ROLE OF THE UNC-82 KINASE IN *C. ELEGANS* STRIATED MUSCLE

[Jason A. Kintzele]{.underline}, Pamela E. Hoppe\*. Western Michigan
University, Department of Biological Sciences,Kalamazoo, MI 49008,
pamela.hoppe@wmich.edu

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.