Phosphorylation of MKL1 at serine 454. (A and B) Serum-starved TO3T3 cells transiently transfected with MKL1 and the indicated mutants in the p3x-FLAG-CMV-7.1 vector (2 μg/35-mm dish) were treated with serum for 30 min and immunoblotted with antisera that recognize phospho-S454 or Flag-MKL1. (C) Serum-starved TO3T3 cells were incubated with serum for the times indicated and detected with anti-phospho-S454 and anti-MKL1 antibodies.

Identification of MKL1 phosphorylation sites. (A) Conserved sequence motifs found between aa 444 and 500 in the MKL/myocardin protein family. (B to E) HeLa cells expressing MKL1 and the indicated mutants were stimulated with serum for 30 min, lysed, and immunoblotted with anti-Flag antibody. The STS/A and STS/E mutants have changes of S449/T450/S454 to alanines and glutamates, respectively.

TPA treatment inhibits serum-induced MKL1 nuclear localization. (A) HeLa cells were transfected with Flag-tagged MKL1, serum-starved, and treated with (+) or without (−) TPA for 30 min, followed by serum induction for 30 min as indicated. The cellular localization of MKL1 was determined by immunofluorescence with anti-Flag antibodies as shown in Fig. 4. (B) As described in the panel A legend above, except that the STS/E phosphomimetic MKL1 mutant was used. C, cytoplasmic; N, nuclear; C/N, both cytoplasmic and nuclear.

MKL1 is subject to serum-inducible phosphorylation at aa 444 to 500. (A) Quiescent HeLa cells transfected with Flag-tagged MKL1 were stimulated as indicated with serum prior to lysis and immunoblotted with anti-Flag antibodies. (B) Lysates from quiescent and serum-stimulated HeLa cells expressing Flag-MKL1 were treated with (+) or without (−) calf intestinal alkaline phosphatase and immunoblotted as above. MKL1 electrophoretic mobility was examined as described in the legend to panel A. (C) The MKL1 derivatives used for mapping of phosphorylation sites are shown. Conserved domains, RPEL domain; B, basic domain; Q, glutamine-rich domain; SAP domain; LZ, leucine zipper-like domain. (D) HeLa cells expressing Flag-MKL1 or its derivatives were left unstimulated or stimulated for 30 min with serum and analyzed by immunoblotting.

The MKL1 phosphorylation site mutant is constitutively localized to the nucleus. (A) HeLa cells were transfected with Flag-MKL1 or the phosphorylation site mutant (STS/A), serum starved, and treated with (+) or without (−) serum for 30 min. The cells were analyzed by immunofluorescence with anti-Flag antibodies. (B) Subcellular localization shown in panel A was scored as predominantly cytoplasmic (C), nuclear (N), or both (C/N). Statistical analyses were carried out for three independent experiments, with 100 to 200 cells per condition; error bars indicate standard errors of the means. (C) Quantitation of immunofluorescence in HeLa cells transiently transfected with MKL1 and the indicated mutants as described in the legend to panel A. (D) Mutants in putative nuclear export sequences were analyzed for cellular localization as described in the legend to panel B.

The phosphorylation site mutant has a weaker affinity for actin. (A) HeLa cells were cotransfected with Flag-tagged MKL1 or the indicated MKL1 mutants together with GFP-actin expression vector (2 μg each). Immunoprecipitates with anti-Flag antibody were immunoblotted with anti-GFP or anti-Flag antibodies. A portion of the cell lysate (1/30) was also directly immunoblotted with anti-GFP antibodies (bottom panel). (B) HeLa cells were transfected with GFP-actin and Flag-MKL1 (lanes 1 and 2) or neither (lane 3), serum-starved, and treated with TPA (100 ng/ml) for 30 min (lane 2). Immunoprecipitates with anti-GFP antibodies were immunoblotted with anti-Flag or anti-GFP antibodies. A portion of the cell lysate (1/30) was also directly immunoblotted with anti-Flag antibodies (bottom panel). (C) The model for serum regulation of MKL1: serum induction results in the activation of the RhoA- and Ras/MEK/ERK pathways. RhoA activation stimulates MKL1 nuclear localization due to the formation of actin stress fibers and a decrease in G-actin levels, while nuclear export is stimulated due to ERK1/2 phosphorylation of MKL1 and increased G-actin binding. Export of actin-bound MKL1 reduces the amount of MKL1 available to bind to SRF and activate transcription (see Discussion for details).

MKL1 phosphorylation is regulated by the ERK1/2 MAPK pathway. (A) HeLa cells were transfected with Flag-MKL1, serum-starved, preincubated with or without the MEK1 inhibitor UO126 (10 μM) for 30 min, and then stimulated with serum for 30 min. Lysates were immunoblotted with anti-Flag antibodies. (B) For immunofluorescence analysis, HeLa cells were transfected with Flag-MKL1, serum starved, and then serum stimulated for the indicated times and quantitated for cellular localization as shown in Fig. 4. (C) As in panel B, except that cells were pretreated for 30 min with 10 μM U0126. Localization of FLAG-MKL1 was scored in 100 to 200 cells; N, nuclear; C, cytoplasmic; C/N, both nuclear and cytoplasmic. Three independent experiments were performed. Error bars, standard errors of the means. (D) Left panel, quiescent HeLa cells transfected with wt MKL1 or the phosphorylation site mutant (STS/A) were incubated with (+) or without (−) TPA (100 ng/ml) for 30 min and immunoblotted as described in the legend to panel A. (D) Right panel, untransfected (lane 1) or wt MKL1-transfected (lanes 2 and 3) cells were treated with or without TPA and immunoblotted using the anti-phospho-S454 MKL or anti-Flag antibodies as indicated. (E) Time course of MKL1 localization in HeLa cells preincubated with TPA for 30 min and stimulated with serum was determined by immunostaining as described in the legends to panels B and C. (F) HeLa cells were transfected with the STS/E MKL1 mutant, where the phosphorylation sites were changed to glutamates and analyzed for cellular localization, as described above.

The nonphosphorylatable mutant is constitutively active. (A) The indicated MKL1 expression vectors in pRevTRE (5 ng) or vector alone (Vec) were transiently transfected into TO3T3 cells with an SRE-dependent luciferase reporter gene (p5xSRE; 100 ng), together with a Renilla luciferase internal control (pRL-SV40P; 50 ng). Two days after cells were transfected, and 3 days after doxycycline was removed, the cells were treated with or without serum (20%) for 3 h. Luciferase assays were performed for firefly luciferase and normalized to the Renilla luciferase control. Data are represented as standard errors of the means of three independent experiments, each with triplicates. STS/A, phosphorylation site mutant; N100, deletion of aa 1 to 100; N100-STS/A, double mutant. (B) TO3T3 cells stably expressing the indicated mutants were transfected with the p5xSRE and pRL-SV40P reporters as described in the legend to panel A. Inset, immunoblot with anti-Flag antibodies of the indicated stably transfected TO3T3 cells. (C) RNA was isolated from the stably expressing MKL1 variant cells shown in panel B that were serum starved and treated with (+) or without (−) serum for 2 h. The abundance of vinculin mRNA was analyzed by quantitative RT-PCR, normalized to 18S rRNA levels, and represented as the means ± standard deviations of three independent experiments. (D) TO3T3 cells were serum starved and treated with TPA or serum, as indicated, or with TPA for 30 min, followed by serum for the indicated times. RNA was then isolated and analyzed for vinculin expression as described above.