Molecular Genetic Analysis of UNC-82 Kinase Function in C. Elegans Muscle

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DESCRIPTION (provided by applicant): Striated muscle contraction is accomplished by a highly ordered cytoskeletal structure consisting of interdigitating actin and myosin filaments, the filament anchoring structures, and a host of regulatory proteins. Over the past several decades, the advanced molecular genetics and cell biology of the model organism C. elegans have provided an important discovery tool for identifying and studying proteins critical for striated muscle cell development and function. We have determined that the C. elegans muscle-affecting gene unc-82 encodes a serine/threonine kinase that is orthologous to human proteins ARK5 (NUAK1) and SNARK (NUAK2). Both ARK5 and SNARK are expressed in striated muscle, but the role that either plays in muscle development or physiology is unknown. Both ARK5 and SNARK were named for their sequence similarity to AMPK kinases, which are activated by stress, including glucose starvation and the resulting high levels of cellular AMP. However, sequence comparisons and biochemical data from human cell lines suggest that the activation of enzyme activity occurs by different mechanisms in ARK5 and SNARK. Whereas ARK5 is activated by phosphorylation at a particular regulatory site by the kinase Akt in conditions of glucose starvation, SNARK does not contain the motif recognized by the Akt enzyme. Interestingly, through alternative splicing, the single C. elegans gene unc-82 also produces protein isoforms that differ in the presence or absence of the Akt regulatory phosphorylation site. Although some isoforms of UNC-82 may be activated in response to stress, we hypothesize that in embryonic muscle UNC-82 is part of a signaling pathway that is required for the organization of thick filaments and membrane-distal components of the M-line in response to changes in cell shape. Experiments outlined in this proposal are designed to test the role of phosphorylation in the activation of UNC-82 in early muscle development. In addition, we hope to exploit the advanced molecular genetics of the C. elegans system to identify other members of the UNC-82 signaling pathway, including UNC-82 substrates and potential kinase partners in a signaling cascade. UNC-82 and the human proteins ARK5 and SNARK are expressed in striated muscle throughout life, indicating perhaps diverse roles in muscle cell development, structural maintenance, or even contractile function in both heart and skeletal muscle. The majority of the published work on ARK5 and SNARK concerns the role of these proteins in cancer, and particularly in regulation of the invasiveness of tumors cells. The change in tumor cell behavior in response to activation of ARK5 or SNARK may imply that the enzymes are affecting invasiveness through regulation of the cytoskeleton. If so, the knowledge gained about the role of UNC-82 in normal muscle development may promote understanding of the role of ARK5 and SNARK in promotion metastasis. PUBLIC HEALTH RELEVANCE: We have identified a new enzyme, UNC-82, that is important for organizing the filaments of the striated muscle cell contractile apparatus in the model organism C. elegans. Interestingly, the human homologs of this enzyme have been shown to be important in metastasis of human cancers. The human homologs of UNC-82 are expressed in heart and skeletal muscle. Understanding the role of UNC-82 in C. elegans will shed light on the role of the human enzymes in normal muscle development and function.