A cGMP‐dependent protein kinase is implicated in wild‐type motility in C. elegans

Abstract

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 ef®cacy. 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 ®rst exon but are similar in length. Northern analysis of C. elegans RNA, performed with a probe designed to hybridize to all three transcripts, con®rmed 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 Sf9 cells and puri®ed. CGK-1C shows a K a of 190^14 nM for cGMP and 18.4^2 mM 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, RNAmediated interference of CGK-1 resulted in movement defects in nematode larvae. These results provide the ®rst demonstration that cGMP-dependent protein kinase is present in neurons of C.elegans and show that this kinase is required for normal motility.

The Importance of cGMP Signaling in Sensory Cilia for Body Size Regulation in Caenorhabditis elegans

“…The gcy-12 mutations, as well as the egl-4 mutations, were classified as class I suppressors, indicating that the che-2 mutation causes the small-body phenotype through altered GCY-12 function. Biochemical analyses showed that EGL-4 indeed requires cGMP for its kinase activity (Stansberry et al 2001;Hirose et al 2003); therefore, the simplest interpretation is that GCY-12 is the guanylyl cyclase that provides cGMP to EGL-4 in the pathway regulating body size. The studies of this article provide several lines of evidence to support this hypothesis.…”

“…Mutations in egl-4 result in the class I Chb phenotype and the expression of egl-4 cDNA in several sensory neurons can rescue this phenotype (Fujiwara et al 2002). The EGL-4 kinase is activated by cGMP (Stansberry et al 2001;Hirose et al 2003); therefore, it is conceivable that cGMP produced by the GCY-12 guanylyl cyclase activates EGL-4 in vivo. To examine this possibility by genetic analyses, we first made a gcy-12;egl-4 double mutant.…”

“…Since EGL-4 kinase activity is dependent on cGMP (Stansberry et al 2001;Hirose et al 2003), it is likely provided by one or more of the guanylyl cyclases encoded in the C. elegans genome. A total of 34 guanylyl cyclases (GC) have been identified in the C. elegans genome, of which 7 are soluble cyclases and 27 are receptor-type cyclases (Ortiz et al 2006).…”

Nuclear entry of a cGMP-dependent kinase converts transient into long-lasting olfactory adaptation

“…3C). Two allosteric cGMP-binding domains are present in the amino one-half of EGL-4 (29,30). In other PKGs, cGMP binding has been shown to relieve an inhibitory interaction between an amino terminal pseudosubstrate region and the kinase domain (31).…”

Dimerization of cGMP-dependent Protein Kinase Iβ Is Mediated by an Extensive Amino-terminal Leucine Zipper Motif, and Dimerization Modulates Enzyme Function

“…All three mammalian PKG isoforms (I␣, I␤, and II) contain a leucine zipper motif in this sequence, although the remainder of the residues in the sequence have limited sequence identity. Other eukaryotic PKGs such as the two Drosophila PKGs (28) and the PKGs found in Caenorhabditis elegans (29), Hydra oligactis, and Apis mellifera (30) also contain a leucine zipper motif in this region (see Fig. 1).…”