Abstract

The overall Aim of this Project is to determine the physiologically relevant mechanisms of signal transduction that regulate contractility by pathways not dependent on cytoplasmic Ca concentration. The domain dependent functions of the upstream regulators of Ca?+-sensitization by the RhoA-Rho kinase pathway, the guanine nucleotide exchange factors (GEFs) PDZ.RhoGEF and LARG, will be determined in conjunction with the structural identification and recombinant expression of GEF domains in Project 2. The role of a membrane associated protein (p120 catenin) as a potential RhoA-binding partner guanine nucleotide dissociations inhibitor (GDI) will be determined. The kinetics of myosin phosphatase regulation by Rho-kinase and its dependence on the expression level of the endogenous phosphatase inhibitor, CP1-17 will be quantitated in order to determine their relative physiological relevance. The dependence of myosin phosphatase activity on the specific long and short splice isoforms of the regulatory subunit (MYPT1) expressed in mammalian smooth muscle will be evaluated, based on the hypothesis that expression levels of the ratio of the two isoforms determine localization of the phosphatase and the mechanism of its regulation through differences in their phosphorylation sites and myosin binding properties. A major, novel, objective of this Project is to determine the functions of a protein, LPP, newly discovered to be selectively expressed in smooth muscle, on cellular phenotypes, including morphology, adhesion to substrate, cytoskeletal structure and motility. Localization and stimulus induced translocation of signaling proteins, co-localization of proteins, such as p120 catenin with RhoA or CP1-17 with myosin phosphatase or telokin and telokin and LPP partners identified in two-hybrid screens will be performed in Project 3. p120 catenin, LPP, recombinant RhoA, telokin, RhoA- and other protein-mutants will be supplied by Core B. Abnormalities of the RhoA pathway are thought to be involved in asthma and hypertension.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Program Projects (P01)
Project #
5P01HL048807-12
Application #
7063232
Study Section
Heart, Lung, and Blood Initial Review Group (HLBP)
Project Start
Project End
Budget Start
2005-04-01
Budget End
2006-03-31
Support Year
12
Fiscal Year
2005
Total Cost
$485,884
Indirect Cost

  Institution

Name
University of Virginia
Department
Type
DUNS #
065391526
City
Charlottesville
State
VA
Country
United States
Zip Code
22904

  Publications

Utepbergenov, Darkhan; Derewenda, Urszula; Olekhnovich, Natalya et al. (2012) Insights into the inhibition of the p90 ribosomal S6 kinase (RSK) by the flavonol glycoside SL0101 from the 1.5 Å crystal structure of the N-terminal domain of RSK2 with bound inhibitor. Biochemistry 51:6499-510
Hoofnagle, Mark H; Neppl, Ronald L; Berzin, Erica L et al. (2011) Myocardin is differentially required for the development of smooth muscle cells and cardiomyocytes. Am J Physiol Heart Circ Physiol 300:H1707-21
Jin, Li; Gan, Qiong; Zieba, Bartosz J et al. (2010) The actin associated protein palladin is important for the early smooth muscle cell differentiation. PLoS One 5:e12823
Khromov, Alexander; Choudhury, Nandini; Stevenson, Andra S et al. (2009) Phosphorylation-dependent autoinhibition of myosin light chain phosphatase accounts for Ca2+ sensitization force of smooth muscle contraction. J Biol Chem 284:21569-79
Jin, Li; Hastings, Nicole E; Blackman, Brett R et al. (2009) Mechanical properties of the extracellular matrix alter expression of smooth muscle protein LPP and its partner palladin; relationship to early atherosclerosis and vascular injury. J Muscle Res Cell Motil 30:41-55
Cierpicki, Tomasz; Bielnicki, Jakub; Zheng, Meiying et al. (2009) The solution structure and dynamics of the DH-PH module of PDZRhoGEF in isolation and in complex with nucleotide-free RhoA. Protein Sci 18:2067-79
Jin, Li; Yoshida, Tadashi; Ho, Ruoya et al. (2009) The actin-associated protein Palladin is required for development of normal contractile properties of smooth muscle cells derived from embryoid bodies. J Biol Chem 284:2121-30
Zheng, Meiying; Cierpicki, Tomasz; Momotani, Ko et al. (2009) On the mechanism of autoinhibition of the RhoA-specific nucleotide exchange factor PDZRhoGEF. BMC Struct Biol 9:36
Jelen, Filip; Lachowicz, Pawel; Apostoluk, Wlodzimierz et al. (2009) Dissecting the thermodynamics of GAP-RhoA interactions. J Struct Biol 165:10-8
Freitas, Maria Regina; Eto, Masumi; Kirkbride, Jason A et al. (2009) Y27632, a Rho-activated kinase inhibitor, normalizes dysregulation in alpha1-adrenergic receptor-induced contraction of Lyon hypertensive rat artery smooth muscle. Fundam Clin Pharmacol 23:169-78

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