MOLECULAR AND CELLULAR BIOLOGY, Sept. 1994, p. 6232-6243 Vol. 14, No. 9 0270-7306/94/$04.00+0 Copyright © 1994, American Society for Microbiology Dimerization through the Helix-Loop-Helix Motif Enhances Phosphorylation of the Transcription Activation Domains of Myogenin JUMIN ZHOU AND ERIC N. OLSON* Department of Biochemistry and Molecular Biology, University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030 Received 6 December 1993/Returned for modification 4 February 1994/Accepted 6 June 1994

The muscle-specific basic helix-loop-helix (bHLH) protein myogenin activates muscle transcription by binding to target sequences in muscle-specific promoters and enhancers as a heterodimer with ubiquitous bHLH proteins, such as the E2A products E12 and E47. We show that dimerization with E2A products potentiates phosphorylation of myogenin at sites within its amino- and carboxyl-terminal transcription activation domains. Phosphorylation of myogenin at these sites was mediated by the bHLH region of E2A products and was dependent on dimerization but not on DNA binding. Mutations of the dimerization- dependent phosphorylation sites resulted in enhanced transcriptional activity of myogenin, suggesting that their phosphorylation diminishes myogenin's transcriptional activity. The ability ofE2A products to potentiate myogenin phosphorylation suggests that dimerization induces a conformational change in myogenin that unmasks otherwise cryptic phosphorylation sites or that E2A proteins recruit a kinase for which myogenin is a substrate. That phosphorylation of these dimerization-dependent sites diminished myogenin's transcrip- tional activity suggests that these sites are targets for a kinase that interferes with muscle-specific .

A variety of transcription factors bind DNA and activate nin, , and MRF4 (reviewed in references 15, 36, 37, and transcription as heterodimers. Interactions among heterolo- 49). These myogenic bHLH proteins bind DNA weakly by gous transcription factors provide opportunities for positive themselves but acquire high affinity for the E box consensus and negative control of gene transcription and dramatically sequence upon heterodimerization with several ubiquitous expand the regulatory potential of different classes of tran- bHLH proteins, referred to as E proteins. Among the E scription factors. In many cases, each component of a het- proteins that have been shown to serve as partners for myo- erodimer binds DNA weakly on its own but shows enhanced genic bHLH proteins are E12 and E47, encoded by the E2A DNA-binding activity when associated with the appropriate gene, and the HEB gene product (9, 21, 25, 34). Activation of partner. While it is conceivable that heterodimerization could muscle-specific transcription ensues when heterodimers of also alter the properties of the transcription factors them- myogenic bHLH proteins and E proteins bind to E boxes in the selves, there is as yet little evidence for this type of cooperat- control regions of muscle-specific . ivity among dimerization partners. Intracellular growth factor signals interfere with the ability The helix-loop-helix (HLH) family of transcription factors of myogenic bHLH proteins to activate muscle-specific tran- includes a wide range of regulatory proteins that control gene scription through several different mechanisms (reviewed in transcription in species ranging from Drosophila melanogaster reference 37). Recently, we showed that protein kinase C to humans (reviewed in reference 23). The HLH motif medi- (PKC) can block through phosphorylation of a site ates dimerization and brings together the basic regions of HLH in the DNA-binding domain of myogenin that is specific to and proteins to form a composite DNA-binding domain that conserved in all myogenic bHLH proteins (28). Phosphoryla- recognizes the consensus sequence CANNTG (where N is any tion of site attenuates the of nucleotide), known as an E box (2, 33). Most HLH proteins this transcriptional activity preferentially form heterodimers with specific HLH protein myogenin by inhibiting its ability to bind DNA. Protein kinase partners (10, 34). The ability of individual basic HLH (bHLH) A (PKA) also phosphorylates the myogenin DNA-binding proteins to recognize distinct half-sites within the dyad sym- domain, but phosphorylation by PKA does not appear to metry of the E box consensus sequence therefore allows influence myogenin's ability to activate muscle transcription different combinations of HLH proteins to discriminate be- (26). In addition to sites for phosphorylation by PKC and PKA, tween different E boxes (2). HLH proteins lacking basic myogenin contains several other phosphorylation sites, the regions can inhibit the activities of bHLH proteins by forming locations of which remain to be determined (26, 28). MyoD, heterodimers that are incapable of binding DNA (1). myf5, and MRF4 are also highly phosphorylated, but the sites gene expression is controlled by a family of for phosphorylation in these proteins have likewise not yet muscle-specific bHLH proteins which includes MyoD, myoge- been mapped (17, 25, 32, 46, 52). Here we show that dimerization with E2A gene products results in an increase in the phosphorylation of myogenin at * Corresponding author. Mailing address: Department of Biochem- sites within its amino- and carboxyl-terminal transcription istry and Molecular Biology, Box 117, The University of Texas M. D. activation domains. Replacement of these sites with neutral Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030. amino acids that prevent phosphorylation enhances the ability Phone: (713) 792-3648. Fax: (713) 791-9478. of myogenin to activate E box-dependent reporter genes and 6232 VOL. 14, 1994 DIMERIZATION-DEPENDENT PHOSPHORYLATION OF MYOGENIN 6233 potentiates the activity of myogenin's transcription activation and loaded onto a 10% polyacrylamide-SDS gel. Following domains when they are fused to the DNA-binding domain of electrophoresis, the gel was dried and processed for autora- the yeast protein GAL4. The ability of E2A products to diography. enhance phosphorylation of myogenin suggests that dimeriza- Phosphopeptide mapping and phosphoamino acid analysis. tion induces a conformational change in myogenin that ex- For phosphoamino acid analysis, 32P-labeled myogenin was poses otherwise cryptic phosphorylation sites or that E2A excised from SDS gel slices, which were then hydrated in water products recruit a kinase for which myogenin is a substrate. and rinsed in 50 mM NH4HCO3. The labeled protein was That phosphorylation of the dimerization-dependent sites di- ground into pieces and boiled for 3 min in 1 ml of 50 mM minishes myogenin's activity also suggests that the dimeriza- NH4HCO3 with 5 ,ul of and 10 p.l of tion-dependent kinase plays a negative role in regulating the ,B-mercaptoethanol 10% myogenic program. SDS. After a 5-h incubation at 37°C, the eluted proteins were precipitated in cold 15% trichloroacetic acid in the presence of 20 p.g of heat-treated RNase A. After overnight incubation on MATERIALS AND METHODS ice, the trichloroacetic acid precipitates were collected by Cell culture and transfections. Transfections were per- centrifugation for 10 min at 4°C and washed with 500 p.l of formed by calcium phosphate precipitation as described before ice-cold absolute ethanol. The pellet was then dried in air and (28). Briefly, COS-1 or 1OT1/2 cells were plated at 50% subjected to peptide mapping as described elsewhere (28) or confluency, and 1 day later, each plate of cells was refed with resuspended in 6 N HCl for phosphoamino acid analysis. Dulbecco's minimal essential medium (DMEM) containing Protein was then hydrolyzed for 2.5 h, and the partial acid 10% fetal bovine serum (2 ml for 60-mm dishes and 4 ml for hydrolysates were mixed with 3 p.g of phosphoserine, phospho- 100-mm dishes) and transfected within 4 h. Calcium phos- threonine, and phosphotyrosine (Sigma) and resolved by two- phate-DNA precipitates were prepared by adding 250 ,ul of 2x dimensional electrophoresis, with the first dimension at pH 1.9 HBS (0.818% NaCl, 0.594% HEPES [N-2-hydroxyethylpipera- and the second dimension at pH 3.5 (acetic acid-pyridine- zine-N'-2-ethanesulfonic acid], 0.02% Na2HPO4 [pH 7.12]) to water, 50:5:945). The thin-layer chromatography plate was first 250 p.l of 250 mM calcium chloride plus DNA (5 pLg per 60-mm visualized by autoradiography, and the phosphoamino acid plate). At 4 to 16 h after transfection, the medium was replaced standards were visualized after the plate was sprayed with with fresh DMEM containing 10% fetal bovine serum. For 0.25% ninhydrin in acetone and incubated for 10 min at chloramphenicol acetyltransferase (CAT) assays, transfected 650C. cells were refed with DMEM containing 2% horse serum 24 h Myogenin and E47 mutants. Site-directed mutagenesis was following transfection and allowed to differentiate for 2 days performed by PCR. All mutations were confirmed by DNA before being harvested. CAT activity was determined on aliquots of cell extract normalized to,-galactosidase activity as sequencing. described before (28). All assays were linear with respect to The following myogenin deletion mutants were made: protein concentration. DM1-33 was made by insertion of a SmaI fragment from the The reporter plasmid MCK-CAT contains the basal pro- myogenin cDNA (14), extending from codon 33 to the 3' end moter and muscle-specific enhancer from the mouse muscle of the transcript, into the SmaI site of the pECE-Flag vector creatine kinase (MCK) gene (45). Plasmid 4R-tkCAT (gener- (IBI), creating a Flag epitope-tagged protein. An NcoI-XbaI ously provided by A. Lassar) contains four tandem copies of fragment that encompassed the myogenin coding region was the high-affinity right-hand E box from the MCK enhancer isolated from this vector, the ends were filled in with de- linked to the thymidine kinase (tk) gene basal promoter (48). oxynucleotide triphosphates and Klenow polymerase, and the The GAL4 reporter plasmid pG5E1bCAT contains five copies fragment was subcloned into a filled EcoRI site of the vector of the GAL4 binding site upstream of the Elb promoter (29). pCDM8* (22). Termination mutants TM158, TM198, and One microgram of the construct RSV-lacZ was included in DM4-77/TM158 were described previously (43). DM159-170 transfections as an internal control for transfection efficiency. and DM159-170/TM198 were created by digesting myogenin Metabolic labeling and immunoprecipitation. For 32p label- or TM198 with NcoI and AccIII, filling in the ends, and ing, transfected cells were transferred into phosphate-free religating to remove codons 159 to 170. TM 168 was created by medium (Sigma) 2 days after transfection and incubated for up digesting the myogenin cDNA with PstI, adding NheI linkers, to 8 h before 32p, (ICN) was added at 1 mCi/ml for another 4 and religating. All myogenin mutant cDNAs were subcloned h. For [35S]methionine labeling, cells were incubated in methi- into the EcoRI site of pCDM8*. onine-free medium (GIBCO) for 8 h and labeled for 1 h with GAL4-myogenin chimeras were constructed by subcloning a L-[35S]methionine (ICN). After labeling, cells were lysed with SmaI fragment of wild-type or mutant RIPA buffer (50 mM Tris [pH 8.0], 0.1% sodium dodecyl myogenin cDNAs sulfate [SDS], 1.0% Nonidet P-40, 0.5% sodium deoxycholate, (extending from codon 33 to the end of the 3' untranslated 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 mM region) into the SmaI site of the vector pSG424 (29). This NaF, 30 mM sodium PP1, 1.0% Trasylol [FBA Pharmaceuti- SmaI site lies immediately downstream of the GAL4 DNA- cals], 150 mM NaCl), and the lysate was homogenized by binding domain (residues 1 to 147). drawing it through a 26-gauge needle attached to a 1-ml The expression plasmid for E47(E2-5) (provided by T. syringe 10 times to reduce viscosity and then given a 10-min Kadesch, University of Pennsylvania) was described previously spin to remove debris. Myogenin monoclonal antibody 5-D (19). The E47 basic region mutant E47/BS2-3 has also been (generously provided by W. Wright) was then added, and after described (10). The E47 deletion mutant E47-bHLH, previ- a 1-h incubation, protein A-agarose beads were added for ously called E47S (33) (provided by C. Murre, University of another 30 min. At the end of the incubation, the beads were California at San Diego), encompasses the codons for amino collected by a quick spin, and the pellet was washed three times acids 459 to 584 (the termination codon). E47-bHLH (33) was with RIPA buffer containing 500 mM NaCl, followed by a final subcloned into the EcoRI site of pCDM8* after filling in the wash in phosphate-buffered saline (PBS). The pellet was then ends of an EcoRI-HindIII fragment containing the coding resuspended in SDS sample buffer, heated to 95°C for 3 min, region. 6234 ZHOU AND OLSON MOL. CELL. BIOL.

-a -a A. 35S-met 32p c c A. 0 B. 0 0cm r N It St Jo :i: n + + EL 0 0 0 0 < < o aL E E E E kD kD lb

... 43- 43- £8.,.:

27.-... .:

.._ ...-. 27- 27-

FIG. 1. Multiple forms of myogenin arise from differential phos- B. phorylation. (A) Differentiated C2 myotube cultures were labeled with S [35S]methionine for 2 h, and myogenin was immunoprecipitated from /iQT cell extracts with an antimyogenin monoclonal antibody, as described in the text. Immunoprecipitates were treated with calf intestinal phosphatase (CIP) or potato acid phosphatase (PAP) or with reaction buffer alone, and labeled species were analyzed by SDS-PAGE and FIG. 2. E47 induces myogenin phosphorylation. (A) COS cells autoradiography. (B) Differentiated C2 myotube cultures were labeled were transiently transfected with a myogenin expression vector (myo) with [35S]methionine or 32p; for 2 h, and myogenin was immunopre- with or without an E47 expression vector, cultures were labeled with cipitated from nuclear extracts and analyzed by SDS-PAGE. The 32p or [35S]methionine, as indicated, and myogenin was immunopre- positions of size markers are indicated to the left. cipitated from cell extracts with an antimyogenin monoclonal antibody, as described in the text. Immunoprecipitates were analyzed by SDS- PAGE. (B) 32P-labeled myogenin immunoprecipitated from COS cells RESULTS transiently transfected with myogenin and E47 expression vectors was subjected to phosphoamino acid analysis. The positions of phospho- Multiple forms of myogenin arise from differential phos- serine (S), phosphothreonine (T), and phosphotyrosine (Y) standards phorylation. When myogenin from myotube nuclear extracts is are indicated. Only [32P]phosphoserine was detected. labeled with [35S]methionine and analyzed by SDS-polyacryl- amide gel electrophoresis (PAGE), two major species of 32 and 34 kDa, in addition to several minor species with interme- indicates that some phosphorylation sites do not alter myoge- diate mobilities, appear (9, 13, 26, 28). To determine whether nin's mobility on SDS-PAGE and suggests that the shift in the higher-Mr species arise from phosphorylation of the 32- mobility may be due to phosphorylation at a specific site (see kDa species, we immunoprecipitated myogenin from extracts below). The small amount of the 34-kDa form observed with of C2 myotubes and treated the immunoprecipitates with the myogenin expression vector alone presumably reflects potato acid phosphatase and calf intestinal phosphatase. As oligomerization of myogenin with endogenous E proteins, shown in Fig. 1A, phosphatase treatment converted the upper which are limiting. form to the 32-kDa species, indicating that it indeed arose The residues in myogenin that were phosphorylated were from phosphorylation of the lower-Mr form. We also labeled determined by phosphoamino acid analysis of the wild-type myotube cultures with 32p; and [35S]methionine and compared protein immunoprecipitated from transiently transfected COS the ratios of 32P and 35S in the two major forms of myogenin. cells exposed to low-serum medium. Only phosphoserine was The 32P/35S ratio was at least five times higher in the 34-kDa detected (Fig. 2B). We also detected only phosphoserine in species than in the 32-kDa species, further indicating that the myogenin immunoprecipitated from differentiated C2 myo- 34-kDa form is more highly phosphorylated (Fig. 1B). tubes (data not shown). This is in contrast to myogenin from E24 gene products induce myogenin phosphorylation. Pre- COS cells exposed to either phorbol dibutyrate or fibroblast vious studies showed that phosphorylation of MyoD was growth factor, in which a small amount of phosphothreonine enhanced in the presence of E47 (25) and that phosphorylation can be detected, which reflects phosphorylation of threonine retarded MyoD's electrophoretic mobility (47). To determine 87 in the basic region (28). whether this was a property shared by other myogenic bHLH E47-dependent phosphorylation of myogenin requires proteins, we examined the effect of E47 on myogenin phos- dimerization but not DNA binding. To begin to define the phorylation. When COS cells were transfected with a myoge- mechanism by which E24 products potentiate myogenin phos- nin expression vector alone, greater than 75% of the myogenin phorylation, we analyzed the effect of E47 on phosphorylation protein was in the more rapidly migrating form following a 1-h of a series of myogenin mutants that were defective for period of labeling with [35S]methionine (Fig. 2A). In contrast, transcriptional activation, DNA binding, and dimerization. To when myogenin and E47 expression vectors were transfected test whether transcriptional activation played a role in the together, more than half of the myogenin protein was in the induction of myogenin phosphorylation, as has been shown for electrophoretically retarded form, suggesting that E47 induced other transcription factors (41), we examined phosphorylation the conversion of myogenin from the faster- to the slower- of the myogenin mutant T-A87, which contains a threonine-to- migrating species. The same shift to the lower-mobility form of alanine substitution at residue 87 in the basic domain. This myogenin was observed in the presence of E12 and HEB (data mutant oligomerizes with E proteins and binds DNA normally, not shown). By 32p labeling, it was apparent that E47 induced but it fails to activate muscle-specific transcription (8). As the phosphorylation of both the 32- and 34-kDa species (Fig. shown in Fig. 3, the phosphorylation of mutant T-A87 was 2A). The increase in phosphorylation of the 32-kDa species induced in the presence of E47 to an extent comparable to that VOL. 14, 1994 DIMERIZATION-DEPENDENT PHOSPHORYLATION OF MYOGENIN 6235

A. ++ HLH s srr - 55 -.5 .. r... myogerun r WIA I

Bc HeN-I Trauonpr E47-Irfu1ed 1 B-2 5-3 Duoenzation DNA BrdnV ANapon Rvsohoryb.on . Wid-tye 74R R R A A T L R E K R R L K K VN E A F E A L K R S T tW ++ + +

TA87 - A ------++ + _ T-D87 ++ - -

BS-3 ------M L G ------+ - - +

F-P10------P------_

B. 32p 6

wed-type T-A87 TD87 BS-3 F P101 F-P1o1

C. .+HLH E47 Inducten of Phosphorylation Dimenzabon DNA &nding of myogenin

*d-type - A N N ARERVR

D. C/)

LU LU LU

E E E E E E

FIG. 3. E47-dependent phosphorylation of myogenin requires dimerization but not DNA binding. (A) Amino acid sequence of the myogenin basic region and helix 1 of the HLH motif is shown; the names of mutants are to the left. B-2 and B-3 denote the second and third clusters, A respectively, of basic amino acids in the basic region that mediate DNA binding. 5, serine-rich region; S/T, serine- and threonine-rich region. dash indicates no change at that position. The ability of each mutant to dimerize with E47, bind DNA, and activate myogenesis is indicated. -, loss of activity. The properties of these mutants were reported previously (8). (B) COS cells were transiently transfected with expression vectors encoding each myogenin mutant with or without an E47 expression vector, cultures were labeled with 32P or [31 S]methionine as indicated, and myogenin was immunoprecipitated from cell extracts with an antimyogenin monoclonal antibody, as described in the text. Immunoprecipitates were analyzed by SDS-PAGE and autoradiography. [35S]methionine-labeled protein is shown for mutant F-Pl01 to confirm the presence of the protein, since it was undetectable with 32p labeling. All other mutants were also labeled with [31 S]methionine, but those results are not shown. (C) Amino acid sequence of the E47 basic region. The potential of the mutant E47/BS2-3 to dimerize with myogenin and its lack of DNA activity have been shown previously (10). (D) COS cells were transfected with expression vectors encoding myogenin (myo) alone or with E47, 3P-lbee mutant E47/BS2-3, or E47-bHLH, as indicated, and myogenin was immunoprecipitated from labeled cell extracts as described in the text. The two E47 mutants induced myogenin phosphorylation to an extent comparable to that by wild-type E47. of wild-type myogenin, indicating that enhanced phosphoryla- and B). The mutant BS-3, which lacks the third cluster of basic tion of myogenin is not dependent on transcriptional activity. residues in the DNA-binding domain and cannot bind DNA The potential importance of DNA binding for E47-depen- (8), also showed enhanced phosphorylation in the presence of dent phosphorylation of myogenin was tested with the myoge- E47, but the level of phosphorylation of the 34-kDa species nin mutant T-D87, which contains a threonine-to-aspartic acid was lower than that of wild-type myogenin or mutant T-D87 substitution in the DNA-binding domain. This mutant dimer- (Fig. 3A and B). This reduction in phosphorylation likely izes normally but cannot bind DNA (8); its level of phosphor- reflects the reduced efficiency with which this mutant dimerizes ylation, however, was comparable to that of the wild-type (8) (see below). protein in the presence and absence of exogenous E47 (Fig. 3A To further examine the influence of DNA binding on 6236 ZHOU AND OLSON MOL. CELL. BIOL.

E47-induced phosphorylation of myogenin, we expressed wild- A. 100 200 type myogenin with an E47 basic-domain mutant (E47/BS2-3), N-TAD C-TAD which can dimerize but cannot bind DNA (10). As shown in r ++ HLH S SIT Fig. 3C and D, this E47 mutant efficiently induced myogenin wild-type phosphorylation. Together, these results demonstrate that DNA binding is not required for E47 to induce phosphoryla- DM1 -33 IL * tion of myogenin. To begin to map the region of E47 that was required to DM4-79 induce myogenin phosphorylation, we expressed myogenin TM1 58 with an E47 deletion mutant lacking sequences amino-terminal DM4-791 to the bHLH region, which is located near the carboxyl TM1 58 UJ terminus. This mutant, designated E47-bHLH, was as efficient as wild-type E47 in inducing myogenin phosphorylation (Fig. B. 32P 3C and D). These results suggest that the ability of E47 to Wild-Type DM1-33 DM4-79 TM158 induce myogenin phosphorylation maps to the bHLH region of E12: - + - + - + ' + E47, although we cannot rule out the possibility that the short * * ' INttz. -' '''' :.s,',:,. ,.s region carboxyl-terminal to the bHLH region may also be ...... r.S: :-...,,,,...... involved. To determine whether it was necessary for E47 to dimerize with myogenin to induce its phosphorylation, we expressed it with myogenin mutant F-P1ol, which contains a phenylalanine-to-proline substitution in helix 1 of the HLH ...... ;...... t...... ,,,,.,.~~.....,...... motif and cannot dimerize. In contrast to the wild-type protein, failed to show enhanced phosphorylation in the pres- .:.''s'w"|Si'W" F-P1lo :::s~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~. ence of E47 (Fig. 3A and B). Mutant was localized to ,~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~..._::::_e_,,. F-P1lo ...... ~~~~~~~~~...... the nucleus (data not shown), indicating that its failure to be ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~...... _'_~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~...... phosphorylated did not reflect a defect in nuclear transloca- tion. Together, these results suggest that E47 must interact directly with myogenin through the HLH motif to induce its C. 35S phosphorylation. Wild-Type DM1-33 DM4-79 TM158 Dimerization-dependent phosphorylation sites lie within E12: - + - + - + - + the N- and C-terminal regions of myogenin. To begin to localize the site(s) for dimerization-dependent phosphoryla- tion, we examined the effect of E2A products on phosphory- lation of several deletion derivatives of myogenin. As shown in Fig. 4, a deletion mutant lacking amino acids 1 to 33 (DM1-33) continued to show enhanced phosphorylation and gave rise to multiple species in the presence of E12. Deletion mutant DM4-79, which lacks the entire amino terminus, the cysteine- rich region, and the first cluster of basic amino acids, also FIG. 4. Sites for phosphorylation lie within the amino- and carbox- showed an increase in phosphorylation in the presence of E12, yl-terminal regions of myogenin. (A) Schematic representations of but the level of phosphorylation was less than with wild-type myogenin deletion mutants. Mutants DM1-33 and DM4-79 lack myogenin. This mutant also did not give rise to the predomi- residues 1 to 33 and 4 to 79, respectively. Mutant TM158 contains a nant electrophoretically retarded species. The diminished termination codon at position 158. DM4-79/TM158 lacks residues 4 to phosphorylation and the absence of the slowest-migrating 79 and 158 to 224. + + +, basic region; 5, serine-rich region; S/T, species with DM4-79 compared with DM1-33 suggested that a serine- and threonine-rich region. N-TAD and C-TAD, N- and major phosphorylation site lay between residues 34 and 79 (see C-terminal transcription activation domains, respectively. COS cells below). However, that phosphorylation of DM4-79 was en- were transiently transfected with expression vectors encoding each myogenin mutant with and without an E12 expression vector, cultures hanced in the presence of E12, albeit to a lesser degree than were labeled with 32P (B) or [35S]methionine (C), and myogenin was with wild-type myogenin, also suggested that the remainder of immunoprecipitated from cell extracts with an antimyogenin monoclo- the protein contained an additional site for dimerization- nal antibody, as described in the text. Immunoprecipitates were dependent phosphorylation. analyzed by SDS-PAGE and autoradiography. The arrows to the right A truncation mutant lacking residues 158 to 224 (TM158) mark the positions of the fast- and slow-migrating forms of TM158. also showed enhanced phosphorylation in the presence of E12 Only the slower form which was induced by E12 contained detectable and gave rise to the major electrophoretically retarded species amounts of 32p, indicating that phosphorylation of the faster-migrating (Fig. 4). Comparison of [35S]methionine- and 32P-labeled form of myogenin normally occurs between residues 159 and 224. forms of TM158 revealed that only the more slowly migrating form of the protein incorporated measurable amounts of 32P (Fig. 4B and C). These results indicate that the phosphoryla- readily detectable with [35S]methionine (data not shown). tion sites normally observed in the faster-migrating form of These results further support the conclusion that the major myogenin under these conditions are contained between resi- sites for phosphorylation of myogenin under these conditions dues 158 and 224 and confirm that phosphorylation of the are contained in the N- and C-terminal portions of the protein. amino-terminal portion of the protein is responsible for the Identification of the dimerization-dependent phosphoryla- shift in mobility on SDS-PAGE. tion site in the amino terminus. Because the 32P labeling We also analyzed the phosphorylation of a mutant that lacks studies of the above deletion mutants suggested that the the N and C termini (DM4-79/TM158) but were unable to dimerization-dependent phosphorylation site responsible for detect any phosphorylation of this mutant even though it was the shift in mobility of myogenin on SDS-PAGE lay between VOL. 14, 1994 DIMERIZATION-DEPENDENT PHOSPHORYLATION OF MYOGENIN 6237

A. electrophoretic mobility. Tryptic peptide mapping of wild-type myogenin and S-A43 confirmed that serine 43 was the dimer- ++ HLH S S/T ization-dependent phosphorylation site that gave rise to the slower-migrating form of myogenin (data not shown). To investigate whether phosphorylation of serine 43 caused wild-type 41 SLSPE 45 a shift in mobility through the introduction of negative charge, S-A41 A---- we created a myogenin mutant in which serine 43 was replaced S-A43 --A-- with aspartic acid (mutant S-D43) to mimic the negative charge S-D43 of a phosphate at that position. When expressed in COS cells, this mutant gave rise only to the faster-migrating form of the protein (Fig. 5B), which suggests that the shift in electro- upon of serine 43 is not due R phoretic mobility phosphorylation 32p 35S-met to the introduction of negative charge. Mapping of the dimerization-dependent phosphorylation wild- @ I type S-A41 S-A43 '; site in the carboxyl terminus. The initial deletion analyses (Fig. E 47: + . 4) suggested that the region of myogenin between residues 158 and 224 contained one or more dimerization-dependent phos- phorylation sites that, in contrast to the N-terminal site, do not affect myogenin's electrophoretic mobility. To further map these sites, we created additional carboxyl-terminal deletion V.,~I mutants and monitored their phosphorylation by examining o the incorporation of 32p into the more rapidly migrating forms of the proteins on SDS-PAGE. As shown in Fig. 6, deletion of residues 198 to 224 of myogenin (TM198), which eliminates a serine- and threonine-rich region near the C terminus, did not FIG. 5. Identification of dimerization-dependent phosphorylation affect dimerization-dependent phosphorylation of the faster- site in the amino-terminal region of myogenin. (A) The amino acid migrating species. However, deletion to amino acid 157 sequence surrounding the dimerization-dependent phosphorylation (TM158 in Fig. 4B) or 168 (TM169 in Fig. 6) led to a complete site (serine 43) is shown~~~~~~~~~~~~~~~~~~~~~~~~~~~~..-beneath a schematic representation of the loss of 32p labeling of this species. Since the rapidly migrating myogenin protein. Names of mutants are to the left. A dash indicates form of mutant TM169 was readily detectable with [35S]me- no change at that position. (B) COS cells were transfected with thionine (data not shown), these results suggest that the expression vectors encoding wild-type myogenin or the indicated decrease in 32p incorporation into this species reflects the mutants, with and without an E47 expression vector, cultures were deletion of phosphorylation sites between residues 169 and labeled with 32p or [35S]methionine, as indicated, and myogenin was 198. of the sequence revealed immunoprecipitated from cell extracts with an anti-myogenin anti- Inspection myogenin primary body, as described in the text. Immunoprecipitates were analyzed by that serine 170 was the only serine in this region (Fig. 6A). SDS-PAGE and autoradiography. The arrowheads to the left mark the Notably, the sequence surrounding serine 170 is similar to that positions of the fast- and slow-migrating forms of wild-type and mutant surrounding serine 43 (SXSPE). To determine whether serine myogenin proteins. Only the faster-migrating form of myogenin was 170 was indeed the site for dimerization-dependent phosphor- observed when serine 43 was replaced with alanine or aspartic acid. ylation in the carboxyl terminus, we replaced this residue with alanine. This mutant (S-A170) showed a decrease in phosphor- ylation of the more rapidly migrating form of the protein when residues 34 and 79, we examined the sequence of this region analyzed by SDS-PAGE (Fig. 6B), suggesting that serine 170 is for serines that might serve as phosphoacceptors. We found indeed a site for phosphorylation. A similar decrease in three within this region, at positions 41, 43, and 77. Serine 77 phosphorylation was observed when residues 158 to 170 were has previously been shown to be phosphorylated by PKA and deleted from myogenin (DM158-170 in Fig. 6). possibly by PKC (26, 28). However, this site is not appreciably Although mutants S-A170 and DM158-170 showed de- phosphorylated under the conditions of these transfection creased incorporation of 32p into the more rapidly migrating assays, nor does it influence myogenin's mobility on SDS- species, we were able to detect 32p incorporation into the PAGE (26, 28). We therefore focused on serines 41 and 43 faster-migrating forms of these proteins, which suggested that (Fig. 5A). there might be additional phosphorylation sites between resi- To determine whether serine 41 or serine 43 was phosphor- dues 168 and 224. To determine whether the serine- and ylated upon dimerization of myogenin with E2A products, we threonine-rich region near the carboxyl terminus (residues 200 replaced these residues individually with alanines and analyzed to 215) might also contain phosphorylation sites, we deleted the phosphorylation of the resulting mutants (mutants S-A41 this region from DM158-170 to yield the mutant DM158-170/ and S-A43). When mutant S-A41 was expressed in COS cells Tm198. As shown in Fig. 6, this second C-terminal deletion with E12, it gave rise to the more slowly migrating species on abolished all detectable phosphorylation of the faster-migrat- SDS-PAGE, suggesting that serine 41 is not responsible for the ing species. Together, these results demonstrate that a dimer- shift in myogenin's mobility on SDS-PAGE. Mutant S-A43 ization-dependent phosphorylation site maps to serine 170 and showed a high level of phosphorylation in the presence of E47, is contained within a region of primary amino acid sequence but this mutant did not exhibit the slower-migrating form of similar to that surrounding the amino-terminal site. In addi- the protein (Fig. SB). These results demonstrate that phos- tion, it appears that other phosphorylation sites lie between phorylation of serine 43 is responsible for the shift in myoge- residues 199 and 224. nin's mobility on SDS-PAGE. The dramatic induction of Dimerization-dependent phosphorylation sites diminish phosphorylation of mutant S-A43 in the presence of E47 also myogenin's transcriptional activity. To begin to assess the confirms that sites in addition to serine 43 are phosphorylated consequences of phosphorylation of serines 43 and 170 on in response to E47, but these sites do not affect myogenin's myogenin's transcriptional activity, we replaced these residues 6238 ZHOU AND OLSON MOL. CELL. BIOL.

200 215 A. SLTSIVDSIrVEDMSV +++ HLH wild-type VPSECNSHSASCSPE 158 172

TM1 98 I mMEM,\NI 9::::I

TM1 69 I ELE: TM158 I~~~~I' I -- "MO I DM158-170

DM1 58-170/ I- TM198 1E~~II

B. 32p DM DM 158--170/ wild-type TM198 TMR 69 158-170 TM111198 S-A1 70 E47: - + + + + + + _ b :: _ _ OEM .: s* i.. :7 ..E _ R ._t _r X X _: _ .'. ;_.'.' _ Y :. _ 9 XJ l _ F ._, _; _ .::. ,.%,S _ T ._,:._ _E' 'S0 :w. .. E W. .,e..... F. W of_18[: :a: w: lig rs.s... sN, .,_,,,) ., tCg. S'' -:. _ .. -g- _t .:>> _ | ._ .... '...... _ | ._." _. :.'.'': _ _ | :_. _ ::^S. _ _ _. _ * ._', .a_, > _ - WF

,' !.m.:,..': .. ' '::__i,._F',1& __.al:.r _.EI!F b ....'S;.._,.:,' FIG. 6. Mapping of dimerization-dependent phosphorylation sites in the carboxyl-terminal region of myogenin by deletion mutagenesis. (A) Schematic representation of myogenin deletion mutants. Mutants TM198, TM169, and TM158 contain termination codons at position 198, 169, and 158, respectively. DM158-170 lacks amino acids 158 to 170, and DM158-170/TM198 lacks amino acids 158 to 170 and 198 to 224. (B) COS cells were transfected with expression vectors encoding wild-type_|._.....myogenin_...or the....indicated..: mutants, with and without an E47 expression vector, cultures were labeled with 32p, and myogenin was immunoprecipitated from cell extracts with an antimyogenin antibody, as described in the text. Immunoprecipitates were analyzed by SDS-PAGE and autoradiography._|._._n...... The arrowheads_::_Sto the left of each set of lanes mark the positions of the fast- and slow-migrating forms of wild-type and mutant _myogenin||....]_:aW_,proteins.ITE::...... _Es.TM169*.=Fand DM158-170/TM198 each gave rise to two species when labeled with [35S]methionine (data not shown), but with _32p,*only....the ,-a.more slowly.^s.migrating.sF*formsor of these proteins were detected. The positions of the faster-migrating [35S]methionine-labeled species are_ indicated|!..._i_i;* by...... _the larger,,t,lowerf:i*s.arrowheads...... with alanines and tested the resulting mutants for their ability phosphorylation sites have enhanced transcriptional activity to transactivate CAT reporters under control of the MCK (30, 43). enhancer (MCK-CAT) or four tandem copies of the high- We also tested whether substitution of serine 43 or 170 with affinity E box from the MCK enhancer (4R-tkCAT). When aspartic acid affected myogenin's transcriptional activity. In introduced into either 1OT1/2 fibroblasts or COS cells, single- contrast to the mutants in which these residues were changed phosphorylation-site mutants (S-A43 and S-A170) showed tran- to alanines, mutants S-D43 and S-D170 showed transcriptional scriptional activities greater than that of the wild-type protein, activities slightly lower than that of the wild-type protein (Fig. and the mutant with both phosphorylation sites mutated 7A). Since we observed an approximately fivefold increase in (S-A43/S-AI70) showed even greater activity (Fig. 7A). The phosphorylation of serine 43 in the presence of E proteins, we enhanced activity of these mutants was observed in the absence would anticipate a greater reduction in transcriptional activity and presence of exogenous E12. These results suggest that of these mutants if phosphorylation reduced myogenin's activ- phosphorylation of serines 43 and 170 diminishes myogenin's ity by the introduction of negative charge. transcriptional activity. This result is consistent with those of Dimerization-dependent phosphorylation sites diminish previous studies, which showed that deletion mutant DM158- transcriptional activity through the GAL4 DNA-binding site. 170 and amino-terminal deletion mutants which lack these Since serines 43 and 170 lie outside the bHLH region, their Relative A. 100 200 Transactivation COS Cells S S srr 1OT1/2 Cells 4R-tkCAT ] MCK-CAT 4R-tkCAT (-E12) (+E12) 100.0 t 28.3 100.0 ± 45.3 100.0 ± 9.3 220.9 ± 39.5 wild-type 41 SLSPEI45 168 SCSPE172 S-A43 --A-- 178.3 ± 30.4 281.1 + 41.5 246.5 + 72.1 568.4 + 181.4 S-A170 - -A- - 180.4 ± 21.7 345.3 ± 128.3 325.6 ± 116.3 776.8 + 346.5 S-A43/S-A170 --A-- - -A- - 421.0 ± 41.3 381.1 + 47..2 416.3 + 197.8 702.6 + 279.1 87.6 ± 9.5 48.2 ± 9.5 73.0 ± 15.2 90.2 ± 4.9

S-D170 89.7 ± 28.9 54.5 + 1.8 74.5 + 30.1 94.5 + 10.9

B. 100 200

S S/T GAL4(1-147) Relative Transactivalion

GAL4-Myo 41 SLSPE45 168 SCSPE172 100.0 + 47.8 GAL4-MyoS-A43 --A-- 165.2 + 21.7 GAL4-MyoS-A170 --A-- 273.9 + 34.8 GAL4-MyoS-A43/S-A170 - -A- - - -A - - 400.0 + 43.5

C. 35S-met 32p 0 rl_ CD C D c1r c _- v

U) U) C) ch c)) U cn O 0 COIU Uo0 O o 0 o N oCl >% 0 >% >% 2 J J 0 < cF <

FIG. 7. Mutagenesis of dimerization-dependent phosphorylation sites increases myogenin's transcriptional activity. (A and B) Amino acid sequences surrounding the dimerization-dependent phosphorylation sites (serine 43 and 170); names of mutants are to the left. A dash indicates no change at that position. (A) Expression vectors encoding each mutant were transfected into 1OT1/2 or COS cells with the indicated reporter genes. Transfections of COS cells were carried out with and without an expression vector encoding E12, as indicated. Values are expressed relative to the CAT activity observed with myogenin with each reporter gene. (B) Expression vectors encoding each GAL4 chimera were transfected into COS cells with pG5E1bCAT as a reporter. CAT activity in extracts from transfected cells was determined as described in the text. Values are expressed relative to the CAT activity observed with GAL4-myo. The results in panels A and B represent the averages of at least three independent experiments. (C) COS cells were transfected with the indicated expression vectors, cultures were labeled with [35S]methionine or 2p, and GAL4-myogenin chimeric proteins were immunoprecipitated from cell extracts with an antimyogenin antibody, as described in the text. Immunoprecipitates were analyzed by SDS-PAGE and autoradiography. The arrows in the left panel point to the two forms of GAL4-Myo. 6239 6240 ZHOU AND OLSON MOL. CELL. BIOL. effects on myogenin's transcriptional activity are likely to be motif near their carboxyl termini (5, 6, 12, 31, 39, 40). Serine 43 mediated by the amino- and carboxyl-terminal transcriptional and the surrounding sequence are conserved in myogenin activation domains. To test this possibility, we investigated proteins from mouse, human, and rat, but this sequence is whether mutation of these residues affected the transcriptional absent from chicken myogenin (4, 14, 16, 53). Other myogenic activity of chimeric proteins in which myogenin was fused to bHLH proteins also contain numerous sites near their amino the DNA-binding domain of the yeast protein GAL4. As termini that conform to the site for dimerization-dependent reported previously, the GAL4-myogenin chimera (GAL-myo) phosphorylation (Ser-Pro) (6, 12, 31, 39, 40). This conservation efficiently activated transcription through the GAL4 binding suggests that these phosphorylation sites play an important site (30, 43). Replacement of serines 43 and 170 with alanine in role in regulating the function not only of myogenin, but also GAL4-myo significantly increased myogenin's transcriptional of other members of this family of myogenic regulators. activity (Fig. 7B). These results support the conclusion that Further evidence to suggest that other myogenic bHLH pro- phosphorylation of these sites in myogenin diminishes myoge- teins are phosphorylated by the dimerization-dependent ki- nin's transcriptional activity through an effect on its activation nase comes from the observation that MyoD becomes hyper- domains. phosphorylated in the presence of E47 (25). We also investigated whether serines 43 and 170 were Phosphorylation of myogenin by the dimerization-depen- phosphorylated in the GAL4-myogenin chimera. GAL4-myo dent kinase appears to diminish transcriptional activity. How migrated as a doublet on SDS-PAGE, whereas the chimera might phosphorylation at the above sites diminish myogenin's containing a serine-to-alanine substitution at residue 43 transcriptional activity? The transcription activation domains (GAL4-myoS-A43) migrated only as a single species (Fig. 7C). of myogenin have been mapped to residues 7 to 77 and 156 to Thus, phosphorylation of serine 43 appears to retard the 224 and therefore encompass the dimerization-dependent migration of the GAL4-myo chimera on SDS-PAGE in the phosphorylation sites (43). Because these phosphorylation same way as it does for wild-type myogenin. Substitution of sites lie outside of the bHLH region and do not influence alanine for serine 170 in the GAL4-myo chimera (GAL4- myogenin's ability to dimerize or bind DNA, we favor the myoS-AI70) did not significantly affect the mobilit' of the possibility that phosphorylation alters the efficiency with which protein on SDS-PAGE but dramatically decreased3 P incor- the transcription activation domains interact with other com- poration, indicating that this residue is also phosphorylated in ponents of the transcriptional machinery to activate target the GAL4 fusion protein. We assume that endogenous E2A muscle genes. One mechanism through which this might occur products are responsible for the phosphorylation of these sites would be a phosphorylation-induced conformational change in in the GAL4 fusion protein. myogenin. In this regard, our results show that phosphoryla- tion of serine 43 leads to a shift in the mobility of myogenin on DISCUSSION SDS-PAGE, which could reflect altered conformation. Nega- tive charge alone does not appear to be sufficient to induce this Myogenin, like other myogenic bHLH proteins, activates change in electrophoretic mobility, since mutant S-D43, which muscle-specific transcription as a heterodimer with ubiquitous contains a serine-to-aspartic acid substitution at this position, bHLH proteins, such as the E2A products E12 and E47 and the showed the same mobility as the unphosphorylated form of HEB gene product (8, 21, 25). Our results demonstrate that myogenin. Moreover, this mutant and another in which serine dimerization with E2A products potentiates the phosphoryla- 170 was replaced with aspartate showed transcriptional activ- tion of myogenin at sites near its amino and carboxyl termini. ities only slightly lower than that of the normal protein, These dimerization-dependent phosphorylation sites are con- suggesting that the negative charge introduced by phosphory- tained within regions of similar amino acid sequence, suggest- lation at these sites probably does not by itself account for the ing that they are phosphorylated by the same kinase. Myogenin apparent inhibitory effects of phosphorylation on myogenin's mutants in which the dimerization-dependent phosphorylation transcriptional activity. sites are replaced with alanines show enhanced transcriptional The finding that serine 170 decreases myogenin's transcrip- activity, consistent with the conclusion that phosphorylation at tional activity is consistent with the finding that deletion of the these sites diminishes the efficiency with which myogenin corresponding region of myf5 increased its myogenic activity activates its target genes. (7, 51). Similarly, deletion mutants of MyoD that lack potential Dimerization-dependent kinase phosphorylates myogenin's dimerization-dependent phosphorylation sites in the amino transcription activation domains. The major sites for dimer- and carboxyl termini show enhanced myogenic activity relative ization-dependent phosphorylation of myogenin are serines 43 to the wild-type protein (46). Two such sites are contained in and 170, both of which are contained within the amino acid the amino-terminal activation domain of MyoD (12, 50). sequence SPE. We do not yet know the identity of the kinase Myogenin deletion mutants lacking the amino- and carboxyl- that phosphorylates these sites or its exact recognition se- terminal dimerization-dependent sites also show greater tran- quence. However, numerous proline-dependent protein ki- scriptional activity than the wild-type protein (30, 43). nases recognize serine or threonine followed by proline (24) Another mechanism through which phosphorylation could and are therefore candidates for the dimerization-dependent diminish the activity of myogenin would be by decreasing its kinase. Among these is the cell cycle-regulated protein kinase half-life. We have compared the half-lives of mutants S-A43 p34cdc2 and the mitogen-activated protein (MAP) kinases (3, and S-A170 as well as mutants with mutations at both of these 38). We note that serines 43 and 170 are contained in a region positions with that of the wild-type protein and found no identical to that of the phosphorylation sites in the N-terminal significant differences (54). The wild-type and mutant proteins activation domain of c-Jun (SPD/E) (44). The kinase that also accumulate to comparable levels during metabolic label- phosphorylates these sites in c-Jun is activated by growth ing experiments. Thus, it does not appear that phosphorylation factors and oncoproteins (20, 44). at the dimerization-dependent sites influences the turnover of Serine 170 and the surrounding amino acid sequence is myogenin. conserved in myogenin proteins from mouse, human, rat, There are only a few other examples, to date, of negative chicken, and quail (4, 14, 16, 39, 53). MyoD, myf5, and MRF4 control of transactivation by phosphorylation. The transcrip- also contain a serine within a similar amino acid sequence tional activity of the yeast ADR1, for VOL. 14, 1994 DIMERIZATION-DEPENDENT PHOSPHORYLATION OF MYOGENIN 6241

myogenin

E-Proteins

++ E12

1 Dimerization- Dependent Kinase

N C

N C C) FIG. 8. Hypothetical model for dimerization-dependent phosphorylation of myogenin. In the monomeric form, serines 43 and 170 may be cryptic. Upon dimerization with E proteins, these sites acquire the potential to be phosphorylated by a dimerization-dependent kinase. When phosphorylated, serines 43 and 170 diminish myogenin's transcriptional activity, suggesting that the transcriptionally active species lacks phosphate at these positions.

example, is normally repressed by PKA independent of DNA essential activator of the kinase. If this is the case, only the binding (11). Repression is associated with phosphorylation of bHLH region and the short carboxyl-terminal domain of E2A a serine in the ADRI transactivation region, whereas mutants products would seem to be required for binding this kinase, lacking this phosphorylation site are constitutively active. The because an E47 deletion mutant containing only these regions ability of c-Fos to transrepress its own expression is also is as efficient as the complete E47 protein in potentiating dependent on phosphorylation sites near its C terminus (35). myogenin phosphorylation. That HEB, which diverges from Replacement of these residues with alanines abolishes transre- E2M products outside the bHLH region, is as efficient as E12 pression, whereas replacement with glutamic acids mimics the and E47 in inducing myogenin phosphorylation also suggests effect of phosphorylation, suggesting that the effects of phos- that the bHLH region is sufficient to induce myogenin phos- phorylation are due to negative charge (35). phorylation (54). Potential mechanisms for dimerization-dependent phos- Previously, Lassar and coworkers reported that E47 en- phorylation. How might E24 products potentiate myogenin hances the phosphorylation of MyoD and that DNA binding phosphorylation? Induction of phosphorylation by E24 prod- was required for this enhancement (25). Our results indicate ucts requires their dimerization with myogenin but does not that phosphorylation of myogenin in response to E47 requires require DNA binding or transcriptional activation. We there- neither DNA binding nor transcription activation. Whether fore favor a model in which serines 43 and 170 are cryptic in these differences are attributable to subtle experimental differ- the monomeric form of myogenin, making them inaccessible to ences or reflect distinct mechanisms through which E2M prod- the dimerization-dependent kinase (Fig. 8). Upon dimeriza- ucts regulate the phosphorylation of myogenin and MyoD tion with EMA products, these sites would presumably become remains to be determined. exposed and phosphorylatable. It is possible that E2A products Inasmuch as myogenic bHLH proteins require EM4 products recruit a kinase to the heterodimeric complex and serve as an to activate myogenesis, it seems paradoxical that the associa- 6242 ZHOU AND OLSON MOL. CELL. BIOL. tion of myogenin with E2A products would diminish its tran- multiplicity of effects of phosphorylation on the activities of scriptional activity. What might be the physiological relevance transcription factors, it is not unreasonable to anticipate that of this type of phosphorylation? From the ratio of the fast- and this type of regulatory interaction may prove to be a common slow-migrating forms of myogenin in differentiated muscle mechanism for cross-talk between transcriptional regulators. cells, it appears that the majority of the protein (-75%) at steady state does not contain phosphate at serine 43 (9, 18, 26, ACKNOWLEDGMENTS 28). We cannot be certain whether myogenin molecules lack- We are grateful to Woody Wright for the generous gift of antimyo- ing phosphate at this site are monomeric or whether only a genin monoclonal antibody and to Tom Kadesch, Case Murre, and fraction of dimerized myogenin becomes phosphorylated at Andrew Lassar for plasmids. this position. It is conceivable, for example, that a fraction of This work was supported by grants from the NIH, the Robert A. the faster-migrating form of myogenin is associated with EMA Welch Foundation, and the Council for Tobacco Research to E.N.O. products and that this is the transcriptionally active form of the protein. In this model, phosphorylation at serine 43, and REFERENCES perhaps at serine 170, might restrict the activity of excess 1. Benezra, R., R. L. Davis, D. Lockshon, D. L. Turner, and H. myogenin molecules and thereby prevent "squelching" as a Weintraub. 1990. The protein Id: a negative regulator of helix- factors. It is loop-helix DNA binding proteins. Cell 61:49-59. result of their interaction with other transcription 2. Blackwell, K. T., and H. Weintraub. 1990. Differences and simi- also possible that dimerization-dependent phosphorylation larities in DNA-binding preferences of MyoD and E2A protein represents a normal step in the processing of myogenin or that complexes revealed by binding site selection. Science 250:1104-1110. it inactivates excess myogenin molecules. 3. Blenis, J. 1993. Signal transduction via the MAP kinases: proceed It is also possible that the dimerization-dependent phos- at your own risk. Proc. Natl. Acad. Sci. USA 90:5889-5892. phorylation sites are targets for a growth factor-inducible 4. Braun, T., E. Bober, G. Buschhausen-Denker, S. Kohtz, K.-H. protein kinase that suppresses myogenin's activity. That the Grzeschik, and H. H. Arnold. 1989. Differential expression of dimerization-dependent phosphorylation sites correspond to myogenic determination genes in muscle cells: possible autoacti- the consensus for MAP and Cdc2 kinases (3, 24), which are vation by the Myf gene products. EMBO J. 8:3617-3625. 5. Braun, T., E. Bober, B. Winter, N. Rosenthal, and H. H. Arnold. activated in mitotic cells in which myogenin and other myo- 1990. Myf-6, a new member of the human gene family of myogenic genic bHLH proteins are inactive, is consistent with this determination factors: evidence for a gene cluster on possibility. If these sites are indeed targets for a growth 12. EMBO J. 9:821-831. factor-inducible protein kinase, the finding that their replace- 6. Braun, T., G. Buschhausen-Denker, E. Bober, E. Tannich, and ment with alanines leads to enhanced transcriptional activity H. H. Arnold. 1989. A novel human muscle factor related to but suggests that this kinase must be active in cultured cells under distinct from MyoD1 induces myogenic conversion in 10T1/2 the conditions of our transfection assays. The kinase(s) must fibroblasts. EMBO J. 8:701-709. also be active in cultures of differentiated C2 myotubes, 7. Braun, T., B. Winter, E. Bober, and H. H. Arnold. 1990. Tran- because the electrophoretically retarded form of myogenin scriptional activation domain of the muscle-specific gene-regula- of serine 43 is present in tory protein myf5. Nature (London) 346:663-665. which arises from phosphorylation 8. Brennan, T. J., T. Chakraborty, and E. N. Olson. 1991. Mutagen- differentiated muscle cells (9, 13, 26, 28). We have attempted esis of the myogenin basic region identifies an ancient protein to determine whether the dimerization-dependent kinase motif critical for activation of myogenesis. Proc. Natl. Acad. Sci. might be downregulated upon exposure of cells to growth USA 88:5675-5679. factor-deficient medium, but thus far we have not been able to 9. Brennan, T. J., and E. N. Olson. 1990. Myogenin resides in the demonstrate its regulation. nucleus and acquires high affinity for a conserved enhancer Myogenin is a target for multiple protein kinases. The element on heterodimerization. Genes Dev. 4:582-595. myogenin protein appears to be a target for regulation by a 10. Chakraborty, T., T. J. Brennan, L. Li, D. G. Edmondson, and E. N. variety of protein kinases. In addition to the dimerization- Olson. 1991. Inefficient homooligomerization contributes to the dependence of myogenin on E2A products for efficient DNA dependent phosphorylation sites, deletion mutations indicate binding. Mol. Cell. Biol. 11:3633-3641. that the serine- and threonine-rich domain near the carboxyl 11. Cherry, J. R., T. R. Johnson, C. A. Dollard, J. R. Shuster, and C. L. terminus (residues 200 to 221) contains one or more phospho- Denis. 1989. Cyclic AMP-dependent protein kinase phosphory- serines. Serines 203 and 207 are contained within the consen- lates and inactivates the yeast transcriptional activator ADRI. Cell sus sequences for casein kinase II, which can phosphorylate 56:409-419. bacterially expressed myogenin (27). It remains to be deter- 12. Davis, R. L., H. Weintraub, and A. B. Lassar. 1987. Expression of mined, however, whether these are the residues in this region a single transfected cDNA converts fibroblasts to myoblasts. Cell that are phosphorylated and whether casein kinase II is the 51:987-1000. kinase that phosphorylates these sites in vivo. It is interesting 13. Edmondson, D. G., T. J. Brennan, and E. N. Olson. 1991. Mitogenic repression of myogenin autoregulation. J. Biol. Chem. that these residues are contained within a region of the 266:21343-21346. carboxyl-terminal activation domain that is required for tran- 14. Edmondson, D. G., and E. N. Olson. 1989. A gene with homology scriptional activity (43). to the similarity region of MyoDl is expressed during Previously, we reported that PKC phosphorylates threonine myogenesis and is sufficient to activate the muscle differentiation 87 in the basic region of myogenin, resulting in inhibition of program. Genes Dev. 3:628-640. DNA binding (26). PKA also phosphorylates serine 79 in the 15. Emerson, C. P. 1990. Myogenesis and developmental control basic region, but the functional significance of this phosphor- genes. Curr. Opin. Cell Biol. 2:1065-1075. ylation is unclear (28). The existence of multiple protein 16. Fujisawa-Sehara, A., Y. Nabeshima, Y. Hosoda, T. Obinata, and kinases that act on different domains of myogenin to inhibit its Y.-I. Nabeshima. 1990. Myogenin contains two domains conserved in cells may ensure that it remains inactive among myogenic factors. J. Biol. Chem. 265:15219-15223. activity proliferating 17. Hardy, S., Y. Kong, and S. F. Konieczny. 1993. Fibroblast growth under conditions in which muscle gene transcription is inhib- factor inhibits MRF4 activity independently of the phosphoryla- ited. tion status of a conserved threonine residue within the DNA- The regulation of myogenin phosphorylation by E2M prod- binding domain. Mol. Cell. Biol. 13:5943-5956. ucts represents a novel mechanism by which one transcription 18. Hasty, P., A. Bradley, J. Morris, D. G. Edmondson, J. M. Venuti, factor can regulate the phosphorylation of another. Given the E. N. Olson, and W. H. Klein. 1993. Muscle deficiency and VOL. 14, 1994 DIMERIZATION-DEPENDENT PHOSPHORYLATION OF MYOGENIN 6243

neonatal death in mice with a targeted mutation in the myogenin ation within the myogenic lineage. Dev. Biol. 154:261-272. gene. Nature (London) 364:501-506. 38. Pelech, S. L., and J. S. Sanghera. 1992. Mitogen-activated protein 19. Henthorn, P., M. Kildejian, and T. Kadesch. 1990. Two distinct kinases: versatile transducers for cell signaling. Trends Biochem. transcription factors that bind the immunoglobulin enhancer Sci. 17:233-238. ~LE5/KE2 motif. Science 247:467-470. 39. Pownall, M. E., and C. P. Emerson, Jr. 1992. Sequential activation 20. Hibi, M., L. Anning, T. Smeal, A. Minden, and M. Karin. 1993. of three myogenic regulatory genes during somite morphogenesis Identification of an oncoprotein- and UV-responsive protein in quail embryos. Dev. Biol. 151:67-79. kinase that binds and potentiates the c-Jun activation domain. 40. Rhodes, S. J., and S. F. Konieczny. 1989. Identification of MRF4: Genes Dev. 7:2135-214. a new member of the muscle regulatory factory gene family. Genes 21. Hu, J.-S., E. N. Olson, and R. E. Kingston. 1992. HEB, a Dev. 3:2050-2061. helix-loop-helix protein related to E2A and ITF2 that can modu- 41. Sadowski, I., D. Niedbala, K. Wood, and M. Ptashne. 1991. GAL4 late the DNA-binding ability of myogenic regulatory factors. Mol. is phosphorylated as a consequence of transcriptional activation. Cell. Biol. 12:1031-1042. Proc. Natl. Acad. Sci. USA 88:10510-10514. 22. James, G., and E. N. Olson. 1992. Deletion of the regulatory 42. Scales, J., E. N. Olson, and M. Perry. 1990. Two distinct Xeniopus domain of protein kinase C a. exposes regions in the hinge and genes with homology to MyoD1 are expressed before somite catalytic domains that mediate nuclear targeting. J. Cell Biol. formation in early embryogenesis. Mol. Cell. Biol. 10:1516-1524. 116:863-874. 43. Schwarz, J. J., T. Chakraborty, J. Martin, J. Zhou, and E. N. 23. Kadesch, T. 1992. Helix-loop-helix proteins in the regulation of Olson. 1992. The basic region of myogenin cooperates with two immunoglobulin gene transcription. Immunol. Today 13:31-36. transcription activation domains to induce muscle-specific tran- 24. Kemp, B. E., and R. B. Pearson. 1990. Protein kinase recognition scription. Mol. Cell. Biol. 12:266-275. sequence motifs. Trends Biochem. Sci. 15:342-346. 44. Smeal, T., B. Binetruy, D. Mercola, A. Grover-Bardwick, G. 25. Lassar, A. B., R. L. Davis, W. E. Wright, T. Kadesch, C. Murre, A. Heidecker, U. R. Rapp, and M. Karin. 1992. Oncoprotein-medi- Voronova, D. Baltimore, and H. Weintraub. 1991. Functional ated signalling cascade stimulates c-Jun activity by phosphoryla- activity of myogenic HLH proteins requires heterooligomerization tion of serines 63 and 73. Mol. Cell. Biol. 12:3507-3513. with E12/E47-like proteins in vivo. Cell 66:305-315. 45. E. W. M. D. T. and 26. Li, L., R. Heller-Harrison, M. Czech, and E. N. Olson. 1992. Cyclic Sternberg, A., G. Spizz, Perry, Vizard, Weil, AMP-dependent protein kinase inhibits the activity of myogenic E. N. Olson. 1988. Identification of upstream and intragenic helix-loop-helix proteins. Mol. Cell. Biol. 12:4478-4485. regulatory elements that confer cell-type-restricted and differen- 27. Li, L., and E. N. Olson. Unpublished observations. tiation-specific expression on the muscle creatine kinase gene. 28. Li, L., J. Zhou, G. James, R. Heller-Harrison, M. P. Czech, and Mol. Cell. Biol. 8:2896-2909. E. N. Olson. 1992. FGF inactivates myogenic helix-loop-helix 46. Tapscott, S. J., R. L. Davis, M. J. Thayer, P.-F. Cheng, H. proteins through phosphorylation of a conserved protein kinase C Weintraub, and A. B. Lassar. 1988. MyoDl: a nuclear phospho- site in their DNA-binding domains. Cell 71:1181-1194. protein requiring a myc homology region to convert fibroblasts to 29. Lillie, J. W., and M. R. Green. 1989. Transcription activation by myoblasts. Science 242:405-411. the adenovirus Ela protein. Nature (London) 338:39-44. 47. Thayer, M. J., S. J. Tapscott, R. L. Davis, W. E. Wright, A. B. 30. Martin, J. F., L. Li, and E. N. Olson. 1992. Repression of Lassar, and H. Weintraub. 1989. Positive autoregulation of the myogenin function by TGF-,B1 is targeted at the basic helix-loop- myogenic determination gene MyoD1. Cell 58:241-248. helix motif and is independent of E2A products. J. Biol. Chem. 48. Weintraub, H., R. Davis, D. Lockshon, and A. Lassar. 1990. MyoD 267:10956-10960. binds cooperatively to two sites in a target enhancer sequence: 31. Miner, J. H., and B. Wold. 1990. Herculin, a fourth member of the occupancy of two sites is required for activation. Proc. Natl. Acad. MyoD family of myogenic regulatory genes. Proc. Natl. Acad. Sci. Sci. USA 87:5623-5627. USA 87:1089-1093. 49. Weintraub, H., R. Davis, S. Tapscott, M. Thayer, M. Krause, R. 32. Mitsui, K., M. Shirakata, and B. M. Paterson. 1993. Phosphory- Benezra, T. Blackwell, D. Turner, R. Rupp, S. Hollenberg, Y. lation inhibits the DNA-binding activity of MyoD homodimers but Zhuang, and A. Lassar. 1991. The myoD gene family: nodal point not MyoD-E12 heterodimers. J. Biol. Chem. 268:24415-24420. during specification of the lineage. Science 251:761- 33. Murre, C., P. S. McCaw, and D. Baltimore. 1989. A new DNA 766. binding and dimerization motif in immunoglobulin enhancer 50. Weintraub, H., V. J. Dwarki, I. Verma, R. Davis, S. Hollenberg, L. binding, daughterless, MyoD, and myc proteins. Cell 56:777-783. Snider, A. Lassar, and S. J. Tapscott. 1991. Muscle-specific 34. Murre, C., P. S. McCaw, H. Vaessin, M. Caudy, L. Y. Jan, Y. N. transcriptional activation by MyoD. Genes Dev. 5:1377-1386. Jan, C. V. Cabrera, J. N. Buskin, S. D. Hauschka, A. B. Lassar, H. 51. Winter, B., T. Braun, and H. H. Arnold. 1992. Co-operativity of Weintraub, and D. Baltimore. 1989. Interactions between heter- functional domains in the muscle-specific transcription factor ologous helix-loop-helix proteins generate complexes that bind Myf-5. EMBO J. 11:1843-1855. specifically to a common DNA sequence. Cell 58:537-544. 52. Winter, B., T. Braun, and H. H. Arnold. 1993. cAMP-dependent 35. Ofir, R., V. J. Dwarki, D. Rashid, and I. M. Verma. 1990. protein kinase represses myogenic differentiation and the activity Phosphorylation of the C terminus of fos protein is required for of the muscle-specific helix-loop-helix transcription factors Myf-5 transcriptional transrepression of the c-fos promoter. Nature and MyoD. J. Biol. Chem. 268:9869-9878. (London) 348:80-82. 53. Wright, W. E., D. A. Sassoon, and V. K. Lin. 1989. Myogenin, a 36. Olson, E. N. 1990. The MyoD family: a paradigm for develop- factor regulating myogenesis, has a domain homologous to MyoD. ment? Genes Dev. 4:1454-1461. Cell 56:607-617. 37. Olson, E. N. 1993. Interplay between proliferation and differenti- 54. Zhou, J., and E. N. Olson. Unpublished observations.