Activated MEK1 Binds the Nuclear MyoD Transcriptional Complex to Repress Transactivation  Robert L.S Perry, Maura H Parker, Michael A Rudnicki  Molecular Cell  Volume 8, Issue 2, Pages 291-301 (August 2001) DOI: 10.1016/S1097-2765(01)00302-1

Figure 1 Activated MEK1 Represses Transcriptional Activity of the Myogenic Factors (A and B) Myogenic factors were cotransfected alone or with wild-type, dominant-negative, or activated MEK1, and their transcriptional activity was assessed using (A) 4RtkCAT or (B) MLC-CAT reporter vectors. The bars represent the mean, and the error bars represent the standard error of the mean (±SEM; n = 9). (C) 10T1/2 mouse fibroblasts were transfected with the vectors shown and induced to undergo terminal differentiation. Differentiation was assessed by immunocytochemical detection of terminally differentiated myotubes with antibody MF20, which is reactive with the myosin heavy chain (black arrows). Coexpression of MyoD with activated MEK1 efficiently repressed differentiation. (D) Immunoblot analysis of transfected 10T1/2 cell lysates probed with the indicated antibodies. Note the reduced myogenin levels when MyoD was transfected with activated MEK1 Molecular Cell 2001 8, 291-301DOI: (10.1016/S1097-2765(01)00302-1)

Figure 2 Activation of MEK1 Inhibits the Switch from Proliferation to Differentiation (A) Stimulation of C2C12 myoblasts with TPA (200 ng/ml, 5 min) resulted in a high proportion of cells exhibiting activated endogenous MEK1 in the nucleus, as detected with anti-phospho-MEK1 antibody 30 min following stimulation. (B) Synchronized C2C12 myoblasts incubated in differentiation medium in the presence of the specific MEK1 inhibitor U0126 (10 μM) exhibited precocious differentiation by 48 h, as assessed by staining with antibody MF20, which is reactive with the myosin heavy chain (arrows). Percent MF20-positive calculations represent mean values calculated using counts of more than 1000 nuclei on multiple experimental culture dishes and are as follows: DMSO 24 hr, 0.20%; DMSO 48 hr, 3.55%; U0126 24 hr, 2.19%; U0126 48 hr, 22.11%. (C) C2C12 myoblasts transiently transfected with activated MEK1 and GFP failed to efficiently form multinucleated myotubes after 5 days in differentiation medium Molecular Cell 2001 8, 291-301DOI: (10.1016/S1097-2765(01)00302-1)

Figure 3 Immunofluorescence Reveals the Presence of Nuclear-Activated MEK1 (A and B) Transfected 10T1/2 fibroblasts were simultaneously cotransfected with a constitutive GFP vector (GFP panels) to examine MyoD and MEK1 localization. MyoD or HA-tagged MEK1 proteins were immunodetected using rhodamine-conjugated secondary antibodies (rhodamine panels). MyoD was strictly localized to the nucleus under all conditions (yellow arrows). Wild-type and dominant-negative (not shown) MEK1 displayed preferential cytoplasmic localization, whereas (B) activated MEK1 was detected in both the nucleus and the cytoplasm (white arrows). Photographs were taken using a 40× objective. (C) Nuclear versus cytoplasmic extracts were subjected to immunoblotting using the indicated antibodies. MyoD was detected at similar levels in both nuclear and cytoplasmic fractions when coexpressed with the three versions of MEK1. Under low serum conditions (2% horse serum), abundant activated HA-MEK1 was present in the nuclear fraction due to the deletion of the nuclear export signal (nuclear lane 10) Molecular Cell 2001 8, 291-301DOI: (10.1016/S1097-2765(01)00302-1)

Figure 4 Activated MEK1 Repression of MyoD Activity Is Independent of Chromatin Remodeling Domains (A) A schematic representation of the MyoD mutants containing deleted domains. (B) 10T1/2 fibroblasts were transfected with mutant MyoD vectors in the presence and absence of activated MEK1, and the transactivation ability was assessed using the MLC-CAT reporter vector. With the exception of MyoDΔ3–56, which did not transactivate, coexpression of activated MEK1 efficiently repressed transcriptional activation of the active MyoD deletion mutants. The bars represent the mean, and the error bars represent the standard error of the mean (±SEM; n = 9 for all but MyoDΔ63–99/Δ216–269, in which n = 6). Abbreviations: TAD, transactiation domain; CRD, chromatin binding domain; b, basic domain; H-L-H, helix-loop-helix domain Molecular Cell 2001 8, 291-301DOI: (10.1016/S1097-2765(01)00302-1)

Figure 5 Repression of MyoD by MEK1 Requires the MyoD N-Terminal Transactivation Domain (A) A schematic representation of the C terminus-truncated mutants of MyoD with and without the VP16 acidic transactivation domain substituted for the transactivation of MyoD. (B) Truncated mutants of MyoD containing or lacking the N-terminal chromatin-remodeling domain transactivated 4RtkCAT but were repressed in the presence of activated MEK1. The substitution of VP16 for the amino-terminal transactivation domain of MyoD abolished repression exerted by activated MEK1. The bars represent the mean, and the error bars represent the standard error of the mean (±SEM; n = 3). Abbreviations: TAD, transactiation domain; CRD, chromatin binding domain; b, basic domain; H-L-H, helix-loop-helix domain. (C) 10T1/2 mouse fibroblasts were transfected with the vectors shown and allowed to undergo terminal differentiation. Differentiation was assessed by immunocytochemical detection of myotubes with the anti-sarcomeric myosin heavy chain antibody MF20 (black arrows). Coexpression of activated MEK1 strongly repressed differentiation induced by MyoD 1-bHLH and reduced myotube size induced by VP16-63bHLH. (D) Immunoblot analysis of 10T1/2 mouse fibroblasts transfected with VP16-63bHLH and mutant versions of MEK1 reveals that activated MEK1 does not repress the expression of myogenin (bottom panel). Levels of HEB, VP16-63bHLH, and HA-tagged MEK1 proteins are similar Molecular Cell 2001 8, 291-301DOI: (10.1016/S1097-2765(01)00302-1)

Figure 6 Coimmunoprecipitation of MEK1 and MyoD Requires the N-Terminal Transactivation Domain of MyoD (A) An immunoblot of 10T1/2 cells transfected with MyoD, HA-tagged activated MEK1, or both. The levels of input MyoD, HEB, and activated MEK1 were similar. (B) Coimmunoprecipitation of 10T1/2 cells transfected with MyoD, HA-tagged activated MEK1, or both. HA-tagged activated MEK1 was readily detected by Western blot analysis after immunoprecipitation of MyoD or HEB only when MyoD protein is present. (C) Immunoblot analysis of 10T1/2 fibroblasts transfected with the deletion mutants of MyoD and activated MEK1. Deletion mutants of MyoD, HEB, and HA-tagged activated MEK1 were expressed at similar levels. (D) Coimmunoprecipitation of MyoD and activated MEK1. Note that coimmunoprecipitation of activated MEK1 with MyoD/HEB dimers required the N-terminal transactivation domain of MyoD (compare lane 3 with lanes 2 and 4–7 in both MyoD and HEB IPs). DM denotes MyoDΔ63–99/Δ216–269. Lane 8 represents 10% of the MyoD and activated MEK1 extract used for immunoprecipitations. (E) GST-MyoD1–95, but not GST-MyoD174–318, is sufficient to bind activated HA-tagged MEK1. Whole-cell extracts from MyoD/activated MEK1-transfected cells were loaded onto glutathione beads bound with GST-MyoD fusion proteins. Western analysis with anti-HA antibody revealed that only the N-terminal transactivation domain of MyoD bound activated MEK1 (compare lanes 1 and 3). Lanes 2 and 4 represent 10% of the cell extract used for the interaction studies Molecular Cell 2001 8, 291-301DOI: (10.1016/S1097-2765(01)00302-1)

Figure 7 Binding of Endogenous MEK1 and MyoD Proteins (A) Coimmunoprecipitation of C2C12 cells in growth medium transfected with HA-tagged wild-type MEK1 after a 30-min stimulation with 200 ng/ml TPA. HA-tagged activated MEK1 (upper band) and endogenous MEK1 (lower band) were readily detected by Western blot after immunoprecipitation of endogenous MyoD. TPA treatment resulted in a significant increase in the amount of transfected wild-type MEK1 and endogenous MEK1 associated with endogenous MyoD. Similarly, immunoprecipitation using α-phospho-MEK1/2 and blotting for MEK1 demonstrated that both transfected and endogenous MEK1 forms are activated by TPA stimulation. The Western blots shown in the right hand panels confirm the identity of the upper and lower bands as HA-MEK1 and endogenous MEK1, respectively. (B) Coimmunoprecipitation of C2C12 cells in growth medium 30 min after stimulation with 200 ng/ml TPA. Western blotting revealed that TPA stimulation led to an increase in MEK1 activation, as detected by the phospho-MEK1/2 antibody, without changes in MEK1 or MyoD levels. Immunoprecipitation of endogenous MyoD resulted in a significant increase in the amount of endogenous MEK1 associated with endogenous MyoD after TPA treatment Molecular Cell 2001 8, 291-301DOI: (10.1016/S1097-2765(01)00302-1)