Three new isoforms of Caenorhabditis elegans UNC-89 containing MLCK-like protein kinase domains.
In Caenorhabditis elegans, the gene unc-89 is required for A-band organization of striated muscle. In mammals, a likely homolog of UNC-89, called obscurin, has been described and found to be localized at both the M-lines and Z-discs of striated muscle. Here, we show that the coding sequence for unc-89 is larger than originally thought, and that the gene encodes at least four major isoforms: UNC-89-A (original isoform, 732 kDa), UNC-89-B (potentially 900 kDa), and UNC-89-C and UNC-89-D (each 156 kDa). UNC-89-C and -D, except for unique N-terminal tails of eight and 11 residues, respectively, are co-linear with the C terminus of UNC-89-B. The unc-89 complex transcription unit contains at least three promoters: one directing UNC-89-A and -B primarily in body-wall and pharyngeal muscle, one internal promoter directing expression of UNC-89-C primarily in body-wall muscle, and one internal promoter directing expression of UNC-89-D primarily in a few muscle cells of the tail. Isoform-specific RNA interference resulted in a muscle structural phenotype similar to a typical unc-89 mutant, but with varying degrees of severity. Antibodies generated to the interkinase region shared by the UNC-89-B, -C and -D isoforms localize to the middle of A-bands, like previously-described UNC-89 antibodies, and detect proteins on immunoblots consistent with the proposed gene organization and additional isoforms. The three new UNC-89 isoforms contain two protein kinase domains, of the myosin light chain kinase (MLCK) family. UNC-89-B contains two complete protein kinase domains, designated PK1 and PK2. UNC-89-C and -D begin with partial kinase domains, PK1-C and PK1-D. Homology modeling suggests that PK2 is catalytically active, PK1 is inactive, and that PK1-C and PK1-D have similar structures at their N termini that may create sites for interaction with other proteins.
Department of Pathology, Emory University, Atlanta, GA 30322, USA.
Tracey M
TM
Small
Kim M
KM
Gernert
Denise B
DB
Flaherty
Kristina B
KB
Mercer
Mark
M
Borodovsky
Guy M
GM
Benian
D000595:Amino Acid Sequence; D000818:Animals; D030801:Animals, Genetically Modified; D017173:Caenorhabditis elegans / Q000166:cytology / Q000235:genetics / Q000378:metabolism; D029742:Caenorhabditis elegans Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005091:Exons; D008958:Models, Molecular; D009124:Muscle Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D009219:Myosin-Light-Chain Kinase / Q000737:chemistry* / Q000235:genetics; D011401:Promoter Regions, Genetic; D020033:Protein Isoforms / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D034622:RNA Interference; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment
D029742:Caenorhabditis elegans Proteins; D009124:Muscle Proteins; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; C098932:Unc-89 protein, C elegans; D009219:Myosin-Light-Chain Kinase