The physiological regulation of vascular tone is critical to the maintenance of normal vascular function. Abnormalities of vascular tone are a hallmark of atherosclerotic cardiovascular diseases and hypertension. Vascular smooth muscle cells (VSMC) are the principal cellular component of blood vessels and their contractile state regulates vascular tone. VSMC contractile state is determined by the degree of myosin light chain (MLC) phosphorylation, the biochemical determinant of contraction. MLC phosphorylation state, in turn, is tightly regulated by the relative activities of the counter-regulatory enzymes myosin light chain kinase (MLCK) and myosin phosphatase (PP1M). PP1M is the critical phosphatase that mediates VSMC relaxation by dephosphorylation of MLCs. It is now understood that PP1M is regulated by both vasodilator pathways that activate PP1M, causing VSMC relaxation, and vasoconstrictor pathways that inhibit the phosphatase, causing VSMC contraction. The RhoA/Rho-kinase pathway, which is activated by vasoconstrictors, inhibits PP1M activity, leading to VSMC contraction. Recent studies have shown that RhoA/Rhokinase activity can influence the pathogenesis of vascular diseases, including hypertension, coronary artery spasm and neointimal formation. Despite the data supporting RhoA/Rho-kinase-mediated PP1M inhibition, no direct interaction between RhoA/Rho-kinase and PP1M has been reported. The mechanism by which RhoA/Rho-kinase localizes to the contractile apparatus and the cellular substrates and events that mediate Rho-dependent inhibition of PP1M are unknown. My long-term goal is to identify molecules in the myofibrillar contractile apparatus and to understand how their interactions regulate PP1M activity in response to vasodilator and vasoconstrictor signaling pathways. I have recently cloned and partially characterized an exciting new molecule that directly binds both RhoA and MBS, assembling a complex of all three proteins. This Myosin Phosphatase-Rho Interacting Protein (M-RIP) is found in the PP1M complex in VSMC. Further preliminary studies show that M-RIP colocalizes with myofibrils in VSMCs, and MRIP itself is a Rho-kinase substrate. The central hypothesis of this proposal is that M-RIP mediates Rho-dependent myosin phosphatase inhibition in response to vasoconstrictors by targeting RhoA/Rho-kinase to the PP1M complex. In SA 1, the complex formation between M-RIP, RhoA and MBS will be investigated. Using in vitro interaction studies and site-specific mutagenesis, the molecular determinants of RhoA-M-RIP-MBS binding will be defined and both disrupting peptides and non-binding M-RIP mutants will be developed. In SA 2, the role of phosphorylation in RhoA/Rho-kinase-dependent PP 1M inhibition will be studied by (a) investigating the role of M-RIP phosphorylation on PP 1M regulation and (b) the role of M-RIP in Rho-kinase phosphorylation of PP 1M. In SA 3, the functional role of MRIP in regulation of PP 1M will be studied in vivo by biochemical analysis of vasoconstrictor-mediated PP 1M inhibition in (a) cells made devoid of M-RIP and in (b) cells in which M-RIP-RhoA or M-RIP-MBS interactions are disrupted. These studies are expected to provide valuable new insights into the molecular mechanism of PP1M inhibition by RhoA/Rho-kinase, a central mediator of VSMC contraction that regulates blood vessel tone. Elucidating the molecular pathways that regulate blood vessel tone is of utmost importance for understanding both normal vascular function and the major vascular diseases in our society.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
1R01HL074069-01A1
Application #
6780551
Study Section
Experimental Cardiovascular Sciences Study Section (ECS)
Program Officer
Rabadan-Diehl, Cristina
Project Start
2004-07-01
Project End
2009-06-30
Budget Start
2004-07-01
Budget End
2005-06-30
Support Year
1
Fiscal Year
2004
Total Cost
$326,000
Indirect Cost
Name
Tufts University
Department
Type
DUNS #
079532263
City
Boston
State
MA
Country
United States
Zip Code
02111
Durham, Jennifer T; Surks, Howard K; Dulmovits, Brian M et al. (2014) Pericyte contractility controls endothelial cell cycle progression and sprouting: insights into angiogenic switch mechanics. Am J Physiol Cell Physiol 307:C878-92
Wang, Guang-rong; Surks, Howard K; Tang, K Mary et al. (2013) Steroid-sensitive gene 1 is a novel cyclic GMP-dependent protein kinase I substrate in vascular smooth muscle cells. J Biol Chem 288:24972-83
Wang, Yuepeng; Zheng, Xiaoyu Rayne; Riddick, Nadeene et al. (2009) ROCK isoform regulation of myosin phosphatase and contractility in vascular smooth muscle cells. Circ Res 104:531-40
Sharma, Alok K; Zhou, Guo-Ping; Kupferman, Joseph et al. (2008) Probing the interaction between the coiled coil leucine zipper of cGMP-dependent protein kinase Ialpha and the C terminus of the myosin binding subunit of the myosin light chain phosphatase. J Biol Chem 283:32860-9
Riddick, Nadeene; Ohtani, Ken-Ichi; Surks, Howard K (2008) Targeting by myosin phosphatase-RhoA interacting protein mediates RhoA/ROCK regulation of myosin phosphatase. J Cell Biochem 103:1158-70
Surks, Howard K (2007) cGMP-dependent protein kinase I and smooth muscle relaxation: a tale of two isoforms. Circ Res 101:1078-80
Kutcher, Matthew E; Kolyada, Alexey Y; Surks, Howard K et al. (2007) Pericyte Rho GTPase mediates both pericyte contractile phenotype and capillary endothelial growth state. Am J Pathol 171:693-701
Surks, Howard K; Riddick, Nadeene; Ohtani, Ken-Ichi (2005) M-RIP targets myosin phosphatase to stress fibers to regulate myosin light chain phosphorylation in vascular smooth muscle cells. J Biol Chem 280:42543-51