The cells of eukaryotic organisms are organized to perform specialized functions. For example, cells lining the stomach wall are specialized to secrete gastric acid while cells that occupy the middle layer of blood vessels are specialized to contract andrelax, thereby closing and opening the vessel. It is now known that one of the major biochemical mechanisms for controlling the activity of the enzymes that regulate these specialized activities is a process called protein phosphorylation. Protein phosphorylation involves one type of enzyme, called a protein kinase, modifying another type of protein, called a substrate, by attaching phosphate groups to its backbone structure. Once the substrate accepts the phosphate group, it is said to be phosphorylatedand its activity or function changes. The newly phosphorylated protein can then change the activity of the cell. In the case of the cells that regulate the blood vessel tone (these are called smooth muscle cells), the protein kinase must act quickly to phosphorylate substrates so that blood flow can be adjusted rapidly. It is not known how protein kinases can act so quickly in the smooth muscle cell, however. This proposal will test one hypothesis on how protein kinases in smooth muscle cells rapidly phosphorylate proteins. This hypothesis is called "targeting" which means simply that the protein kinase and the substrate are localized very near each other so that the process may occur rapidly and efficiently. We will utilize the techniques of modern recombinant DNA technology and confocal laser scanning microscopy (CLSM) to study how protein kinases situate themselves in the cell so that they can be close by the substrate which they phosphorylate. This hypothesis is a new idea on how cells are structured, and challenges previous notions that the cell is merely a "bag of enzymes" which "swim around" in search of substrate molecules. The results of this study will shed light on the architecture of living cells and will hopefully provide a basis for studying cellular events other than phosphorylation.
Our laboratory is interested in the role of nitric oxide (NO) signaling and the mechanisms by which the second messenger, cyclic GMP, regulates vascular smooth muscle cell function. NO increases cyclic GMP that, in turn, activates a serine/threonine protein kinase, the cyclic GMP-dependent protein kinase (PKG). We have identified several proteins whose phosphorylation is catalyzed by PKG in smooth muscle cells. More recently, we have found that NO and PKG regulate gene expression in vascular smooth muscle cells. Stable transfection or adenoviral gene delivery of the Type I PKG gene into vascular smooth muscle cells induces the expression of contractile proteins such as smooth muscle specific myosin and actin and repress the expression of extracellular matrix proteins such as osteopontin. DNA microarray analysis shows that over 100 genes appear to be regulated by PKG in smooth muscle. These results are pathophysiologically important because arterial vascular smooth muscle cells, in response to injury and atherosclerosis, lose their contractile phenotype and secrete extracellular matrix proteins. Hence, PKG appears to suppress the development of the atherosclerotic phenotype in vascular smooth muscle cells. More recently, we have observed that in response to injury and inflammatory cytokines, endogenous PKG mRNA expression is suppressed resulting in the loss of PKG protein in the vascular smooth muscle cells. These inflammatory conditions promote the modulation of vascular smooth muscle cells to the atherosclerotic phenotype. Restoration of PKG expression by adenoviral gene transfer restores the contractile, non-atherosclerotic phenotype. Therefore, one possible link between inflammation and fibroproliferative behavior of vascular smooth muscle cells in the suppression of PKG expression. We are currently studying the molecular mechanisms that control PKG mRNA and protein expression in vascular smooth muscle cells and would like to identify pharmacologic agents that increase PKG expression in these cells to prevent the modulation to the atherosclerotic phenotype. Clearly, adenoviral gene transfer of PKG cDNA into vascular lesions in vivo would be one mechanism using gene therapy for such vascular diseases as atherosclerosis, restenosis and inflammatory lesions.
The role of nitric oxide (NO) signaling and the mechanisms by which the second messenger, cyclic GMP, regulates vascular smooth muscle cell function.
Animal Systems Physiology, Health, Life Science Biological
