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 and relax, 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 phosphorylated and 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.
