The goal of this project is to understand the mechanism by which the Rho/MLCP (myosin light chain phosphatase) pathway regulates myosin light chain (MLC) phosphorylation and contraction in vascular smooth muscle. In the present proposal, we will clarify the function of p116Rip, the novel regulatory protein of the RhoA/MLCP pathway, based upon our recent findings of this MBS/RhoA binding molecule. While a number of studies have been done on the regulatory role of Rho kinase in smooth muscle, nothing is known about the function of PKN, another RhoA down-stream kinase. Our PRELIMINARY STUDIES raise the possibility that PKN plays a role in the regulation of the RhoA/MLCP pathway, and we will study the function and regulation of PKN in the agonist induced regulation of smooth muscle contraction. Based upon our PRELIMINARY STUDIES, we propose the following hypothesis of the regulatory function of PKN and p116Rip. Upon agonist stimulation, RhoA translocates to the membrane and cytosolic PKN, which has a binding affinity for active RhoA, is recruited to the membrane. PKN becomes activated and sustains the membrane binding of RhoA, thus prolongs RhoA activity. On the other hand, p116Rip associates with myosin and MBS at the actomyosin fiber and activates MLCP reaction, thus facilitating MLC dephosphorylation. P116Rip, at the actomyosin filaments, interacts with cytosolic RhoA to prevent translocation to the membrane, thus attenuating RhoA activation. These effects result in the increase in MLCP activity and down-regulation of MLC phosphorylation in smooth muscle. In this proposal, we will verify this hypothesis. We will first use a siRNA approach to eliminate p116Rip and PKN, respectively, and study the effects of the specific siRNAs in the agonist induced change in MLC20 phosphorylation and muscle contraction. Once we identify the function of p116Rip and PKN in the regulation of MLC20 phosphorylation, we will study the regulatory mechanism of p116Rip and PKN activity. It has been known that the activation of Rho and its downstream molecules are closely related to the translocation of these molecules to the membrane. To further address our hypothesis, we will study the spatio-temporal change in the localization of these molecules after stimulation by using 3D digital imaging analysis of the differentiated smooth muscle cells. To achieve this, we will introduce fluorescent protein (FP)-tagged regulatory proteins using the protein delivery technique. We will also use our newly developed total internal reflection fluorescence (TIRF) microscope to monitor the change in the near-membrane domain of the cells. Furthermore, we will study the binding of p116Rip and PKN with their target molecules by using FRET analysis, thus monitoring the spatio-temporal change in the interaction of the molecules in living cells. The proposed project will clarify the mechanism by which agonists induce vascular smooth muscle contraction, which should provide novel insights into the regulation of vascular constriction and contribute to the pathogenesis of cardiovascular diseases.

Public Health Relevance

Smooth muscle plays an important role in arterial constriction/dilation thus controlling blood pressure and its malfunction causes various vascular diseases such as high blood pressure. In vascular smooth muscle, the external stimuli induce the sustained contraction and this is critical for the prolonged constriction of the blood vessels. The proposed project will clarify the regulatory function of the newly found protein components in the mechanism linking the external stimuli and the contraction of smooth muscle, thus will improve our understanding of vascular constriction and the dilation mechanism.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL073050-09
Application #
8287162
Study Section
Vascular Cell and Molecular Biology Study Section (VCMB)
Program Officer
Olive, Michelle
Project Start
2003-04-01
Project End
2014-06-30
Budget Start
2012-07-01
Budget End
2013-06-30
Support Year
9
Fiscal Year
2012
Total Cost
$407,138
Indirect Cost
$159,638
Name
University of Massachusetts Medical School Worcester
Department
Physiology
Type
Schools of Medicine
DUNS #
603847393
City
Worcester
State
MA
Country
United States
Zip Code
01655
Zhang, Cheng-Hai; Lifshitz, Lawrence M; Uy, Karl F et al. (2013) The cellular and molecular basis of bitter tastant-induced bronchodilation. PLoS Biol 11:e1001501
Sakai, Tsuyoshi; Umeki, Nobuhisa; Ikebe, Reiko et al. (2011) Cargo binding activates myosin VIIA motor function in cells. Proc Natl Acad Sci U S A 108:7028-33
Umeki, Nobuhisa; Jung, Hyun Suk; Sakai, Tsuyoshi et al. (2011) Phospholipid-dependent regulation of the motor activity of myosin X. Nat Struct Mol Biol 18:783-8
Ressmeyer, Anna-Rebekka; Bai, Yan; Delmotte, Philippe et al. (2010) Human airway contraction and formoterol-induced relaxation is determined by Ca2+ oscillations and Ca2+ sensitivity. Am J Respir Cell Mol Biol 43:179-91
Tanaka, Hiroto; Homma, Kazuaki; White, Howard D et al. (2008) Smooth muscle myosin phosphorylated at single head shows sustained mechanical activity. J Biol Chem 283:15611-8
Lu, Yuan; Zhang, Haiying; Gokina, Natalia et al. (2008) Uterine artery myosin phosphatase isoform switching and increased sensitivity to SNP in a rat L-NAME model of hypertension of pregnancy. Am J Physiol Cell Physiol 294:C564-71
Koga, Yasuhiko; Ikebe, Mitsuo (2008) A novel regulatory mechanism of myosin light chain phosphorylation via binding of 14-3-3 to myosin phosphatase. Mol Biol Cell 19:1062-71
Takizawa, Norio; Ikebe, Reiko; Ikebe, Mitsuo et al. (2007) Supervillin slows cell spreading by facilitating myosin II activation at the cell periphery. J Cell Sci 120:3792-803
Komatsu, Satoshi; Ikebe, Mitsuo (2007) The phosphorylation of myosin II at the Ser1 and Ser2 is critical for normal platelet-derived growth factor induced reorganization of myosin filaments. Mol Biol Cell 18:5081-90
Takamoto, Norio; Komatsu, Satoshi; Komaba, Shigeru et al. (2006) Novel ZIP kinase isoform lacks leucine zipper. Arch Biochem Biophys 456:194-203

Showing the most recent 10 out of 11 publications