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Skeletal muscle CaMKII enriches in nuclei and phosphorylates myogenic factor SRF at multiple sites
M Flück1, F W Booth, M N Waxham
1Department of Integrative Biology and Pharmacology, University of Texas Medical School, Houston, Texas 77030, USA. flueck@mem.unibe.ch
Abstract:
We characterized the activity of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) in homogenates and nuclear extracts of skeletal muscle and analyzed their capacity to phosphorylate the myogenic factor SRF. Isoforms of CaMKII enriched from skeletal muscle phosphorylated SRF in vitro to high stoichiometries and produced multiple forms on SDS-PAGE, suggesting that SRF was phosphorylated at multiple sites. Phosphopeptide-mapping experiments using truncated SRF proteins located the residues of SRF phosphorylated by recombinant CaMKII within amino acids 1-171, with at least one site residing in amino acids 142-171. Microsequencing of these phosphorylated peptides identified that both Ser-103 and a novel residue, Thr-160 in the MADS box of SRF, were sites of phosphorylation. CaMKII activity was enriched in nuclear extracts relative to crude homogenates from skeletal muscle and similarly phosphorylated the nuclear transcription factor SRF in vitro. The location of Thr-160 in the 3-D structure of SRF suggests that its phosphorylation by nuclear CaMKII may directly influence DNA binding of SRF and other MADS box factors.
Insights
Calcium/calmodulin-dependent protein kinase II (CaMKII) phosphorylates the myogenic factor SRF at multiple sites, including novel Thr-160 in its MADS box. This phosphorylation by nuclear CaMKII may regulate SRF
Area of Science:
- Molecular biology
- Cell signaling
- Muscle physiology
Background:
- Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) plays crucial roles in cellular signaling.
- SRF is a key myogenic factor regulating muscle gene expression.
- Understanding CaMKII's role in skeletal muscle is vital for muscle development and function.
Purpose of the Study:
- To characterize CaMKII activity in skeletal muscle.
- To investigate the phosphorylation of SRF by CaMKII.
- To identify specific phosphorylation sites on SRF.
Main Methods:
- Skeletal muscle homogenates and nuclear extracts were used.
- In vitro phosphorylation assays with recombinant CaMKII and SRF.
- SDS-PAGE and phosphopeptide mapping.
- Microsequencing of phosphorylated peptides.
Main Results:
- CaMKII isoforms from skeletal muscle efficiently phosphorylated SRF in vitro.
- Multiple phosphorylation sites were identified on SRF within amino acids 1-171.
- Ser-103 and a novel site, Thr-160 in the MADS box, were identified as phosphorylation sites.
- CaMKII activity was enriched in nuclear extracts, phosphorylating nuclear SRF.
Conclusions:
- CaMKII phosphorylates SRF at multiple sites, including Thr-160 in the MADS box.
- Nuclear CaMKII activity targets SRF for phosphorylation.
- Phosphorylation of Thr-160 may directly impact SRF DNA binding and function.