This is a revision of a competitive renewal application to fund the next 5 years of a longstanding research program to investigate the mechanisms and physiologic significance of Ca/CaM-dependent signaling pathways in vascular smooth muscle (VSM). The next period of support will focus on determining the mechanisms of calmodulin (CaM) trafficking in VSM and the roles of 2 important, but little understood, smooth muscle CaM targets, Myristylated Alanine-Rich C-Kinase Substrate (MARCKS) and CaMKII.
The specific aims are: (1) to test the hypothesis that MARCKS is a significant, PKC-dependent regulator of CaM availability and targeting in VSM; (2) to test the hypothesis that MARCKS is a regulator of contractility in differentiated VSM; (3) to test the hypothesis that 6 gamma CaMKII variants identified as cDNAs from an aorta library are physiologically important in differentiated VSM; (4) to determine the targets of the gamma variants of CaMKII in smooth muscle and to test the hypothesis that targets are variant-specific; (5) to test the hypothesis that the memory function of the gamma CaMKII isoform is physiologically significant in VSM and that the presence of memory is variant-specific. The experiments outlined involve the use of contractile tissues in organ culture and freshly isolated vascular cells as well as purified and recombinant proteins. The techniques to be used include quantitative confocal microscopy, chemical loading of decoy peptides and antisense oligos, photoaffinity crosslinking in situ, and a range of biochemical, physiological and molecular techniques to test protein-protein interactions. All techniques are established in the principal investigator's laboratory or home institution and results are expected to significantly advance our understanding of how CaM availability and trafficking, as well as vascular tone, are regulated. Additionally, novel information will be gained on targeting mechanisms of broad relevance to the signal transduction community.
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