Novel Phosphorylation Target in the Serum Response Factor MADS Box Regulates α-Actin Transcription

Abstract

Serum response factor (SRF) is a phosphoprotein that regulates skeletal and cardiac alpha-actin gene transcription. Myotonic dystrophy protein kinase (DMPK), a muscle- and neuron-restricted kinase, enhanced SRF-mediated promoter activity of the skeletal and cardiac alpha-actin genes in C2C12 myoblasts as well as in nonmyogenic cells. DMPK phosphorylated SRF in vitro in the alphaI coil of the DNA-binding domain in the MADS box, a highly conserved region required for DNA binding, dimerization, and co-activator interaction in COS and CV1 cells. Threonine 159 in the MADS box alphaI coil was a specific phosphorylation target in vitro as well as in vivo of both DMPK and protein kinase C-alpha. Substitution of threonine 159 with the nonphosphorylatable residue alanine markedly diminished activation of the cardiac alpha-actin promoter in the presence of kinase, while its substitution with aspartic acid, to introduce a negative charge and mimic phosphorylation, restored activation completely. Phosphorylation of the MADS box may constitute a novel mechanism for regulation of SRF-dependent actin gene transcription.

Serum response factor MADS box serine -162 phosphorylation switches proliferation and myogenic gene programs

“…Previous experiments (24) and data from x-ray crystallographic analysis of SRF's MADS box bound to its cognate serum response element (SRE) (ref. 25 and Fig.…”

“…36). SRF MADS box mutants were made by PCR mutagenesis and cloned into the pCGN vector as described (24). Construction of a GST mouse myocardin fusion protein in pGEX-4T-1 was as described (14).…”

“…MEK-1 and Erk-1 in the vector pMT3-HA were from Zhijun Luo (Boston University Medical Center, Boston). GST-SRF (amino acid residues 1-171) in pGEX 4T3 was as described (24).…”

“…Synthesis of anti-SRF peptide antibody, anti-SRF threonine 159 phospho-specific antibody, and anti-SRF serine-162 phospho-specific antibody have been described (24). Briefly, the antibodies were raised in rabbits against the following peptides representing amino acids 152-169 of SRF, DNKLR-RYTTFSKRKTGIM, DNKLRRYT(-PO 4 )TFSKRKTGIM, and DNKLRRYTTFS(-PO 4 )KRK TGIM (Bethyl Laboratories, Montgomery, TX), and affinity-purified before use.…”

SRF is a nuclear repressor of Smad3-mediated TGF-β signaling

“…To confirm the activation of SRF by the addition of 20% serum after 24 h serum starvation, we examined the phosphorylation of SRF at Ser-103. SRF contains a number of phosphorylation sites , and it is a target of several kinase pathways (Manak and Prywes, 1991;Janknecht et al, 1992;Liu et al, 1993;Rivera et al, 1993;Iyer et al, 2003Iyer et al, , 2006. The physiological importance of these modifications remains unsettled.…”