Yuen, Samantha L.

The participation of nonmuscle myosin in force maintenance is controversial. To participate in force maintenance, nonmuscle myosin activity must be regulated in smooth muscle. To investigate this question, phosphorylation of the regulatory light chains of smooth muscle myosin (SMLC) and nonmuscle myosin (NMLC) were examined by two dimensional SDS‐PAGE. Intact mouse aortic smooth muscle strips were stimulated with either KCl depolarization or angiotensin II, and 4 distinct LC spots were resolved by two dimensional SDS‐PAGE. Using mass spectroscopy, these spots were identified as four distinct proteins; phosphorylated NMLC, non‐phosphorylated NMLC, phosphorylated SMLC and non‐phosphorylated SMLC. During KCl depolarization, inhibition of Rho kinase or PKC did not affect the increases in SMLC and NMLC phosphorylation. During angiotensin II stimulation, inhibition of PKC or Rho kinase reduced SMLC phosphorylation, while only inhibition of Rho kinase reduced NMLC phosphorylation. These data suggest that in smooth muscle, nonmuscle myosin is regulated by Ca 2+ ‐calmodulin activated MLCK during depolarization, and a Rho kinase mediated pathway during agonist stimulation.

During nitric oxide (NO) signaling, cGMP activates type Iα protein kinase G (PKGIα), which subsequently activates myosin light chain phosphatase (MLCP). The increase in MLCP activity decreases myosin phosphorylation which leads to smooth muscle relaxation. MLCP is a trimeric enzyme consisting of a myosin targeting subunit (MYPT1), a catalytic subunit and a subunit of unknown function. MYPT1 has two isoforms; alterative mRNA splicing of a 3’ exon results in the presence or absence of a C‐terminal leucine zipper (LZ) domain. Further, the MYPT1 LZ+ is required for PKGIα mediated activation of MLCP. Moreover, recent studies have demonstrated PKGIα phosphorylates MYPT1. This study examined the affect of MYPT1 structure for PKGIα phosphorylation. Two MYPT1 fragments differing only by the LZ domain (aa 1010–1039) were used: MYPT1FL (aa 500–1039, LZ+) and MYPT1TR2 (aa 500–1010, LZ−). Both the rate (0.0216±0.005 min −1 vs 0.0596±0.005 min −1 ) and extent of PKGIα mediated phosphorylation of MYPT1TR2 were lower when compared to MYPT1FL. In addition, two‐dimensional SDS‐PAGE demonstrated that following PKGIα phosphorylation, MYPT1TR2 existed primarily as the non‐phosphorylated protein whereas MYPT1FL was diphosphorylated. These results demonstrate that MYPT1 structure is important for determining the sensitivity of smooth muscle to NO mediated vasodilatation.