Inappropriate responses by vascular smooth muscle to the complex neural and hormonal mechanisms governing contraction occur in most cases of morbidity and mortality in the United States. Hypertension and vasospasm are examples of failures of the regulation of smooth muscle contraction. Therapy frequently targets critical steps in the signaling events linking receptors in vascular cell membranes to intracellular second proteins or genetic material. Therefore, it is of considerable importance to understand these mechanisms and elucidate their normal operation the major mechanism governing contractile activity in smooth muscle and in many non-muscle cells is phosphorylation of the 20 kDa regulatory light chain (LC20) of myosin. In vivo, the protein kinase, myosin light chain kinase (MLCK) and myosin light chain phosphatase (SMPP-1M). SMPP-1M is the target of several opposing signaling pathways that either activate or inhibit the enzyme to effect the contractile state of smooth muscle. In intact smooth muscle we have observed that the 110 kDa regulatory increased in phosphate content in response to agonists that cause Ca2+ sensitization as well as Ca2+ desensitization. A major aim of Project 2 will be to sequence these phosphorylation sites and determine their functional significance in the regulation of smooth muscle.
A second Aim will determine the molecular mechanisms by which cyclic nucleotides at fixed [Ca2+] causes muscle relaxation by activating SMPP-1M.
The final aim will define protein kinase mediated signal transduction pathways that regulate SMPP-1M in smooth muscle. Central to the project are technologies newly developed in our laboratory mixed peptide sequencing and proximity cross-linking. ATP derivatives, that enable regulatory proteins to be studied in single smooth muscle fibers. To achieve the proposed Aims we will combine our biochemical approaches with the physiological and biophysical expertise of Project 1, the molecular biological expertise of Project 3 and the unique synthetic chemistry offered by Core A.
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