A cGMP-dependent protein kinase is implicated in wild-type motility in C. elegans.
In mammals, cyclic GMP and cGMP-dependent protein kinases (cGKs) have been implicated in the regulation of many neuronal functions including long-term potentiation and long-term depression of synaptic efficacy. To develop Caenorhabditis elegans as a model system for studying the neuronal function of the cGKs, we cloned and characterized the cgk-1 gene. A combination of approaches showed that cgk-1 produces three transcripts, which differ in their first exon but are similar in length. Northern analysis of C. elegans RNA, performed with a probe designed to hybridize to all three transcripts, confirmed that a major 3.0 kb cgk-1 transcript is present at all stages of development. To determine if the CGK-1C protein was a cGMP-dependent protein kinase, CGK-1C was expressed in SF:9 cells and purified. CGK-1C shows a K(a) of 190 +/- 14 nM for cGMP and 18.4 +/- 2 microM for cAMP. Furthermore, CGK-1C undergoes autophosphorylation in a cGMP-dependent manner and is inhibited by the commonly used cGK inhibitor, KT5823. To determine which cells expressed CGK-1C, a 2.4-kb DNA fragment from the promoter of CGK-1C was used to drive GFP expression. The CGK-1C reporter construct is strongly expressed in the ventral nerve cord and in several other neurons as well as the marginal cells of the pharynx and intestine. Finally, RNA-mediated interference of CGK-1 resulted in movement defects in nematode larvae. These results provide the first demonstration that cGMP-dependent protein kinase is present in neurons of C. elegans and show that this kinase is required for normal motility.
Stansberry
J
J
Department of Biological Chemistry, Neuroscience Graduate Program, University of Michigan, Ann Arbor, Michigan, USA.
Baude
E J
EJ
Taylor
M K
MK
Chen
P J
PJ
Jin
S W
SW
Ellis
R E
RE
Uhler
M D
MD
D000818:Animals; D017173:Caenorhabditis elegans / Q000201:enzymology*; D017869:Cyclic GMP-Dependent Protein Kinases / Q000235:genetics* / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D018507:Gene Expression Regulation, Developmental; D049452:Green Fluorescent Proteins; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics; D008845:Microinjections; D008969:Molecular Sequence Data; D009043:Motor Activity / Q000187:drug effects / Q000502:physiology*; D009474:Neurons / Q000166:cytology / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D012330:RNA, Double-Stranded / Q000008:administration & dosage; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D007527:Isoenzymes; D008164:Luminescent Proteins; D012330:RNA, Double-Stranded; D012333:RNA, Messenger; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D017869:Cyclic GMP-Dependent Protein Kinases