Caenorhabditis elegans unc-82 Encodes a Serine/Threonine Kinase Important for Myosin Filament Organization in Muscle During Growth

Abstract

Mutations in the unc-82 locus of Caenorhabditis elegans were previously identified by screening for disrupted muscle cytoskeleton in otherwise apparently normal mutagenized animals. Here we demonstrate that the locus encodes a serine/threonine kinase orthologous to human ARK5/SNARK (NUAK1/NUAK2) and related to the PAR-1 and SNF1/AMP-Activated kinase (AMPK) families. The predicted 1600-amino-acid polypeptide contains an N-terminal catalytic domain and noncomplex repetitive sequence in the remainder of the molecule. Phenotypic analyses indicate that unc-82 is required for maintaining the organization of myosin filaments and internal components of the M-line during cellshape changes. Mutants exhibit normal patterning of cytoskeletal elements during early embryogenesis. Defects in localization of thick filament and M-line components arise during embryonic elongation and become progressively more severe as development proceeds. The phenotype is independent of contractile activity, consistent with unc-82 mutations preventing proper cytoskeletal reorganization during growth, rather than undermining structural integrity of the M-line. This is the first report establishing a role for the UNC-82/ARK5/SNARK kinases in normal development. We propose that activation of UNC-82 kinase during cell elongation regulates thick filament attachment or growth, perhaps through phosphorylation of myosin and paramyosin. We speculate that regulation of myosin is an ancestral characteristic of kinases in this region of the kinome.

The Role of the UNC-82 Protein Kinase in Organizing Myosin Filaments in Striated Muscle ofCaenorhabditis elegans

“…A previously isolated extrachromosomal array expressing wild-type UNC-82::GFP (phEx22; Hoppe et al 2010a) was used in this study. The transgene was generated by recombination in vivo between two overlapping linear DNAs, a 17-kb genomic PCR fragment, and a linearized plasmid encoding the C-terminus of UNC-82 fused to GFP, following the method of Yuan et al (2000).…”

“…The plasmid contains genomic DNA spanning exons 29-35, with GFP inserted prior to the stop codon in exon 35. The numbering of the exons within the unc-82 gene has changed from that in the original publication of this construct (Hoppe et al 2010a), due to the discovery of minor transcripts that contain five additional 59 exons that were not included in the previously published gene structure.…”

“…The GFP sequences in the plasmid-encoding exons 29-35 (Hoppe et al 2010a) were removed by digestion with AgeI and BsmI, and replaced with a similarly digested 1240 bp fragment containing RFP, amplified from pKFMbRFP using Platinum Pfx DNA Polymerase (Invitrogen, Carlsbad, CA) and oligonucleotides ATTTTCAGGGCAGGG and GTTAGTAGAACTCAG. Prior to injection, plasmids were linearized with HindIII, extracted with phenol/chloroform, and ethanol precipitated.…”

“…Antibody staining of the putative null mutant unc-82(e1323) revealed that the thick filament components myosin A and paramyosin and the M-line protein UNC-89/obscurin were severely affected, and that these defects arose during body elongation of the embryo. A rescuing UNC-82::GFP fusion protein is detected at the M-line in body-wall muscle (Hoppe et al 2010a). The protein product of unc-82 is an AMPK-related kinase orthologous to human ARK5/NUAK1 and SNARK/NUAK2 (Hoppe et al 2010a).…”

“…A rescuing UNC-82::GFP fusion protein is detected at the M-line in body-wall muscle (Hoppe et al 2010a). The protein product of unc-82 is an AMPK-related kinase orthologous to human ARK5/NUAK1 and SNARK/NUAK2 (Hoppe et al 2010a). Both orthologs are expressed in mammalian striated muscle (Lefebvre and Rosen 2005).…”