(Verbatim from the application): This is a competitive renewal application to fund the next five years of a longstanding research program whose general goal is the determination of the mechanisms and functional significance of PKC dependent contraction of differentiated vascular smooth muscle. The next period of support will focus on the interaction of PKC dependent signaling cascades with two actin-binding proteins, caldesmon and calponin; and the mechanisms by which members of the signaling cascades are targeted to subcellular locations.
The specific aims : (1) to test the hypothesis that calponin is a physiologically important regulator of smooth muscle contractility and to choose between the subhypotheses that: (a) calponin functions as an adaptor protein that links ERK1/2 to PKC versus (b) calponin directly regulates actomyosin interactions; (2) to test the hypothesis that CaP regulates the activation and/or substrate specificity of PKC in differentiated vascular smooth muscle cells.
This aim i s based on preliminary data showing that CaP can trigger phospholipid-independent autophosphorylation of PKC in vitro and that autophosphorylation is regulated in viva; (3) to further test the hypothesis that CaD is a physiologically important regulator of contractility and to test the sub-hypotheses that: (a) ERK1/2 regulates contractility in differentiated vascular smooth muscle via direct phosphorylation of CaD or (b) phosphorylation of CaD by ERK1/2 decreases the CaM requirement for disinhibition of CaD; (4) to investigate the mechanisms of ERK1/2 targeting by testing the hypotheses: (a) that MEK plays a role in the targeting of ERK1/2 in differentiated smooth muscle; (b) that phosphorylation-induced dimerization of ERK1/2 regulates its localization and (C) that CaP may act as an adaptor protein; (5) to test the specific hypothesis that caveolin acts as a scaffolding protein in a PKC-dependent pathway that regulates vascular tone by directing the subcellular targeting of members of this pathway. The experiments outlined in the proposal involve the use of multicellular strips, freshly, enzymatically isolated single cells from ferret aorta and ferret portal vein and purified proteins. The techniques to be used involve a newly developed application of an antisense approach for differentiated contractile smooth muscle as well as high-resolution digital confocal microscopy, microforce recording from single permeabilized cells, analytical ultracentrifugation, and a range of standard biochemical and molecular techniques. All techniques are established in the principal investigator's laboratory or home institution and the results are expected to significantly advance our understanding by which vascular tone is maintained. By working on differentiated rather than cultured vascular smooth muscle, the results on regulation of contractility will have direct relevance to cardiovascular disease. Additionally, novel information will be gained on targeting mechanisms that will be of broad relevance to the signal transduction community.
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