Proline Isomerase Pin1 represses terminal differentiation and Myocyte Enhancer
Factor 2C function in Skeletal Muscle Cells
Alessandro Magli*°, Massimo Ganassi*, Fiorenza Baruffaldi*, Sara Badodi*, Cecilia Angelelli*, Renata Battini*, Giannino Del Sal**, Susanna Molinari*
*Department of Biomedical Sciences, University of Modena and Reggio Emilia, Via Campi 287,41100, Modena, Italy. E-mail:
molinari.susanna@unimore.it
°present address:
Lillehei Heart Institute and Dept. of Medicine University of Minnesota, Minneapolis, MN 55455,
**Laboratorio Nazionale CIB, Area
Science Park, University of Trieste, 34100 Trieste, Italy.
This work is supported with grants from the EU programme Optistem and from the Fondazione Cassa di Risparmio di Modena.
This work is dedicated to the memory and legacy of our friend Stefano Ferrari who recently passed away.
0
60
120
180 aa
WW domain
(binding)
PPiase Domain
(catalytic activity)
NH
2COOH
W34 C113FUNCTIONAL DOMAINS OF Pin1
Fig. 2. A. Schematic representation of the Pin1 functional domains: a WW domain, that mediates the interaction with target phosphoproteins and the PPiase catalytic domain that promote the isomerisation reaction of a peptide bond between a
phosphorylated Serine or Threonine and a Proline (shown in B). Also shown are
critical aminoacids that, when mutated, hamper the binding (W34A) or the catalytic (C113A) activity of Pin1, respectively..
B
Pin1 IS EXPRESSED IN MUSCLE CELLS AND IS SUBJECTED TO
NUCLEAR-TO-CYTOPLASM
TRANSLOCATION DURING MUSCLE DIFFERENTIATIONITS
Fig. 3. A Analysis of Pin1, Myosin Heavy Chain (MyHC) and actin protein levels during
myogenic differentiation. Total protein extracts from proliferating (Mb) or differentiating (Mt 0h, 6h, 24h, and 48h) C2C7 muscle cells were analyzed by Western blot (WB) with the indicated antibodies. B, subcellular localization of Pin1 during skeletal muscle
differentiation. Cytoplasmic, nuclear and total protein extracts from proliferating (Mb) or
differentiating (Mt 0h and Mt 24h) C2C7 muscle cells were analyzed by Western blot using the antibody against Pin1; furthermore, we checked the quality of the subcellular protein extracts with antibodies against the glycolytic enzyme enolase and the nuclear
transcription factor NF-YB.
A B
ABSTRACT
. MEF2 (myocyte enhancer factor 2) transcription factors (MEF2A-D) are highly expressed in skeletal muscle cells, they bind to a conserved AT rich DNA sequence through their N-ter MADS and MEF2 domains and activate transcription via their C-ter transcriptional activation domains (TAD), the functional domains of MEF2C are indicated in Figure 1. MEF2proteins interact with members of the MyoD family of basic helix–loop–helix (bHLH) proteins to establish a unique transcriptional code for skeletal muscle gene activation. Recent studies have revealed multiple signaling systems that stimulate and inhibit myogenesis by altering MEF2
phosphorylation and its association with other transcriptional cofactors. We show that the Pin1 isomerase, which catalyzes the isomerization of phosphorylated Ser/Thr-Pro peptide bonds, interacts with phosphorylated MEF2C in muscle cells. This interaction requires two novel phospho-Ser-Pro motifs in MEF2C: Ser(98) and Ser(110), which are phosphorylated in vivo.
Overexpression of Pin1 decreases MEF2C stability and activity and its ability to cooperate with MyoD to activate myogenesis. Furthermore Pin1 modulates the skeletal muscle differentiation program because down-regulation of Pin1 markedly promotes myogenic differentiation. We suggest that Pin1 is a novel regulator of MEF2C function and muscle differentiation, it is
expressed in muscle cells and a significant proportion of Pin1 in myotubes but not in myoblasts is excluded from the nucleus. We observed a reduction of phosphorylation of the Ser(98) and Ser(110) Pin1 binding sites in differentiated myocytes. Based on these results we propose a model in which, in proliferating myoblasts, Pin1, upon binding to phosphorylated nuclear MEF2C, leads to decreased levels and transcriptional activity of MEF2C. Upon induction of terminal
differentiation, to establish a full activity of MEF2 proteins, a reduced Pin1-MEF2C association is required, possibly due to the relegation of Pin1 to the cytoplasm and to a reduced level of phosphorylation of Ser98 and Ser110.
A
Fig. 1 Functional domains of MEF2C. NLS is the Nuclear Localization Signal.
0 100 200 300 400 aa
NLS
MADS MEF2 TAD1 TAD2
N term C term
Pin1 INTERACTS WITH PHOSPHORYLATED MEF2C
Fig.4. A. MEF2C and Pin1 proteins coimmunoprecipitate from muscle cells: C2C7 cells ectopically expressing FLAG-MEF2C alone or in association with HA-Pin1 were
cross-linked and then lysed and immunoprecipitated (IP) with anti-FLAG antibody. Western blot was performed with anti-MEF2 (polyclonal) and anti-HA antibodies.
B. The interaction between Pin1 and MEF2C is dependent on the phosphorylation of MEF2C as shown in a Far Western assay. COS1 cells were transfected with the empty FLAG vector (lane 1) or the FLAG MEF2C expression vector (lanes 2 and 3). Cells were then lysed and were treated with λ phosphatase (λ PPase) were indicated (lane 3). Anti-FLAG immunoprecipitation was performed on the lysates and immunoprecipitated proteins were resolved on SDS-PAGE, blotted onto PVDF membrane, incubated with bacterially purified GST, GST-Pin1 or GST-Pin1 W34A and then analyzed by Western Blot with the anti-GST antibody.
A
B
THE INTERACTION BETWEEN MEF2C AND Pin1 IS
PHOSPHO-SPECIFIC AND pSer98 AND pSer110 ARE THE PROMINENT
Pin1 BINDING SITES
N term C term
S388
S240
NLS
MADS MEF2 TAD1 TAD2
S98S110
Fig. 5. A Schematic representation of the functional domains of MEF2C (MADS box, MEF2 domain, TAD, Transcriptional Activation Domains 1 and 2 and NLS, Nuclear
Localization Signal), blue triangles indicate the positions of four putative Pin1 binding sites, they are phosphorylated serines that we identified by mass spectrometry analysis of
MEF2C protein (S98, S110, S240 and Ser388, the last two were described previously in Cox et al. 2003 and Zhu et al. 2004). B The interaction between Pin1 and MEF2C is
primarily dependent on the phosphorylation of Ser98 and Ser110 but also on the Ser240 and Ser388 phosphoacceptors, as we observed in a GST pull down experiment where GST or GST-Pin1 were incubated with protein lysates of cells transfected with FLAG MEF2C wild type (wt) or the mutant proteins where the indicated serines were substitude with alanines (S→A). The products of the pull down were analysed in western blot with the anti-FLAG and the anti-GST antibodies.
A B
MEF2C IS PHOSPHORYLATED ON THE Ser98 AND Ser110
RESIDUES SELECTIVELY IN MYOBLASTS
Anti-MEF2C Mt Anti-pSer98 Anti-pSer110 Mb 0 h 24 h 57 200 117 Anti-MyHC Anti-vinculin 57 57 - + 57 Anti-MEF2C phosphatase: Anti-pSer98 Anti-pSer110 Anti-vinculin 117 57 57 KDa KDa
A
B
WB WBFig. 6. Level of phosphorylation of MEF2C on Ser98 and Ser110 in skeletal muscle cells as determined with phospho-specific antibodies. A.
Characterization of phosphospecific antibodies directed against phosphoSer98 and phospho Ser110: lysates of COS cells transfected with the expression
vector of MEF2C were treated with phosphatase (+) or a control buffer (-) and analysed for the presence of MEF2C with a specific antibody and for the level of phosphorylation of MEF2C on Ser98 and Ser110 with polyclonal
antibodies developed against phosphorylated peptides. The quantity of loaded proteins was evaluated with anti vinculin antibody. B. The level of
phosphorylation of MEF2C on the relevant Pin1 binding sites in total lysates of C2C7 myoblasts (Mb) or C2C7 cells induced to differentiate in low serum for 0h or 24h were analysed with the phosphospecific antibodies characterized in A.
Pin1 OVEREXPRESSION IN C2C7 MUSCLE CELLS INHIBITS
MEF2C PROTEIN STABILITY AND MEF2-DEPENDENT TRANSCRIPTION
A
B
Fig 7 Pin1 repressses MEF2C stability and function. A. the stability of transfected FLAG MEF2C was measured in the absence or the presence of overexpressed HA Pin1 in C2C7 cells. After 0h, 6h and 12h of treatment with cycloheximide, cells were lysed and proteins analysed by western blot with the anti FLAG and anti HA antibodies. B Transactivation assay where the transactivation potential of endogenous MEF2 proteins alone or in the presence of increasing amounts of Pin1 (+, ++) in C2C7 cells was evaluated measuring the transcription of the luciferase reporter gene under the control of three MEF2 sites. The luciferase activity was normalized with the activity of co-transfectd beta galactosidase. The activity is expressed as luciferase units relative to the mock transfected sample taken as one. The level of the Pin1, MEF2 and actin proteins were evaluated with the specific antibodies.
PIN1 AFFECTS MUSCLE CELL DIFFERENTIATION
Fig. 8. The role of Pin1 in muscle cell differentiation was investigated by knocking down the expression of Pin1 with a specific short hairpin (sh Pin1) in C2C7 cells that were subsequently induced to differentiate for 24 hours. As a control, cells were infected with lentivirus containing a control short hairpin (sh CTRL). A. Cell lysates were analysed for the presence of MEF2 proteins, Pin1, actin and the muscle
marker Myosin Heavy Chain (MyHC). B. Histogram reporting the number of nuclei in MyHC positive cells of the experiment shown in C. Immunofluorescence where the C2C7 cells are stained for the muscle marker MyHC and nuclei with DAPI
• Pin1 is a negative regulator of skeletal muscle terminal differentiation;
• Pin1 negatively regulates the stability of the MEF2C protein and MEF2 function in skeletal muscle cells suggesting that the inhibitory effect of Pin1 on myogenesis could be due to inhibition of MEF2C function;
• The interaction between MEF2C and Pin1 takes place in the nucleus and depends on the phosphorylation of MEF2C on the two residues Ser98 and Ser110;
• The nuclear localization of Pin1 and the phosphorylation of MEF2C on the relevant Pin1 binding sites is restricted to proliferant myoblasts;
On the basis of the above observations we propose a model in which Pin1, upon binding to phosphorylated nuclear MEF2C leads to decreased levels and
transcriptional activity of MEF2C. Upon induction of myogenesis, to establish a full activity of MEF2 proteins, the level of Ser98 and Ser110 phosphorylation decreases and Pin1 is excluded from the nuclear compartment.
FUTURE PERSPECTIVES
1. Identification of the signalling pathways responsible for the phosphorylation of MEF2C on the Pin1 binding sites and of the nuclear translocation of Pin1.
2. Investigation of the relevance of this regulatory mechanism in in vivo
models of myogenesis, during development and in skeletal muscle regeneration
CONCLUSIONS
pSer98 and pSer110 Activating PTM
