The main goal of this project is to understand the molecular mechanism of myosin phosphatase function and its regulation in vascular smooth muscles by the inhibitor protein CPI17. The human genome encodes about 20 times fewer Ser/Thr protein phosphatases than Ser/Thr protein kinases. During the past decade it has become apparent that a way for the phosphatases to match such diversity among protein kinases is for a single catalytic subunit of a protein phosphatase to interact with many different regulatory subunits. These regulatory subunits control phosphatase localization, substrate specificity and specific activity. In smooth muscle and non-muscle cells, a kinase (MLCK) and a phosphatase (MP) turn the ATPase activity of myosin II on and off, thereby regulating contraction. While MLCK has been studied quite extensively, the molecular mechanism of MP regulation remains to be elucidated. MP is a trimeric holoenzyme composed of a PP18-type catalytic subunit (MPc), a targeting subunit (MPt) and a small subunit of unknown function (MPs). Binding of MPt to MPc is thought to confer higher affinity and increased activity toward the phosphorylated myosin regulatory light chain (RLC). In addition, MP is itself regulated by phosphorylation and phosphatase inhibitors such as CPI17. CPI17 is a protein expressed predominantly in vascular smooth muscle cells. Upon phosphorylation at Thr 38, CPI17 becomes a very potent inhibitor of MP. Our working hypothesis is that binding of MPt to MPc induces a conformational change in MPc and contributes to the formation of an extended substrate-binding interface, thereby providing increased contacts with its phosphorylated substrate (P-RLC) and inhibitor (P-CPI17). This hypothesis will be tested by: 1) Completing the determination of the crystal structure of a complex between MPc and a 34kDa N-terminal fragment of MPt (MPc-MPt34kDa), for which crystals and a preliminary structure (at 4 A resolution) are already available. 2) Determining the structure of a ternary complex between MPc-MPt34m, and thio-P-CPI17. 3) Determining the structure of a ternary complex between MPc-MPt34kDa and thio-P-RLC fragments. At this stage, the determination of one of the key structures is well under way, and all the necessary proteins have been expressed in large quantities for crystallization. This project shall benefit enormously from collaborations with three well-recognized laboratories in the myosin phosphatase field, those of Drs. T. Tao, T. Kitazawa and M. Eto. The findings emerging from this study will have an enormous impact in our understanding of smooth muscle regulation and will serve as a model for our understanding of some of the general principles governing phosphatase targeting and regulation in nature. This knowledge will prove invaluable for the development of new drugs to treat cardiovascular diseases such as hypertension.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL075404-05
Application #
7333312
Study Section
Special Emphasis Panel (ZRG1-SSS-B (01))
Program Officer
Gao, Yunling
Project Start
2003-12-15
Project End
2009-11-30
Budget Start
2007-12-01
Budget End
2009-11-30
Support Year
5
Fiscal Year
2008
Total Cost
$373,801
Indirect Cost
Name
University of Pennsylvania
Department
Physiology
Type
Schools of Medicine
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
Lee, Sung Haeng; Hayes, David B; Rebowski, Grzegorz et al. (2007) Toxofilin from Toxoplasma gondii forms a ternary complex with an antiparallel actin dimer. Proc Natl Acad Sci U S A 104:16122-7
Borrego-Diaz, Emma; Kerff, Frederic; Lee, Sung Haeng et al. (2006) Crystal structure of the actin-binding domain of alpha-actinin 1: evaluating two competing actin-binding models. J Struct Biol 155:230-8