Western Michigan University, Kalamazoo, MI 49008
UNC-82 is a serine/threonine kinase required for maintenance of thick filament and M-line organization in the striated body-wall muscle of the nematode C. elegans. Consistent with these defects, an UNC-82::GFP fusion protein has been shown to localize at or near the M-line. To understand UNC-82 function in muscle, we would like to identify the target substrates of UNC-82, and the mechanism by which UNC-82 catalytic activity is activated. Because the contractile apparatus of muscle is a well-characterized and highly ordered structure, determining the location of the UNC-82 protein within this structure will provide clues to help identify possible interacting proteins. We are taking two approaches to determine the location of the UNC-82 protein with more precision. First, we have created an RFP-tagged UNC-82 construct to enable co-localization of UNC-82 with existing GFP-tagged protein markers in living animals. To make this construct, an existing UNC-82::GFP construct was restriction digested and a PCR fragment containing the RFP sequence was ligated in its place. Second, antibodies have been raised against two UNC-82 epitopes: one adjacent to the N-terminal kinase domain, and one that includes the native C-terminus. Two GST fusion proteins were created to perform affinity purification of the antisera. The GST fusion proteins were expressed in bacteria, affinity purified, and covalently bound to a matrix. The matrix was used to make affinity columns to purify the anti-UNC-82 antibodies. These purified antibodies will be used in immuno-EM experiments to determine the precise location of the UNC-82 protein in muscle. The antisera may also be used for immuno-precipitation experiments to identify other proteins that interact with UNC-82. To investigate the activation of UNC-82 by other kinases in muscle, we are identifying candidate kinases from the literature and testing for a possible role in organization of thick filaments. Additionally all of the UNC-82 homologues were run through the phosphorylation prediction programs Phospho.ELM BLAST and Motif Scan to identify predicted phosphorylation motifs. Candidates with conserved phosphorylation motifs in UNC-82 and its human homologues ARK5 and SNARK are being investigated. UNC-82 and it’s mouse, rat, human, fish, and Drosophila homologues contain a conserved LKB1/PAR-4 motif. Further, it has been shown that LKB1/PAR-4 phosphorylation stimulates SNARK activity. Additionally, activation of the human homologue ARK5 is mediated by AKT and NDR2/SAX-1 phosphorylation both in vitro and in vivo. UNC-82 and the human, mouse, rat, and Drosophila homologues all contain the RXRXX(S/T) AKT phosphorylation domain. All UNC-82 homologues contain a semiconserved NDR2/SAX-1 activation motif within the kinase domain. Mutant C. elegans strains carrying knock-out mutations in these candidate kinase genes are being examined for a muscle phenotype using polarized light microscopy. Where available, we are testing whether GFP-tagged candidates colocalize with UNC-82::RFP.
[Abstract (DOC)]