PMC

Coimmunoprecipitation of SRF with MKL1 mutants. HA-tagged SRF and the indicated Flag-tagged MKL1 constructs were transfected into HeLa cells and immunoprecipitated (IP) with anti-Flag antibodies as described for Fig. . Two separate sets of MKL1 mutant proteins that were examined for their ability to associate with SRF in independent experiments are shown in panels A and B. IB, immunoblot.

Inhibition of RhoA induction of SRE reporter genes by dominant negative MKL1. HeLa cells were transfected with or without DN-MKL1 C630 and with an activated form of RhoA (RhoA-Val14) (A and B) or the combination of activated Raf (RafBXB) and wild-type ERK2 (1 μg each) (C) with the indicated reporter genes as described for Fig. . (D) GAL4-ELK1 and the 5×GAL4 site reporter were transfected with activated Raf, ERK2, and DN-MKL1 as indicated.

RNA interference of MKL1 and MKL2 inhibits serum and RhoA activation. (A) HeLa cells were transfected with control or MKL1-specific pSUPER plasmids or GFP- or MKL2-specific double-stranded RNA oligonucleotides. The cells were metabolically labeled with [³⁵S]methionine and immunoprecipitated with anti-MKL1 or MKL2 serum (A, top and bottom, respectively). (B) HeLa cells were transfected with a c-fos enhancer reporter gene (pm18GL3) with control (pSUPER and GFP-siRNA), pSUPER-MKL1 (MKL1-RNAi), MKL2 siRNA, or pSUPER-MKL1 and MKL2-RNAi. The cells were serum starved and induced with serum, as indicated, and luciferase activities were measured as for Fig. . (C) As in B, except that the indicated samples were cotransfected with a RhoAV14 expression vector as in Fig. .

RBM15-MKL1 fusion protein has a markedly increased ability to activate SRE reporter genes. (A) HeLa cells were transfected with the indicated reporter genes (0.5 μg), together with pRL-SV40P (50 ng) as an internal control, and vector pcDNA3, 1×Flag-RBM15, 1×Flag-MKL1 or 1×Flag-RBM15-MKL1 (1 μg). Cell lysates were assayed for firefly luciferase activity and normalized for Renilla luciferase activity. The averages of three determinations ± the standard deviation are shown. (B and C) HeLa cells were transfected with the c-fos promoter-luciferase gene with or without increasing amounts of 1×Flag-MKL1 or 1×Flag-RBM15-MKL1 expression plasmids, as indicated. Cell lysates were assayed for luciferase activity (B) and for protein expression by immunoblotting with anti-Flag antibodies (C).

Domains of MKL1 required for SRE promoter activation. (A) Diagram of MKL1 mutants. All MKL1 mutants contain a 3×Flag epitope at the amino terminus. MHD, MKL homology domain; B, basic domain; Q, glutamine-rich domain. (B) The indicated MKL1 constructs were tested for SRF activation with the 1×SRE luciferase reporter as in Fig. . (C) HeLa cells were transiently transfected with expression vectors (0.2 μg) encoding the indicated regions of MKL1 fused to the GAL4 DNA binding domain (amino acids 1 to 147) and the 5×GAL4-E1b-luciferase reporter (0.5 μg), which contains binding sites for the GAL4 DNA binding domain. Luciferase activity is expressed as the increase above that observed with the GAL4 DNA binding domain alone and is the mean ± standard deviation of two independent experiments.

Dominant negative MKL1 inhibits serum and LPA induction of the SRE. (A) Expression plasmids for MKL1 and MKL1 C-terminal deletion mutants (1 μg each) were transfected with the cardiac α-actin promoter luciferase reporter plasmid and pRL-SV40P. (B and C) HeLa cells were transfected with the c-fos enhancer reporter gene or the c-fos enhancer with a mutated TCF site (pm18) in the presence or absence of the MKL1 dominant negative mutant C630 (1 μg). The transfected cells were serum starved and induced with serum (B) or LPA (C) for 3 h. (D) Cells were transfected with a c-fos enhancer reporter gene that includes the c-fos AP1 site (FAP) in addition to the SRE and TCF sites. All three sites are required for TPA induction (). Cells were transfected with or without the DN-MKL1 C630 mutant and stimulated with TPA. (E) Cells were transfected with GAL4-ELK1 and the 5×GAL4 site reporter gene with or without DN-MKL1. Cell lysates were assayed for luciferase activity and normalized for Renilla luciferase activity as described for Fig. .

Inhibition of endogenous SRF target genes by dominant negative MKL1. (A) HeLa cells were transfected with vector pcDNA3 or MKL1 dominant negative mutant C630 (4 μg) under conditions yielding greater than 90% transfected cells as confirmed by cotransfection with 0.5 μg of the GFP expression vector pEGFP-N1 (data not shown). Cells were serum starved for 24 h before stimulation with 20% newborn calf serum for the times indicated. Total RNA was prepared, and mRNAs for c-fos, c-jun, and cyclophilin detected by RNase protection assays. (B) The c-fos RNase protection bands in A were quantitated with a Phosphorimager and normalized to the cyclophilin expression levels. (C to G) Cell lines were generated with an empty retroviral vector (pBabe-puro) or the vector driving expression of C630 in NIH 3T3 mouse fibroblasts. The cells were serum starved and treated with 20% serum for the indicated times, and c-fos (C) or SRF and vinculin (E) and control acidic ribosomal phosphoprotein-P0 (ARPP-P0) mRNAs were detected by RNase protection. The signals were quantitated with a Phosphorimager, with c-fos (D), SRF (F), and vinculin (G) expression levels normalized to that of ARRP-P0. The quantitated results are the averages of three determinations ± standard deviation.

Transcriptional activation of SRE-dependent promoters by MKL1 and binding of SRF to MKL1. (A) SRE-dependent or -independent luciferase reporter genes (0.5 μg), together with pRL-SV40P (50 ng) as an internal control, were transiently transfected into HeLa cells with vector (pcDNA3) or an MKL1 expression plasmid, p3×Flag-MKL1 (1 μg). Two days after transfection, luciferase assays were performed and normalized to the Renilla luciferase control. The values are expressed as the increase with MKL1 compared to the vector. Data are represented as the mean ± standard deviation of three independent experiments. α-SA, smooth muscle α-actin; α-CA, cardiac α-actin; ANF, atrial natriuretic factor. (B) Binding of MKL1 to SRF in gel mobility shift assays. Gel mobility shift assays were performed with ³²P-labeled oligonucleotide probe for a high-affinity SRF binding site (XGL) with bacterially expressed SRF(114-508) and/or in vitro translation products of MKL1 (5 and 8 μl in lanes 2 and 3, respectively) or control translation lysates (5 μl) as indicated. (C) Coimmunoprecipitation of MKL1 and SRF. HA-tagged SRF and 3×Flag-tagged MKL1 (2 μg each) were transfected into HeLa cells alone (with 2 μg of control plasmid pEGFP-N1) or together as indicated. Immunoprecipitates (IP) with anti-Flag antibodies were immunoblotted (IB) for SRF with anti-HA antibodies (top) and reprobed with anti-Flag antibodies to determine the presence of Flag-tagged MKL1 (bottom). One-thirtieth of the cell lysates was directly immunoblotted with anti-HA antibodies to detect HA-tagged SRF (middle). The positions of HA-SRF, Flag-MKL1, and the immunoglobulin heavy chain (Ig) are indicated. (D) Coimmunoprecipitation of endogenous SRF and MKL1. HeLa cell lysates were immunoprecipitated with nonspecific rabbit serum (NS) or rabbit antiserum to SRF or MKL1 and immunoblotted with anti-MKL1 serum. For lane 3, 1/10 as much lysate was used. MKL1 migrated at 160 kDa relative to markers.