Mirk phosphorylates transfected Flag-p21 in vivo. (Top panel) Autoradiogram after SDS-PAGE of in vivo phosphorylation of wild-type Flag-p21 cotransfected into NIH3T3 cells with either wild-type or kinase inactive Mirk, and allowed to co-express for 24 hours. After 4 hours of labeling, Flag-p21 was immunoprecipitated with anti-Flag M2 antibody, and the immunoprecipitates were resolved on SDS-PAGE with Flag-p21 migrating at the expected position. Flag-p21 was phosphorylated only in the co-transfections with wild-type Mirk. The nonspecific doublet seen at the top of each lane serves as the loading control. (Bottom panel) Anti-Flag western blot showed that equal amounts of Flag-p21 were immunoprecipitated. The upper band represents light chain, and the lower Flag-p21. There appears to be a slight decrease in mobility in the p21 phosphorylated by Mirk/dyrk1B.

Fluorescence microscopy demonstrated that the Mirk-phosphorylation site phosphomimetic mutant p21-S153D is found in both the cytoplasm and the nucleus, while wild-type p21 and the non-phosphorylatable p21-S153A are localized exclusively in the nucleus in the majority of cells. GFP-p21 wild-type, GFP-p21-S153A which is not phosphorylated by Mirk, and GFP-p21-S153D, the Mirk site-phosphomimetic construct, were transiently expressed in C2C12 cells (panel A, 400X magnification) and in NIH3T3 cells (panel B, 1000X magnification). Nuclei were stained with DAPI and identical fields were photographed for GFP and DAPI. Images were merged with SPOT RT software. Scale bars = 50 um.

When Mirk is upregulated as a part of the myoblast differentiation program, both wild-type p21, as well as the Mirk-phosphorylation site phosphomimetic mutant p21-S153D, are translocated to the cytoplasm as shown by fluorescence microscopy. In contrast, the non-phosphorylatable p21-S153A is localized exclusively in the nuclei of the multinucleated myotubes. GFP-p21 wild-type, GFP-p21-S153A, and GFP-p21-S153D were transfected into C2C12 cells, which were then allowed to differentiate for 3 days. Similar data was seen in each of 4 experiments.

p21 localizes in both the cytoplasm and nucleus of adult skeletal muscle. Frozen sections of human muscle were analyzed for the localization of p21 by immunofluorescence using the polyclonal C-19 antibody (1:200) and goat anti-rabbit IgG Alexa Fluor 594 (1:500); nuclei were counterstained with DAPI. Scale bars = 50 um.

Transient expression of a p21 construct, phosphomimetic at the Mirk phosphorylation site, increases the viability of C2C12 cells. C2C12 myoblasts were transfected with Flag-p21 wild-type, Flag-p21-S153A, Flag-p21-S153D, or vector for 4 hours, allowed to express for 24 hours (0 point) then swtiched to serum-containing growth medium for 0–13 days. A. Each construct was expressed for only about 1 day in growth medium following the transfection and initial 24 hour expression period, as determined by western blotting for the Flag epitope. The zero time points for each construct were examined in parallel to the zero time points for the other two constructs on each blot as internal controls. Ct, cross-reacting protein used as a loading control. B. After 24 hours, the cells were trypsinized and plated at either 500 or 1000 cells in 100 mm tissue culture dishes, each in triplicate. Data shown is the mean of two such experiments, normalized to the number of colonies per 1000 cells plated.

Mirk mediates myoblast survival. A. Mirk is expressed in cycling C2C12 myoblasts. Fluorescence microscopy demonstrates that Mirk expression is greatest in C2C12 cells in mitosis and in cells in early G1. Affinity-purified anti-peptide antibody to the Mirk C-terminus was conjugated to Alexa Fluor 594 and used to detect endogenous Mirk in C2C12 cells in growth medium. Nuclei were stained with DAPI, and identical fields were merged with SPOT RT software. Similar results were obtained with antibody directed to Mirk’s unique N-terminus. B. (top) Ectopic Mirk maintains the survival of differentiating C2C12 cells. Cells were transiently transfected with wild-type Mirk or kinase-inactive mutant YF-Mirk, allowed to express for 18 hours, then transferred to differentiation medium for up to 48 hours. Lysates were analyzed for expression of Mirk and ß-tubulin by western blotting. (bottom) To determine whether the amount of Mirk remaining in cells expressing ectopic YF-Mirk after 48 hours was either ectopic or endogenous Mirk, cells were transiently transfected with kinase-inactive mutant YF-Mirk, or vector (Vc), allowed to express for 18 hours, then transferred to differentiation medium for up to 48 hours. Lysates were analyzed for expression of Mirk and ß-tubulin by western blotting. The YF-Mirk and vector blots were exposed for the same amount of time to allow direct comparison. One of duplicate experiments with similar results is shown. C. Depletion of Mirk in cycling myoblasts by RNA interference reduced C2C12 cell viability in colony formation assays. Cells were plated at 5x10⁵ per 60 mm dish, 3 dishes per point for 24 hours, then were transfected for 4 hours in serum-free medium with 5 ug pSilencer DNA encoding either RNAi to Mirk (RNAi), a mutant RNAi (Mt) or vector (Vc) together with 0.5 ug of neomycin resistance plasmid, using 10 ul each of Lipofectamine and Plus reagent. Fetal bovine serum was then added to 10% to maintain cell viability during expression. Cells were incubated for 20 hours before trypsinization, then lysed for western blotting. Ct, control, cross-reacting band showing similar loading. D. Same experiment as in panel C, but transfected cells were reseeded at the same cell density and selected in 400 ug/ml G418 for 3 weeks. Mean colony number +/SE is shown. E. Mirk knockdown increases apoptosis measured by fluorescence microscopy using an activated caspase 3 inhibitor. C2C12 myoblasts were plated overnight in LabTek 2-well chamber slides (2 x 10⁵ cells per well) and then co-transfected with 0.5 ug pDsRed and 1.5 ug of pSilencer vector (Vec), Mutant si or RNAi to Mirk (4 ul PLUS, and 4 ul LipofectAMINE per well). Cells were transfected in serum free media for 4 hours, then an equal volume of 20% FBS/DMEM was added. Following 24 hours of expression, cells were incubated with differentiation media for 24 hours. Activated caspase was then labeled by a 30 minute incubation with 10 uM of CaspACE FITC-VAD-FMK in situ marker (Promega) diluted in DM. This is a fluorescent conjugate of the permeable caspase inhibitor VAD-FMK that was used as an in situ marker for apoptosis. Slides were washed and mounted with Biomedia GelMount. At least 200 DsRed expressing cells were observed in each of 4 separate preparations and the number of cells labeled for both DsRed and the FITC-VAD-FMK marker was determined using a Green/Orange V2 filter set (Chroma) that allowed simultaneous visualization of both fluorophores. Combined counts were analyzed by the Chi square test to determine the significance of differences between the RNAi constructs. Efficiency of cotransfection was determined to be greater than 85% in parallel experiments using a combination of GFP and DsRed. The number of cells scored per assay conditions is shown. F. Knockdown of endogenous Mirk by RNA interference inhibits the activation of caspase 3 in differentiating C2C12 cells. C2C12 cells (5 x10⁵ per 60 mm dish) were transfected with pSilencer plasmid encoding RNAi to Mirk (RNAi) or mutant RNAi (Mt) for 24 hours, then switched to differentiation medium for 1, 2 and 3 days, as noted. The relative abundance of Mirk/tubulin and of activated caspase 3/tubulin in lysates were determined by western blotting on the upper and lower sections of same blot which was cut in half and then reunited for the exposure, with tubulin used as the loading control. The relative abundance of either protein in cells treated with RNAi to Mirk were then normalized to their relative abundance in cells treated with mutant RNAi. Mean +/− SE (if greater than 5%) is shown from data from 3 experiments. G. Apoptosis induced by differentiation medium or Mirk knockdown blocked to a similar extent by a caspase inhibitor. C2C12 myoblasts were plated, transfected and cultured as in panel E, except they were co-transfected with 0.5 ug pEGFP. A parallel set of cultures was incubated with DM containing 20 uM of the cell-permeable caspase inhibitor z-VAD.fmk (Promega). After 24 hours in DM, DNA breaks in apoptotic cells were labeled with tetramethyl-rhodamine-dUTP by TdT-mediated in situ end labeling (TUNEL) using the Roche In Situ Cell Death Detection Kit. At least 300 GFP expressing cells were observed in each of 4 separate preparations, so that an average of 1250 cells were scored per point. The number of GFP expressing cells labeled with the TUNEL marker was determined using a Green/Orange V2 filter set (Chroma) that allowed simultaneous visualization of both fluorophores. Combined counts were analyzed by the Chi square test. Efficiency of cotransfection was determined to be greater than 85% in parallel experiments using a combination of GFP and DsRed.

Mirk phosphorylates p21 at S153 in vitro and in differentiating myoblasts. A. Mirk phosphorylates p21 at S153 in vitro as shown by peptide mapping with trypsin. Wild-type (Wt) and mutant p21-S153A constructs were phosphorylated in vitro by purified recombinant Mirk, digested with trypsin and then subjected to two-dimensional peptide mapping. The peptide containing S153 and consisting of amino acids 143–154 is shown by a short arrow in both panels. The peptide identified by the longer arrow may be a partial digestion product containing S153, due to steric interference of the enzyme by the phosphate group. Similar data was seen in two additional peptide maps. B. Mirk phosphorylates p21 at S153 in vivo as shown by peptide mapping with chymotrypsin. Wild-type Flag-p21, mutant p21-S153A, and mutant p21-S130A were each cotransfected with wild-type Mirk into C2C12 cells. Constructs were allowed to express overnight, then cells were placed in myoblast differentiation medium for 24 hours, the last 4 hours of which cells were labeled with [³²P]-orthophosphate. In one dish transfected with wild-type p21, 10 uM SB203580 was added at the same time as the label. Flag-p21 was immunoprecipitated with anti-Flag M2 antibody, the immunoprecipitates were digested with chymotrypsin, and then subjected to two-dimensional peptide mapping. The p21-S153A mutant incorporated about 40% as much label as wild-type p21 when normalized to the total amount of p21 immunoprecipitated. Chymotrypsin was used because it yields a simpler map with a broader distribution of peptides. The peptide indicated by the arrow is lost in the p21-S153A mutant, and is the presumed 152-159 amino acid peptide. C. Depletion of endogenous Mirk by RNA interference blocks the phosphorylation of transfected wild-type p21, but not p21-S153A mutant at the Mirk phosphorylation site. Cells were plated at 5x10⁵ per 60 mm dish, cultured for 16 hours, then were transfected for 4 hours in serum-free medium with 2.5 ug of either Flag-p21 or Flag-p21-S153A, and 2.5 ug of pSilencer DNA encoding either RNAi to Mirk (RNAi) or vector. As controls, Flag-p21 was co-transfected with either wild-type Mirk (M) or kinase-inactive YF-Mirk (YF). Fetal bovine serum was then added to 10% to maintain cell viability during overnight expression, and cells were switched to OptiMEM differentiation medium for 24 hours, with 325 uCi ³²P-orthophosphate added to each dish for the last 4 hours following 1 hour in phosphate-free medium. Anti-Flag epitope immunoprecipitates were separated by SDS-PAGE and the Flag-p21 bands were detected by autoradiography (top lanes), then by western blotting for Flag. The amount of p21 and Mirk in the lysates were determined by western blotting (lower 2 bands). D. SB203580 inhibits myoblast differentiation as measured by the level of expression of the myogenic regulatory factor myogenin which controls the differentiation program. C2C12 cells were placed in differentiation medium +/− 10 uM SB203580 for 1 and 2 days and the abundance of myogenin determined by western blotting. Ct, cross-reacting protein used as a loading control. E. Relative stability of mutant p21 constructs. C2C12 cells were transfected with either mutant Flag-p21-S153D, Flag-p21-S153A or wild-type Flag-p21, and constructs were allowed to express for 24 hours. Cycloheximide (CH) at 20 ug/ml was added to each culture and Flag-p21 and tubulin abundance were determined at the indicated times by western blotting, and normalized to the 0 time values. Mean +/− SE shown of 2 separate experiments.

Overexpression of Mirk causes a portion of the wild-type p21 population to translocate to the cytoplasm in a Mirk kinase-dependent manner, but cannot translocate p21 mutated at the Mirk phosphorylation site. NIH3T3 fibroblasts were plated overnight in Labtek 2-well chamber slides (1.5 x 10⁵ cells per well) and then transfected (2 ug plasmid DNA and 4 ul LipofectAMINE 2000 per well) with GFP-p21(p21) or the non-phosphorylatable GFP-p21(p21-S153A), together with either wild-type (M) or kinase inactive (YF-M) Mirk. Cells were transfected in DMEM containing 10% bovine calf serum. After 24 hours of expression, cells were fixed with 4% paraformaldehyde and the GFP-p21 protein was labeled by a 30 minute incubation with mouse anti-GFP monoclonal antibody (Santa Cruz 8334; 1:500), followed by anti-mouse Alexa Fluor 488. Mirk was visualized using rabbit polyclonal antibody to the Mirk C-terminus at 1:500, followed by anti-rabbit Alexa Fluor 594. An average of 1050 GFP-p21 expressing cells were observed for each transfectant. Cells were scored in 8–10 random fields in each of 3 separate preparations and the number of cells labeled for both GFP-p21 and transfected Mirk was determined using a Green/Orange V2 filter set (Chroma) that allowed simultaneous visualization of both fluorophores. Combined counts were analyzed by the Chi square test (Minitab) to determine the significance of differences between conditions. The total number of cells scored per condition is labeled on each bar.

Endogenous p21 is exclusively nuclear in cycling C2C12 cells in growth medium (GM), but a portion of the endogenous p21 protein is translocated to the cytoplasm when C2C12 cells are treated for 48 hours with differentiation medium (DM). Endogenous p21 was visualized with the F-5 monoclonal antibody and goat anti-mouse antibody conjugated to Alexa Fluor 488, the nuclei were stained with DAPI, and actin within the entire cell body was visualized with phalloidin conjugated to Alexa Fluor 594. Identical fields were photographed for each fluorochrome (first three rows), then merged with SPOT RT software. Identical results were obtained with the C-19 polyclonal antibody. Scale bar = 50 um.

A p21 construct, phosphomimetic at the Mirk phosphorylation site, is twice as effective as wild-type p21 in blocking the activation of caspase 3. C2C12 myoblasts were transfected with Flag-p21 wild-type, Flag-p21-S153A, Flag-p21-S153D, or vector for 24 hours, cultured in differentiation medium for 16 hours, and then analyzed by SDS-PAGE and western blotting for activated caspase 3, normalized by western blotting for tubulin and the expression of exogenous Flag-p21 by western blotting for Flag. Mean +/− SD shown of 2 experiments.