- Poster presentation
- Open Access
Differential phosphorylation of LZ+/LZ- MYPT1 isoforms by PKGIα: implication for vascular reactivity
© Yuen et al; licensee BioMed Central Ltd. 2011
- Published: 1 August 2011
- Smooth Muscle
- Alternative Splice
- Unknown Function
- Muscle Relaxation
- Phosphorylation Site
MLC phosphatase is a trimeric enzyme composed of a catalytic subunit, a 20-kDa subunit of unknown function, and a myosin targeting subunit (MYPT1). During NO stimulation, PKGIα mediated phosphorylation of MYPT1 increases MLC phosphatase activity, which produces a decrease in force. Further, alternative splicing of a 3’ exon produces two MYPT1 isoforms, which differ by the presence or absence of a leucine zipper (LZ); a LZ+ MYPT1 isoform is required for PKGIα induced smooth muscle relaxation.
To examine the influence of MYPT1 structure on the ability of PKGIα to phosphorylate the protein, we used two MYPT1 fragments, which differed only by the presence (MYPT1LZ+) or absence (MYPT1LZ-) of the LZ. Purified PKGIα phosphorylated MYPT1LZ+, but not MYPT1LZ-. Following phosphorylation, MYPT1LZ+ predominantly existed as a di-phosphorylated protein, and mass spectrometry identified S668 and S695 as PKGIα-mediated phosphorylation sites. To examine the relative rates of S668 vs S695 MYPT1 phosphorylation, these residues were mutated to either A or D. The rates of D668 and A668 MYPT1 phosphorylation were similar and slow. The D695 MYPT1 mutant had the highest rate of phosphorylation, while the rate of phosphorylation of the A695 MYPT1 mutant was intermediate between that for the D695 MYPT1 and either A668 or D668 MYPT1.
These results suggest that PKGIα-mediated phosphorylation of S695 is slower than S668, and could suggest that PKGIα-mediated phosphorylation of S668 is physiologically significant for the regulation of MLC phosphatase activity. Further, MYPT1 structure has an important role in the regulation of vascular tone, and differential tissue expression of LZ+/LZ- MYPT1 isoforms contributes to the diversity in the sensitivity of smooth muscle to NO mediated vasodilatation.
This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.