| The mammalian calcium/calmodulin-dependent protein kinase kinases (CaMKKs) have been shown to phosphorylate the calcium/calmodulin-dependent protein kinases I and IV (CaMKI and CaMKIV), leading to biochemical activation in vitro and in vivo, and enhanced transcription in transfection assays. We have cloned and characterized Caenorhabditis elegans homologues to the pathway components, ceCaMKI and ceCaMKK, in order to investigate the proposed cascade in the context of a well-defined multicellular model organism. Using recombinant proteins, C. elegans and mammalian CaMKs could be interchangeably activated by the C. elegans and mammalian CaMKKs through phosphorylation of a conserved activation loop threonine. By generating transgenic nematodes with reporter proteins driven the ceCaMKK upstream promoter region, expression was observed in amphid sensory neurons, the excretory cell, and the vulval muscles of adult hermaphrodites; hypodermal cells of L1/L2 larvae; and several male-specific tail cells. cDNA-GFP fusions were used to examine subcellular localization in C. elegans by transgenic methods and in HEK293 fibroblasts by transfection. In both cases, ceCaMKK was primarily cytoplasmic, but treating the fibroblasts with the nuclear export inhibitor leptomycin B caused a partial shift of fluorescence to the nucleus.; To compare the biochemical characteristics of ceCaMK with those of mammalian CaMKI and CaMKIV, differential phosphorylation of a novel CaMK substrate p300(1–117) was studied, revealing a CaMKI-specific site at Ser89, also phosphorylated by ceCaMKI, and a distinct CaMKIV site at Ser24. Using complex protein mixtures obtained from soluble cell extracts as a source of substrates for in vitro kinase reactions, substrate preferences were compared more generally. In this assay, CaMKI, II, and IV phosphorylated distinct sets of proteins, with ceCaMKI generally preferring the same substrates as CaMKI. Using fractionated extract, several potential substrates were identified and microsequenced. One substrate, HSP25, was further characterized using purified proteins to validate it as an in vitro substrate of the CaMKs and thereby corroborate our methods for substrate comparison. Through these studies, we have confirmed a biochemical homology between the mammalian and C. elegans CaMK cascade members, established the expression pattern of the kinases in the nematode, and advanced the search for potential effectors of the pathway. |
