In mammals, cyclic GMP (cGMP) has been shown to regulate smooth muscle contraction, platelet aggregation and calcium channel function. Furthermore, in some neurons cGMP has been implicated in the modulation of synaptic plasticity, long-term potentiation and long-term depression. It appears that cGMP functions predominately by activating the cGMP dependent protein kinase (cGK), a member of the serine/threonine kinase family. To learn how this kinase controls such a wide variety of cellular functions, we are studying this protein in C. elegans . We used degenerate PCR to isolate a cDNA fragment with similarity to mammalian cGMP-dependent kinases. To obtain the complete cDNA, we screened a library with this fragment, and isolated a clone with a 2.6 kb insert. Although this clone lacked the 5' end of the message, we isolated these sequences using an SL1 primer and reverse primers from within the gene. Northern analysis of RNA prepared from a mixed population of males and hermaphrodites confirmed that this gene produces a single transcript of the predicted size. Because our cDNA encodes a protein that resembles mammalian and Drosophila cGKs, we named it
cgk-1 . To determine if CGK-1 behaves like a cGMP-dependent protein kinase, we expressed it in Sf-9 cells and purified it by affinity chromatography. We find that CGK-1 has a 700 nM K a for cGMP, and a 7 microM K a for cAMP, which shows that it preferentially binds cGMP. Furthermore, CGK-1 undergoes autophosphorylation and can phosphorylate known cGK target peptides. Thus, in vitro assays show that CGK-1 functions much like other cGMP-dependent protein kinases. Northern analyses reveal that
cgk-1 is expressed throughout development. We do not yet have antibodies to CGK-1, so we used a fusion gene to begin elucidating where
cgk-1 is expressed. In this construct, transcription of GFP is driven by 2.4 kb of DNA from the
cgk-1 promoter. Our reporter construct is strongly expressed in the marginal cells of the pharynx, the intestine, body wall muscles and sex muscles, the spermatheca, and a small set of neurons that includes cells in the amphids and phasmids. We are now using RNA-mediated inactivation to learn what functions
cgk-1 might play.