Abstract

The goal of the work outlined in this grant is to understand the role of mitogen-activated protein kinase (MAPK) in smooth muscle contractility. During smooth muscle contraction, MAPK is activated, co-localizes with actin, and phosphorylates the thin filament protein caldesmon (CaD). Although the only smooth muscle substrate for MAPK identified thus far os CaD, the functional biochemical effects of the phosphorylation remain controversial. In conflicting studies using permeabilized smooth muscles, Nixon, et.al., found that MAPK had no effect on contractility, while Gerthoffer, et.al., found that MAPK induced a contraction. In the current proposal, the fundamental hypothesis tested is that: MAPK activation and the subsequent phosphorylation of its substrates in the contractile apparatus results in altered smooth muscle contractility. This hypothesis will be studied by performing the following specific aims:
Aim 1. To determine the biochemical effects of ERK1 phosphorylation in an aortic actomyosin preparation. Specifically, alterations in actomyosins ATPase activity due to ERK1, and unique substrates for ERK1 in the actomyosin domain, will be identified.
Aim 2. To determine the contractile effects of ERK1 in permeabilized arterial smooth muscles. Effects of purified ERK1 on the calcium sensitivity of contraction will be tested.
Aim 3. To characterize the binding of wild type (wt) ERK1 or a mutant ERK1 (ERK1k67R) to CaD in order to test the sub-hypothesis that ERK1k67R can act as a dominant negative MAPK in smooth muscle.
Aim 4. To test the in vivo function of MAPK in vascular smooth muscle by eliminating MAPK activity in a mouse transgenic model system. Transgenic mice overexpressing ERK1k67R specifically in smooth muscle will be developed and measurements will be made of biochemical and psychological changes that occur in the animals. The mechanisms responsible for determining the level of force attained in smooth muscle are not completely understood and may involve MAPK. The proposed studies will identify the role of this kinase in regulating contraction, the primary function of smooth muscle.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Program Projects (P01)
Project #
3P01AR041637-09S1
Application #
6434900
Study Section
Project Start
2001-04-01
Project End
2001-11-30
Budget Start
Budget End
Support Year
9
Fiscal Year
2001
Total Cost
$276,287
Indirect Cost

  Institution

Name
Boston Biomedical Research Institute
Department
Type
DUNS #
058893371
City
Watertown
State
MA
Country
United States
Zip Code
02472

  Publications

Guo, Hongqiu; Huang, Renjian; Semba, Shingo et al. (2013) Ablation of smooth muscle caldesmon affects the relaxation kinetics of arterial muscle. Pflugers Arch 465:283-94
Mabuchi, Yasuko; Mabuchi, Katsuhide; Stafford, Walter F et al. (2010) Modular structure of smooth muscle Myosin light chain kinase: hydrodynamic modeling and functional implications. Biochemistry 49:2903-17
Gali?ska, Agnieszka; Hatch, Victoria; Craig, Roger et al. (2010) The C terminus of cardiac troponin I stabilizes the Ca2+-activated state of tropomyosin on actin filaments. Circ Res 106:705-11
Hayes, David; Napoli, Vanessa; Mazurkie, Andrew et al. (2009) Phosphorylation dependence of hsp27 multimeric size and molecular chaperone function. J Biol Chem 284:18801-7
Mudalige, Wasana A K A; Tao, Terence C; Lehrer, Sherwin S (2009) Ca2+-dependent photocrosslinking of tropomyosin residue 146 to residues 157-163 in the C-terminal domain of troponin I in reconstituted skeletal muscle thin filaments. J Mol Biol 389:575-83
Greenberg, M J; Wang, C-L A; Lehman, W et al. (2008) Modulation of actin mechanics by caldesmon and tropomyosin. Cell Motil Cytoskeleton 65:156-64
Coulton, Arthur T; Koka, Kezia; Lehrer, Sherwin S et al. (2008) Role of the head-to-tail overlap region in smooth and skeletal muscle beta-tropomyosin. Biochemistry 47:388-97
Sumida, John P; Wu, Eleanor; Lehrer, Sherwin S (2008) Conserved Asp-137 imparts flexibility to tropomyosin and affects function. J Biol Chem 283:6728-34
Wang, C L Albert (2008) Caldesmon and the regulation of cytoskeletal functions. Adv Exp Med Biol 644:250-72
Lee, Eunhee; Hayes, David B; Langsetmo, Knut et al. (2007) Interactions between the leucine-zipper motif of cGMP-dependent protein kinase and the C-terminal region of the targeting subunit of myosin light chain phosphatase. J Mol Biol 373:1198-212

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