The long-term objectives of this research are to understand the mechanisms by which the gene product of the myotonic dystrophy (DM) locus, a specific serine/threonine protein kinase, acts in the regulatory networks of muscle and nerve. Understanding these actions is significant because of the potential roles that the DM protein kinase may play in signal transduction both during development and in mature function and because of the strong possibility that alteration of the activities of such a protein kinase may be critical in the pathogenesis of DM. The activities include not only intrinsic catalytic activity, but regulation by ligands, other kinases and phosphatases, and molecular association with specific sites in muscle fibers and neurons. Alteration of these activities of a protein kinase with key regulatory actions in signal transduction could be closely related to the alterations of membrane excitability, contractility, and insulin sensitivity in muscle and the mental retardation observed in individuals affected by DM. Substantial progress in the development of reagents and tools required to pursue these studies has been made. Bacterially expressed recombinant DM protein kinase is enzymatically active. Polyclonal and monoclonal antibodies with high affinity and specificity have been produced to recombinant protein kinase and characterized. To accomplish these objectives, I propose:
Specific Aim 1) In order to characterize the enzymatic, structural, and regulatory properties of potentially native DM protein kinase, the enzyme will be expressed in and purified from insect and mammalian cell culture systems;
Specific Aim 2) In order to characterize DM protein kinase in terms of distinct states of modification, different isoforms, and specific sites of association, a comprehensive library of monoclonal antibodies will be produced to recombinant enzymes and characterized;
Specific Aim 3) The biological activities and interactions of potentially native, recombinant DM protein kinase will be characterized by biochemical, immunochemical, and genetic experiments including two hybrid complementation in yeast and recombinant constructs in mammalian cells;
and Specific Aim 4) Tissue- specific and compartment-specific isoforms of DM protein kinase will be identified immunologically in mammalian tissues and cell types affected by DM and these isoforms will be localized by visualization of antibody reaction at the cellular and subcellular levels.
|Muranyi, A; Zhang, R; Liu, F et al. (2001) Myotonic dystrophy protein kinase phosphorylates the myosin phosphatase targeting subunit and inhibits myosin phosphatase activity. FEBS Lett 493:80-4|