phosphorylation-dependent degradation of MyoD (48), and the second mechanism, via a physical interaction, stabilizes MyoD independently of its phosphorylation state during the course of myogenic differentiation.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Plasmids-- pEMSV-MyoD, pEMSV-MyoD mutants, and pEMSV-E12 were generous gifts from the Weintraub laboratory. pEMSV-MyoDAla200 mutant was obtained from M. Kitzmann (45). Expression vectors pCMV-HA-MyoD was generated by cloning three hemagglutinin epitope tags (3Tag HA) at the amino terminus of cDNA insert in pcDNA3 (Invitrogen). The reporter plasmid MCK-Luc (p1256MCK), generously provided by S. Hauschka, contains the promoter-enhancer region from the mouse muscle creatine kinase. pEX10X-p57Kip2 was kindly supplied by J. Massagué. CMV-p16Ink4a was a kind gift from B. Heinglein. Cyclin D1 and Cdk4 were kindly supplied by C. Sherr. To create expression vectors, fragments containing the complete coding sequences were cloned into pcDNA3 expression vectors (Invitrogen) and/or in pEMSV scribe.

Expression vectors pCMV-HA-p57Kip2, pCMV-HA-Cdk4, and pCMV-HA-cyclin D1 were generated by cloning three hemagglutinin epitope tags (3Tag HA) at the amino terminus of cDNA inserts in pcDNA3.

pGEX-2TK-p57Kip2 was obtained by inserting in frame the NcoI-HindIII fragment from pEX10X-57Kip2, into the NcoI-HindIII sites of plasmid pGEX-2TK expression plasmid (Amersham Pharmacia Biotech). pGEX-2T-p57Delta CKI was constructed by inserting in frame the SmaI-PvuII fragment from pGEX-2TK-p57Kip2, at the SmaI site of pGEX-2T expression plasmid. pGEX-2TK-p57Delta CKI was constructed by deleting a NcoI-BglII fragment, of the pGEX-2TK-p57Kip2. pGEX-3X-p57Delta PAC was obtained by deleting the PvuII-SmaI fragment of pEX10X-p57Kip2, the plasmid at this stage was closed, amplified, and used to generate a deleted p57-containing fragment NcoI-HindIII inserted in-frame into pGEX-2TK expression vector. pGEX-3X-p57PAC was obtained by inserting in-frame the PvuII-SmaI fragment, obtained from pGEX-2TK-p57Kip2, at the SmaI-filled site of pGEX-3X. pGEX-2TK-p57Delta QT was generated by inserting in-frame the BlpI-filled-in HindIII fragment, obtained from pEX10X-p57Kip2, at the SmaI site of pGEX-2TK. pGEX-2TK-p57QT was generated by inserting in frame the SmaI fragment, obtained from pGEX-2TK-p57Kip2, at the SmaI site of pGEX-2TK. p57Kip2 mutant R33L was generated by polymerase chain reaction with a 5' primer (5'-GAGCTGGGCCTCGAGCTGCGGATGC-3') and a 3' primer (3'-GCATCCGCAGCTCGAGGCCCAGCTC-3') using pEMSV-scribe p57Kip2 wild type as template and the QuickChangeTM site-directed mutagenesis kit (Stratagene, Ozyme) as instructed by the manufacturer. The Rsa mutated insert was then subcloned in-frame in pGEX-3X expression plasmid at the EcoRI site filled in by the Klenow polymerase to create pGEX-p57R33L.

Cell Cultures, DNA Transfection, and Luciferase Assays-- The mouse skeletal muscle cell line C2C12 and the fibroblastic cell line C3H10T1/2 were maintained in growth medium supplemented with antibiotics (a mixture of penicillin and streptomycin (Life Technologies, Inc.) and with 20% and 15% of fetal calf serum in Dulbecco's modified Eagle's medium, respectively. C2C12 cells were transfected by the calcium phosphate procedure as described previously (49). C3H10T1/2 fibroblasts were transfected by using polyethylenimine essentially as described (50). Briefly, 3 × 104 cells/well were plated onto 24-well plates. On the following day, cells were transfected with various combinations of plasmids, as indicated in legends of the figures. The total amount of DNA used for each plate was normalized with the respective empty expression vector. Luciferase activity was determined in aliquots of cell extracts from harvested cells 48 h after transfection in growth medium. One hundred nanograms of the pEGFP-C1 plasmid (Invitrogen) was included in transfections as an internal control for transfection efficiency. All luciferase activities were determined with equivalent quantities of proteins in triplicate and repeated at least twice.

Cycloheximide Treatment-- C3H10T1/2 cells were transfected with either pCDNA3-HA-MyoD or pCDNA3-HA-MyoDAla200 alone or with pEMSV-p57Kip2 in six-well plates as described above. Transfected cells were treated with cycloheximide (Sigma) at 15 µg/ml for the indicated times and harvested for Western blot analyses. HA-MyoD and HA-MyoDAla200 were detected using anti-HA antibodies (12CA5, Roche Molecular Biochemicals). For each experiment, alpha -tubulin was used as an internal control. Western blots were scanned and quantified by using a GelScan (Amersham Pharmacia Biotech).

Protein Expression, Purification, and GST Pull-down Assay-- Bacterial expression of proteins was performed in Escherichia coli BL21. Protein induction, cell lysis, and affinity purification with glutathione-agarose beads (Sigma) were done as described previously (51). In brief, GST-MyoD and GST-p57Kip2 fusion proteins were prepared and the fusion proteins were not eluted but washed four times at 4 °C in NTEN buffer (20 mM Tris, pH 8, 100 mM NaCl, 1 mM EDTA, 0, 5% Nonidet P-40) containing protease inhibitors and phosphatase inhibitors. Fusion proteins were collected on glutathione-Sepharose 4B (Amersham Pharmacia Biotech), and then the purity of the GST and GST fusion proteins were analyzed by SDS-PAGE and estimated to be 70-80% of purity by Coomassie Brillant Blue staining of the gels. 35S-Labeled proteins were prepared by coupled in vitro transcription-translation using the TnT-coupled rabbit reticulocyte lysate system (Promega). GST pull-down assays were performed as described previously (51). The programmed lysates (1-10 µl) were incubated with GST alone and GST fusion proteins overnight at 4 °C. Beads were washed four times in NTEN buffer at room temperature and then mixed with one volume of 2× SDS loading buffer, and bound proteins were analyzed by SDS-PAGE by using standard procedures.

For the competition/association assays, GST-p57Kip2-covered beads were first incubated with 35S-labeled in vitro translated MyoD for 2 h at 4 °C. Then, unbound 35S-labeled MyoD protein was removed by three wash cycles of binding buffer. Increasing amounts of labeled cyclin D1, Cdk4, or cotranslated cyclin D1-Cdk4 complexes were then added to the binding reactions, and the resulting mixtures were subjected to a GST pull-down assay. The reaction products were separated on SDS-PAGE. Bound proteins were detected by autoradiofluorography and quantified by using PhosphorImager.

Antibodies, Immunoprecipitation, and Western Blot Analyses-- Whole cell extracts from cultured cells were prepared in ice-cold radioimmune precipitation EGTA buffer (50 mM Hepes, pH 7.6, 150 mM NaCl, 2.5 mM EGTA, 1 mM EDTA, 10 µM beta -glycerophosphate, 0.1 mM sodium orthovanadate, 1 mM NaF, 0.1% Tween 20, 10% glycerol, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, 1 µg/ml aprotinin, and 1 µg/ml pepstatin). Lysates were centrifuged at 4 °C for 10 min in a microcentrifuge set at maximum speed, and the supernatant was stored at -80 °C in small aliquots. For immunoprecipitation, precleared cell lysates in immunoprecipitation buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 10% glycerol, 0.5% Nonidet P-40, 0.5 mM sodium orthovanadate, 50 mM NaF, 80 µM beta -glycerophosphate, 10 mM sodium pyrophosphate, 1 mM dithiothreitol, 1 mM EGTA, 10 µg/ml leupeptin, 10 µg/ml pepstatin, and 10 µg/ml aprotinin) were incubated with the indicated antibodies for 2-3 h at 4 °C with gentle agitation. Immunocomplexes bound to protein G-Sepharose were collected by centrifugation and washed several times in immunoprecipitation buffer. Immunoprecipitated proteins were resolved by 10% SDS-PAGE, followed by autoradiofluorography (35S-labeled proteins) and/or autoradiography.

For immunoblot analysis, total cell extracts or immunoprecipitates were solubilized in radioimmune precipitation EGTA buffer and processed as described previously (52). Analyses were performed on 10% polyacrylamide gels with a 5% polyacrylamide stacking gel. Electrophoretic transfer of proteins from SDS-PAGE gels to nitrocellulose membranes were blocked with 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 0.05% Tween 20 containing 5% skim milk and incubated overnight at 4 °C with primary antibodies: polyclonal C20 anti-MyoD diluted 1/500, polyclonal E-17 anti-mouse p57Kip2 diluted 1/250, polyclonal C-22 anti-Cdk4 diluted 1/1000, and polyclonal M-156 anti-p16 diluted 1/250 were provided by Santa Cruz Biotechnology (Santa Cruz, CA). The monoclonal 12CA5 anti-HA antibody was provided by Roche Molecular Biochemicals. Monoclonal anti-myosin light chain 1 clone MY-21 diluted 1/200, monoclonal anti-myosin heavy chain clone MY-32 diluted 1/400, and anti-alpha -tubulin clone DM1A diluted 1/500 were supplied by Sigma. Membranes were washed and incubated 1 h with a peroxidase-conjugated secondary antibody (Sigma) at a dilution of 1/10,000 with polyclonal antibodies and 1/4,000 with monoclonal antibodies. After several washes, membranes were incubated with an enhanced chemiluminescence system (ECL, Amersham Pharmacia Biotech) according to the manufacturer's instructions. Exposure was done with Agfa Curix RP2 films and intensifying screens.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

p57Kip2 Increases Muscle-specific Gene Transactivation by MyoDAla200-- Phosphorylation of MyoD is one of the crucial mechanisms that control its activity in eukaryotic cells, and recent reports show that in proliferating myoblasts phosphorylation of MyoD at serine 200 (44) by Cdk1 or Cdk2 (45) appears to play a major role in modulating MyoD half-life and myogenic activity. We have recently shown that p57Kip2 can stabilize MyoD protein by inhibiting cyclin E-Cdk2 kinase activity in growing myoblasts (48). Transient transfection assays were performed to compare the effects of p57Kip2 on MyoD wild type (MyoDwt) and MyoDAla200-mediated expression of muscle specific genes. C3H10T1/2 cells were transiently transfected with expression vectors encoding p57Kip2 and/or MyoDwt and MyoDAla200 along with a skeletal muscle reporter construct containing 1256 base pairs from the muscle creatine kinase promoter driving expression of luciferase (MCK-Luc). MCK-Luc was not expressed in C3H10T1/2 cells when transfected alone, but it was efficiently activated by cotransfection with a MyoDwt or MyoDAla200 expression vectors (Fig. 1, lanes 1-3). Co-expression of p57Kip2 not only increased the transactivation of MCK-Luc by MyoDwt but also by MyoDAla200 in a dose-dependent manner. In our experiments, we observed, respectively, a 15- and 6-fold increase in the level of Luc expression driven by the MCK-Luc construct in the presence of p57Kip2 (Fig. 1, lanes 5 and 6). The control plasmid MCK-Luc alone (Fig. 1, lane 1) or p57Kip2 alone were inactive on MCK activity (Fig. 1, lane 4). During the course of differentiation, other muscle specific proteins were likewise up-regulated in cells cotransfected with p57Kip2 and MyoDAla200 compared with the transfection of MyoDAla200 alone. These include myosin heavy chain and myosin light chain 1 and, surprisingly, MyoDAla200 (Fig. 1B). Expression of Cdk4, a stable protein in muscle cell, was not modified after p57Kip2 transfection. These results suggest a positive effect of p57Kip2 on MyoD activity outside of its Cdk inhibitory function on MyoD phosphorylation.


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  		Fig. 1.   Effect of ectopically expressed p57Kip2 on MyoDwt or MyoDAla200-dependent transcriptional transactivation of muscle-specific genes. A, C3H10T1/2 cells were co-transfected with 0.5 µg of MCK-Luc reporter plasmid (lanes 1-6) together with 1.5 µg of an expression vector encoding MyoDwt (lanes 2 and 5), MyoDAla200 (lanes 3 and 6), or 3 µg of pEMSV-p57Kip2 alone (lanes 4) or 1.5 µg in combination (lanes 5 and 6). pEMSV expression vector without insert were included to normalize DNA in all transfections. Luciferase levels were determined 48 h after transfections in high serum medium (15% fetal calf serum). Protein concentrations were equalized by Bradford assay. B, C3H10T1/2 cells were transfected with pcDNA-HA-MyoDAla200 alone (lanes 1 and 3) or in combination with pEMSV-p57Kip2 (lanes 2 and 4) as described above. Transfected cells were grown in proliferative medium (Dulbecco's modified Eagle's medium containing 15% fetal calf serum) for 48 h (P) and placed in differentiation medium for 48 h (48 h). Cells were collected either before (P) or after (48 h) myogenic conversion and analyzed by Western blotting for MyoD, p57Kip2, myosin heavy chain (MHC), myosin light chain 1 (LC1), and Cdk4 expression.

p57Kip2 Increases the Stability of MyoDAla200-- We next compared the influence of ectopic expression of p57Kip2 and p16Ink4a on the level of co-expressed MyoDAla200 in C3H10T1/2 fibroblasts in transient transfectants. As previously observed (Fig. 1B), immunoblotting analyses confirmed that p57Kip2 increased the level of co-expressed MyoDAla200 (Fig. 2A, lanes 2 and 3) in a dose-dependent manner while co-expression of empty vector or p16Ink4a did not affect the expression level of MyoDAla200 (Fig. 2A, lanes 4-7). Our data show that MyoDAla200 expression is specifically augmented in presence of the p57Kip2 proteins.


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  		Fig. 2.   Differential accumulation of MyoDAla200 protein by ectopic Cki expression. A, I, C3H10T1/2 cells were transiently transfected with 0.5 µg of expression vector encoding HA-tagged MyoDAla200 (lanes 1-7) plus 0.5 and 1.5 µg of empty expression vector (lanes 6 and 7) or 0.5 and 1.5 µg of pCMV-p57Kip2 (lanes 2 and 3) or p16Ink4a expression vector (lanes 4 and 5). Whole cell lysates (10 µg) were separated by SDS-PAGE. Proteins were transferred to nitrocellulose and immunoblotted using the 12CA5 monoclonal antibody (Roche Molecular Biochemicals) and visualized by ECL (Amersham Pharmacia Biotech). A, II, 50 µg of total cell lysates from transfected cells were analyzed for expression of exogenous p16Ink4a and p57Kip2 by Western blots using anti-p16Ink4a or anti-p57Kip2 antibodies from Santa Cruz. B, stabilization of MyoD Ala200 by p57Kip2 co-expression. C3H10T1/2 fibroblasts were transiently transfected with HA-MyoDAla200 without or with pEMSV-p57Kip2 vector were grown for 24 h in proliferative medium before addition of cycloheximide (15 µg/ml) to the medium for 0, 1, 3, 5, and 8 h. MyoD and alpha -tubulin protein levels were determined by immunoblots analysis at the indicated times after cycloheximide addition. C, immunoblots were quantified by densitometric scanning, and MyoD protein levels (corrected with respect to the alpha -tubulin expression) were expressed relative to that observed before cycloheximide treatment, set as 100%. Data from Reynaud et al. (48) (dashed lines) are added for comparison.

Because MyoDAla200 is nonphosphorylatable by cyclin-Cdk complexes and is much more stable than MyoDwt (45), the stability of MyoDAla200 compared with MyoDwt in the presence of p57Kip2 was also investigated. We transfected MyoDwt and MyoDAla200 in C3H10T1/2 cells and determined their half-life following cycloheximide treatment (Fig. 2B). We previously showed that the half-life of MyoDwt was found to be about 50 min, in agreement with previous reports (45, 53, 54) and as recently reported, MyoDAla200 has a half-life 5-fold higher than MyoDwt (44, 45). In the presence of p57Kip2, the half-life of MyoDwt was extended to 3 h (48). In the same conditions, the half-life of MyoDAla200 was extended over 10 h (Fig. 2, B and C). Expression level of alpha -tubulin, a stable protein, was not modified until 8 h after cycloheximide addition. These results strongly suggest that p57Kip2 stabilizes MyoDAla200 by a biochemical mechanism that is independent of MyoD phosphorylation by Cdks.

In Vivo Detection of p57Kip2-MyoD Complexes-- Recently, degradation of MyoD by the ubiquitin-proteasome pathway has been shown to be regulated by specific DNA binding involving homodimerization or formation of E47-MyoD heterodimers in vitro and in vivo independently of the phosphorylation of MyoD (47). These data suggest that specific binding of MyoD prevents its degradation and causes the accumulation of MyoD in cultured cells. One possibility involved in the stabilization of MyoD could be its interaction with p57Kip2. To test this hypothesis, C3H10T1/2 cells were transiently transfected with pCMV-HA-MyoD and pCMV-HA-p57Kip2 alone and/or in combination and total cellular proteins were immunoprecipitated with anti-MyoD antibodies. The immune complexes were then analyzed by Western blotting using the anti-HA monoclonal antibodies. p57Kip2 was only immunoprecipitated with anti-MyoD antibodies in cells cotransfected with pCMV-HA-MyoD and pCMV-HA-p57Kip2 (Fig. 3, A and B, lanes 5). To ensure that the interaction between MyoD and p57Kip2 was not due to overexpression of transfected expression plasmids, this physical interaction between MyoD and p57Kip2 was confirmed in myogenic cells. Total C2C12 cell extracts were immunoprecipitated with anti-p16Ink4 or anti-p57Kip2 antibodies, and the immunoprecipitates were examined for the presence of MyoD by Western blots. Immunoreactive bands for MyoD were clearly seen in the lanes where anti-p57Kip2 antibodies were used for the immunoprecipitation (Fig. 3C, lanes 3 and 4). The coimmunoprecipitation of MyoD-p57Kip2 was specific because it was not observed with anti-p16 antibodies (Fig. 3C, lanes 1 and 2). We have recently shown that p57Kip2 and MyoD are up-regulated in the course of muscle differentiation (48). Indeed, in differentiating C2C12 myotubes, higher levels of associated p57Kip2 and MyoD proteins were observed than in proliferating C2C12 myoblasts (Fig. 3C, lanes 3 and 4). Altogether, our results show that p57Kip2 can bind to MyoD in vivo.


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  		Fig. 3.   p57Kip2 can associate with MyoD in vivo. A, C3H10T1/2 cells were transfected with either the empty pCMV-HA (lane 2), pCMV-HA p57Kip2 (lane 3), pCMV-HA-MyoD (lane 4) alone or together (lane 5) and 10 µg of whole cell extracts from the transfected cells were subjected to immunoblotting with the anti-HA antibodies. B, 600 µg of total proteins were immunoprecipitated using affinity-purified MyoD antibodies and immune complexes were subjected to immunoblotting using anti-HA monoclonal antibodies. C, lysates (500 µg) from C2C12 myoblasts (lanes 1 and 3) and C2C12 myotubes (lanes 2 and 4) were immunoprecipitated with p16Ink4a antibodies (lanes 1 and 2) or anti-p57Kip2 antibodies (lanes 3 and 4). The immunoprecipitates were subjected to Western blotting with MyoD antibodies (lanes 1-4), stripped, and reprobed with anti-p16Ink4a (lanes 1 and 2) and anti-p57Kip2 antibodies (lanes 3 and 4).

MyoD but Not E12 Binds to p57Kip2-- To determine if the interaction observed between MyoD and p57Kip2 is direct or mediated by a third partner, a biochemical approach was used. GST or GST-p57Kip2-covered beads were incubated with 35S-labeled in vitro translated MyoD, E12, or cyclin D1 (used as positive control). MyoD could bind efficiently to the p57Kip2 protein, while no binding was observed with E12 (Fig. 4A) and/or MyoD-E12 heterodimers (data not shown). To ensure that the direct binding of MyoD with the p57Kip2 protein was not mediated through a particular conformation of GST-p57Kip2 fusion protein, the converse experiments, in which beads were coated with MyoD and incubated with 35S-labeled in vitro translated p57Kip2 or p16Ink4a, were undertaken. E12 was used as a positive control. Data shown in Fig. 4B confirmed that p57Kip2 bound significantly to GST-MyoD. Altogether, these data show that in vitro p57Kip2 but not p16Ink4a is able to bind with MyoD.


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  		Fig. 4.   A, MyoD specifically bind to p57Kip2 in vitro. Expression vectors encoding MyoD, E12, and cyclin D1 were in vitro translated by programmed reticulocyte lysates and 2 µl of the products were analyzed by SDS-PAGE (lanes 1-3). Equimolar amounts of labeled proteins were incubated with beads covered with GST (lanes 4-6) or GST-p57Kip2 (lanes 7-9). Binding assays were carried out in stringency condition with 150 mM NaCl and 0.5% Nonidet P40 in binding buffer. Bound proteins were analyzed by SDS-PAGE and autofluorography. As a control for specific binding in the GST pull-down assay, cyclin D1 was used. B, binding of p57Kip2 but not p16Ink4a protein to GST-MyoD. Expression vectors encoding E12, p16Ink4a, and p57Kip2 were in vitro translated by programmed reticulocyte lysates, and 2 µl of the products were analyzed by SDS-PAGE (lanes 1-3). Similar amounts of labeled proteins were incubated with beads covered with GST-MyoD (+) or GST alone (-). The binding assays were carried out as described in A. Bound proteins were analyzed by SDS-PAGE and autofluorography.

Mapping the Binding Domains of MyoD and p57Kip2-- The domains of each polypeptide required for this interaction were mapped by in vitro protein binding experiments (Figs. 5 and 6). Full-length in vitro translated MyoD efficiently bound to GST-p57Kip2 but not to GST alone (Fig. 4A and 5). Removing amino acids 63-99 from the NH2 terminus or amino acids 218-269 from the COOH terminus of MyoD did not affect the interaction with GST-p57Kip2 (Fig. 5, B and C, lanes 2 and 5). In contrast, MyoD mutants in which the basic region (DM: 102-114), or the basic region and the helix 1 (DM: 102-135), or the mutants MyoD E12 basic and or MyoD T4 basic (mutants of MyoD in which the basic domain has been replaced by the E12 basic and/or the T4 basic domain, respectively) did not bind to GST-p57Kip2 beads (Fig. 5, B and C, lanes 3 and 4 and lanes 6 and 7). These data indicate that the basic domain of MyoD mediates its binding to p57Kip2.


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  		Fig. 5.   The basic domain of MyoD mediates interaction with p57Kip2. MyoDwt and various MyoD mutants were translated by programmed reticulocyte lysates, and 2 µl of the products were analyzed by SDS-PAGE (A). Similar amounts of various [35S]methionine-labeled MyoD proteins were incubated with GST-p57Kip2 (B) or GST alone (C). Bound proteins were analyzed by SDS-PAGE and autofluorography. D, summary of the results shown in panel B.


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  		Fig. 6.   The NH2-terminal portion of p57Kip2 is sufficient for binding to MyoD. A, various p57Kip2-GST deletion mutants were produced in E. coli and similar amounts of the various GST-p57Kip2 fusion proteins bound to glutathione-agarose were incubated with [35S]methionine-labeled MyoD (A) or cyclin D1 (B), and the bound proteins were analyzed by SDS-polyacrylamide gel electrophoresis and autoradiofluorography. C, summary the results observed in A and B.

To determine the domain of p57Kip2 involved in the binding to MyoD, various GST-p57Kip2 fusion proteins containing either the wild type p57Kip2 protein (wt), the complete Cdk inhibition domain (Cki), the prolin-rich and acidic repeat domains (PAC), the QT domain (QT) or the wild type p57Kip2 deleted of the Cdk inhibitor domain (Delta CKI), deleted of the QT domain (Delta QT) or deleted of the prolin-rich plus acidic repeat domains (Delta PAC) were tested for binding to in vitro translated MyoD (Fig. 6). Cyclin D1 was used as a positive control. The results are shown in Fig. 6 (A and B) and summarized in Fig. 6C. They show that the NH2 domain, which contains the cyclin-Cdk binding sites, is necessary and sufficient for p57Kip2 binding to MyoD.

Interaction between MyoD and p57Kip2 Is Competed by the Cyclin D1-Cdk4 Complexes but Not by Cyclin D1 or Cdk4 Alone-- The experiments described above suggest that MyoD could bind either to cyclin D1 or Cdk4 binding sites and/or both, which are located in the NH2 domain of p57Kip2. To test this hypothesis, we exploited an in vitro association/competition assay. GST-p57Kip2-covered beads were first incubated with 35S-labeled in vitro translated MyoD (Fig. 7, lanes 6-15) and after binding, increasing amounts of labeled cyclin D1 (Fig. 7, lanes 7-9), Cdk4 (Fig. 7, lanes 13-15) or cotranslated cyclin D1-Cdk4 complexes were added to the binding reactions (Fig. 7, lanes 10-12) and the resulting mixtures were subjected to a GST pull-down assay. Neither increasing amounts of cyclin D1 nor Cdk4 alone affected the level of MyoD bound to p57Kip2, strongly suggesting that MyoD binds to p57Kip2 independently of Cdk4 and/or cyclin D1 (Fig. 7, lanes 7-9 and lanes 13-15). Surprisingly, when increasing amounts of cyclin D1-Cdk4 complexes were added to the mixture, MyoD-p57Kip2 complexes were dissociated (Fig. 7, lanes 10-12). These results suggest that MyoD does not directly interact to the cyclin-Cdk binding sites but probably with a particular conformation in the NH2 domain of p57Kip2.


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  		Fig. 7.   Cdk4-cyclin D1 complex inhibits p57Kip2 association with MyoD. MyoD was in vitro translated, and 5 µl were preincubated with GST alone (lane 3) or GST-p57Kip2 fusion protein (lanes 6-15) for 2 h at room temperature. Cyclin D1 and Cdk4 were synthesized in reticulocyte lysates separately or together, and then 1, 3, and 6 µl of extracts were added to the binding reaction and incubation was continued for another 1 h. Cyclin D1 alone (lanes 7-9), Cdk4 alone (lanes 13-15) or cyclin D1-Cdk4 translated together (lanes 10-12) were analyzed by SDS-PAGE and autofluorography. IVT (in vitro translation) represents 2 µl of individually translated MyoD, cyclin D1, and Cdk4, which were loaded in the same track (lane 2).

The Integrity of the alpha -Helix in the NH2 Region Is Indispensable for p57Kip2-MyoD Association-- Physical interaction between p57Kip2 and MyoD may occur via a flexible domain outside the cyclin-Cdk binding sites in the amino terminus of the Cki. A domain covering amino acids 26-47 of p57Kip2, a region located between the cyclin and the Cdk binding sites, has a considerable tendency to take an alpha -helical conformation, indicating that this domain is in a coiled conformation in the native protein. Such a coiled conformation is also observed in p21Cip1 and p27Kip1 proteins (55). In an attempt to test the hypothesis that the alpha -helix (amino acids 26-47) is required for p57Kip2-MyoD association, we generated a replacement of the highly conserved arginine (basic residue) at position 33 by a leucine (neutral residue) (mutant R33L) in the alpha -helix domain of the GST-p57Kip2 fusion protein and tested its ability to associate with 35S-labeled MyoD proteins by GST pull-down. As shown in Fig. 8A, substitution of arginine for leucine in the p57Kip2 molecule enhances the stability of the alpha -helix by modifying the three-dimensional structure of p57Kip2 molecules. Substitution of arginine at position 33 for a leucine dramatically reduced the binding of 35S-labeled MyoD, while this mutant retained the ability to bind cyclin D1 or Cdk4 and/or cyclin D1-Cdk4 complexes (Fig. 8B). These data suggest that the alpha -helix domain seems to play a major role in p57Kip2-MyoD interaction.


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  		Fig. 8.   Ponctual mutation in the alpha -helix domain of NH2-terminal sequence of p57Kip2 prevents its interaction with MyoD. A, amino acid sequence comparison of the NH2-terminal domain of mouse p57Kip2 and human p21Cip1 and p27Kip1. Boxed regions indicates the defined sites of binding for Cyclin and Cdk subunits conserved between the members of Cip/Kip family. The corresponding secondary structure elements is schematized above. The arrow indicates the point mutation in the alpha -helix. Diagram showing the predicted helicity per residue for wild type peptide (wt) and the mutant (R33L) was calculated using the program AGADIR (61). B, GST-p57 wt and GST-p57 (R33L) fusion proteins were produced in E. coli, and GST pull-down assays with increasing amounts of [35S]methionine-labeled cyclin D1, Cdk4, cotranslated cyclin D1-Cdk4, or MyoD were carried out as described in Fig. 5.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

It has been recently proposed that phosphorylation of MyoD by Cdk1 and Cdk2 is required for its rapid degradation by the ubiquitin-proteasome pathway, and that phosphorylation of MyoD at serine 200 plays a crucial role in modulating its half-life and transcriptional activity during myoblast proliferation (44, 45). These findings were corroborated by our recent results showing that, in proliferating myoblasts, overexpression of p57Kip2 can reverse mitogen-mediated repression of MyoD functions. We showed that the NH2-terminal domain of p57Kip2 stabilizes MyoD by inhibiting cyclin E-Cdk2 kinase activity in growing myoblasts (48).

The data presented here support an additional role for p57Kip2, independent of its kinase inhibitory activity, in the positive regulation of MyoD activity.

Increased Stability and Myogenic Activity of MyoDAla200 by the p57Kip2 Protein-- We found that the mutant MyoDAla200 in the presence of p57Kip2 was more efficient than MyoDAla200 alone in converting C3H10T1/2 fibroblasts to muscle cells. p57Kip2 protein expression leads to the accumulation of MyoDAla200, a MyoD mutant that is not phosphorylatable by the cyclin-Cdk complexes in vivo (45). This increased level of MyoD is due to a highest half-life time induced by p57Kip2 co-expression in C3H10T1/2 (Fig. 2). In the presence of overexpressed p57Kip2, the half-life of MyoDAla200 protein is about 2 times longer than that of MyoDAla200 alone. Recently it has been shown that degradation of MyoD in vitro and in vivo by the ubiquitin-proteasome pathway was regulated by specific DNA binding of the homo- and/or heterodimers (47). Such a protection could be achieved also by direct interaction between p57Kip2 and MyoD. Outside the NH2-terminal domain, the COOH-terminal sequence (also termed QT box) is a structural motif conserved with p27Kip1. QT box is likely to function in protein-protein interactions and at first sight to be the best motif for p57Kip2-MyoD association. We demonstrate a physical interaction between the basic domain of MyoD and the NH2-terminal region of p57Kip2 which contains the cyclin and Cdk binding sites. This domain of p57Kip2 encompassing amino acids 1-105, is necessary and sufficient for binding to MyoD (Fig. 6). This result is strengthened by the fact that the COOH-terminal domain of p57Kip2 is not required for cell cycle arrest in SAOS2 cells (17) nor to act positively on the transactivation of the MCK promoter by MyoD (48). Thus, these data indicate that p57Kip2, without its QT box, can function to arrest cell cycle in G1 and/or to stabilize MyoD.

Implication of the alpha -Helix Structure in p57Kip2-MyoD Interaction-- An in vitro association/competition binding assay reveals that MyoD remains associated with p57Kip2 even after addition of increasing amounts of cyclin D1 or Cdk4 alone, although these two ligands have higher affinity for p57Kip2 than MyoD. However, p57Kip2-MyoD complexes are dissociated in the presence of increasing amounts of cyclin D1-Cdk4 complexes (Fig. 7). Even if, probably, a portion of GST-p57Kip2 is not fully saturated by MyoD and can associate with cyclin D1 and/or Cdk4 alone or both, altogether our data strongly suggest that MyoD does not bind to the cyclin nor the Cdk binding domain. This shift of MyoD by the cyclin D1-Cdk4 complexes supposes another model of interaction, implying a conformational change of p57Kip2. The crystal structure of p27Kip1 kinase inhibitory domain indicates that the cyclin and the Cdk binding sites, which are located in an extended structure, are jointed by an amphipathic helix highly conserved among the Cip/Kip proteins (55). This should explain how the Cip/Kip family inhibitors can physically interact with the isolated Cdk subunits. Binding to the cyclin-Cdk complex is significantly tighter, consistent with cooperative binding to the two subunits (55). Similar results have been reported for p57Kip2 and cyclin D1-Cdk4 complex interaction (17). In this context, our results strongly suggest that MyoD associates with p57Kip2 probably with a particular conformation which implicates the conserved amphipathic helix (Fig. 8, A and B). It would thus appear that specific structural constraints and affinities, different from constraints in the association of p57Kip2 with cyclin D1-Cdk4 complexes, govern interactions between p57Kip2 and MyoD. The stabilization of MyoD associated with p57Kip2 can be due to a change in the conformation of MyoD protein.

The alpha -helix domain spanning amino acids 25-45 in p21Cip1, a secondary structure relatively well conserved in p57Kip2 (amino acids 26-47), has been shown to a potential multimerization domain (56). We have recently evidenced that homodimerization of p57Kip2 via its alpha -helix is a prerequisite for the inhibition of cyclin D1-Cdk4 kinase activity (57). Such a structure is well known to create a surface that allows binding of ligand. In the case of the Ckis, it has been proposed that the helix axis is submitted to kink and disorder as its binds cyclin and then it becomes well ordered as it reaches Cdk2 (55). The variable expression of MyoD protein in growing myoblasts (58) suggested that, if the autoregulatory loop of MyoD is involved in the maintenance of myogenic potential (53), an additional back-up system must also be functioning to account for the fact that cells with undetectable levels of MyoD can nevertheless regain their myogenic potential as shown by using bromodeoxyuridine treatment. These fluctuations of MyoD in growing myoblasts have recently been precisely correlated to cell cycle progression in a study showing that MyoD protein peaks during G1 and drop abruptly just before entry into the S phase (59). This suggest that a mechanism takes place to reduce the level of MyoD protein when cells progress from G1 into S. The concomitant increase of active MyoD protein and p57Kip2 amounts in myoblasts during early myogenic differentiation could be linked by the second action of Cki upon MyoD stabilization. We show that the alpha -helix domain p57Kip2 interacts with the basic domain of MyoD, allowing masking of the included potential degradation signal revealed by Abu Hatoum et al. (47).

Taken together, our present data and the recent reports from others, strongly suggest that p57Kip2 should have two additive and successive functions during the course of muscle differentiation. First, we demonstrate that association between MyoD and p57Kip2 is weaker than that observed between p57Kip2 and cyclin-Cdk complexes. These data support the conclusion that in late G1 phase the main function of p57Kip2 is to inhibit Cdk activities preventing the phosphorylation/degradation of MyoD and to allow to the myoblasts to exit from the cell cycle (60). This mechanism regulates positively the turn over of MyoD in order to reach a threshold of transcriptional activity that triggers cell cycle withdrawal and myogenic differentiation. Second, we show a concomitant increase in the levels of MyoD and p57Kip2 during early myogenesis and an increasing amount of MyoD bound to p57Kip2 in myotubes versus proliferating myoblasts. In addition we found that the increase in half-life of the nonphosphorylatable MyoD mutant is less than that observed with MyoD wild type in the presence of p57Kip2. Altogether, our results strongly suggest that the protection of the basic domain in MyoD by the alpha -helix located in the NH2 region of p57Kip2 should be one of its major function upon MyoD (and probably myogenin and MRF4 since these two other MRFs physically interact with p57Kip2 in vitro; data not shown) and because the G1-cyclin-Cdk complexes that are totally absent in myotubes raise the question of the persistence of the Ckis in fully differentiated cells.

These new data show that in the NH2 region of p57Kip2, the highly conserved alpha -helix domain is implicated in protein-protein interaction that may confer a new specific regulatory mechanism by which the Cip/Kip protein family can act positively on myogenic differentiation. These new data argue for the persistence and the accumulation of the Cip/Kip proteins in arrested differentiated cells.

    FOOTNOTES

* This work was supported in part by INSERM, the Centre National de la Recherche Scientifique, and grants from Association Française contre les Myopathies, Ligue Nationale contre le Cancer, Association pour la Recherche sur le Cancer Grant 6829, and the Institut Gustave Roussy.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Fellow of the Fondation pour la Recherche Médicale. Present address: Centre National de la Recherche Scientifique UPR 9051, Hôpital Saint-Louis, 75475 Paris Cedex 10, France.

§ These authors contributed equally to this work.

Fellow of the Ministère de la Recherche et de la Technologie.

|| To whom correspondence should be addressed. Tel.: 33-1-42-11-45-16; Fax: 33-1-42-11-52-61 or 33-1-42-11-52-44; E-mail: leibovit@igr.fr.

Published, JBC Papers in Press, April 11, 2000, DOI 10.1074/jbc.M907412199

    ABBREVIATIONS

The abbreviations used are: Cdk, cyclin-dependent kinase; Cki, cyclin-dependent kinase inhibitor; GST, glutathione S-transferase; bHLH, basic helix-loop-helix; PAGE, polyacrylamide gel electrophoresis; HA, hemagglutinin; MRF, muscle-specific transcription factor.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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