Skeletal muscle CaMKII enriches in nuclei and phosphorylates myogenic factor SRF at multiple sites

We characterized the activity of Ca2+/calmodulin-dependent protein kinase I I (CaMKII) in homogenates and nuclear extracts of skeletal muscle and analy zed their capacity to phosphorylate the myogenic factor SRF. Isoforms of Ca MKII enriched from skeletal muscle phosphorylated SRF in vitro to high stoi chiometries and produced multiple forms on SDS-PAGE, suggesting that SRF wa s phosphorylated at multiple sites. Phosphopeptide-mapping experiments usin g truncated SRF proteins located the residues of SRF phosphorylated by reco mbinant CaMKII within amino acids 1-171, with at least one site residing in amino acids 142-171. Microsequencing of these phosphorylated peptides iden tified that both Ser-103 and a novel residue, Thr-160 in the MADS box of SR F, were sites of phosphorylation. CaMKII activity was enriched in nuclear e xtracts relative to crude homogenates from skeletal muscle and similarly ph osphorylated the nuclear transcription factor SRF in vitro. The location of Thr-160 in the 3-D structure of SRF suggests that its phosphorylation by n uclear CaMKII may directly influence DNA binding of SRF and other MADS box factors. (C) 2000 Academic Press.