The long term objective of this research is to understand the role of protein kinases in the regulation of smooth muscle contraction. A number of reports have suggested the effects of various protein kinases on the function of various proteins involved in stimulation-contraction coupling of smooth muscle cells. In this proposal, we focus our attention on the three protein kinases, protein kinase C (PKC) and calmodulin dependent protein kinase II (CaMPKII). While a bulk of reports have suggested that both PKC and CaMPKII play a role in the modulation of smooth muscle contractile response, it is unclear how they modulates contraction in intact cells, It may act on Ca2+ regulating proteins to decrease cytosolic Ca2+ or directly influence the regulatory proteins of myosin phosphorylation such as myosin light chain phosphatase. The complexity in identifying the role of protein kinases in smooth muscle regulation is at least partly due to the fast that there are multiple isoforms expressed in the cells. We hypothesize that each kinase isoform has a specific target site and that this is reflected by specific subcellular localization and translocation after the stimulation. The proposed project will address this problem by using ultrafast 3D digital imaging microscope (UFM) developed by us. Once we identify the subcellular localization of PKC/CaMPKII/PKG isoforms, we will study the function of these protein kinases on the Ca2+ signaling and contraction of smooth muscle cells. The effect of the kinases on contractility will be monitored by force measurement as well as single cell shortening velocity. The role of the kinases on Ca2+ homeostasis will be primarily studied by Ca2+ imaging of single cells loaded by various Ca2+ indicators using UFM. To clarify the role of protein kinases on smooth muscle contraction, we will employ genetic manipulation using cultured smooth muscle cells showing contractile phenotype, as well as by use of biochemical probes such as caged peptides, constitutively active kinases etc using freshly isolated strips/cells. The localization/translocation data will be utilized for the use of probes and the design of experiments. The following itemized specific aims and experiments will be carries out.
Specific aim 1. To define the mechanism and signals which initiate the translocation of PKCs and CaMPKII.
Specific aim 2. To define the role of PKC and CaMPKII on Ca2+ signaling and contraction of smooth muscle cells.
Specific aim 3. To define the protein targets phosphorylated by the PKC and CaMPKIIcascade in smooth muscle cells.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL061426-02
Application #
6125975
Study Section
Experimental Cardiovascular Sciences Study Section (ECS)
Program Officer
Lymn, Richard W
Project Start
1998-12-01
Project End
2002-11-30
Budget Start
1999-12-01
Budget End
2000-11-30
Support Year
2
Fiscal Year
2000
Total Cost
$428,621
Indirect Cost
Name
University of Massachusetts Medical School Worcester
Department
Physiology
Type
Schools of Medicine
DUNS #
660735098
City
Worcester
State
MA
Country
United States
Zip Code
01655
Miyazaki, Koji; Komatsu, Satoshi; Ikebe, Mitsuo (2006) Dynamics of RhoA and ROKalpha translocation in single living cells. Cell Biochem Biophys 45:243-54
Miyazaki, Koji; Komatsu, Satoshi; Ikebe, Mitsuo et al. (2004) Protein kinase Cepsilon and the antiadrenergic action of adenosine in rat ventricular myocytes. Am J Physiol Heart Circ Physiol 287:H1721-9
Komatsu, Satoshi; Ikebe, Mitsuo (2004) ZIP kinase is responsible for the phosphorylation of myosin II and necessary for cell motility in mammalian fibroblasts. J Cell Biol 165:243-54
Takizawa, Norio; Schmidt, David J; Mabuchi, Katsuhide et al. (2003) M20, the small subunit of PP1M, binds to microtubules. Am J Physiol Cell Physiol 284:C250-62
Li, Xiang-dong; Ikebe, Mitsuo (2003) Two functional heads are required for full activation of smooth muscle myosin. J Biol Chem 278:29435-41
Niiro, Naohisa; Koga, Yasuhiko; Ikebe, Mitsuo (2003) Agonist-induced changes in the phosphorylation of the myosin- binding subunit of myosin light chain phosphatase and CPI17, two regulatory factors of myosin light chain phosphatase, in smooth muscle. Biochem J 369:117-28
Komatsu, Satoshi; Miyazaki, Koji; Tuft, Richard A et al. (2002) Translocation of telokin by cGMP signaling in smooth muscle cells. Am J Physiol Cell Physiol 283:C752-61
Takizawa, Norio; Koga, Yasuhiko; Ikebe, Mitsuo (2002) Phosphorylation of CPI17 and myosin binding subunit of type 1 protein phosphatase by p21-activated kinase. Biochem Biophys Res Commun 297:773-8
Takizawa, Norio; Niiro, Naohisa; Ikebe, Mitsuo (2002) Dephosphorylation of the two regulatory components of myosin phosphatase, MBS and CPI17. FEBS Lett 515:127-32
Miyazaki, Koji; Yano, Takeo; Schmidt, David J et al. (2002) Rho-dependent agonist-induced spatio-temporal change in myosin phosphorylation in smooth muscle cells. J Biol Chem 277:725-34

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