PKC2/KIN11 is the only diacylglycerol (DAG) and Ca 2+ protein kinase C isoform in C.elegans .
pkc-2 null worms wander randomly at temperatures higher and lower than their cultivation temperature when placed in a thermal gradient. These athermotactic worms were rescued with the 30 kbp
kin-11 gene (containing 3 promoters previously characterized in this lab). Expression of PKC2 protein at the wild type level (in stable transgenic integrants) normalized the mutant phenotype. High level PKC2 expression caused worms to migrate to regions where temperatures are lower than their cultivation temperature (cryophilic phenotype).
pkc-2 null animals are unable to transmit signals generated by binding of various receptors with classical model odorants. High-level expression of PKC2 in worms carrying an inactivating mutation [
tax-6 (
p695)] in the catalytic domain of PP2B (calcineurin) converts thermophilic worms to cryophiles. Overexpression of PKC2 might cause chronic phosphorylation of a target-effector, thereby locking temperature perception circuitry in a one state and bypassing
tax-6 . Another possibility is that TAX-6 may be involved in up-regulation of a channel or receptor that elevates DAG and/or Ca 2+ . A high concentration of PKC2 could overcome inactive TAX-6 by enabling kinase activation at basal levels of DAG and Ca 2+ .
tax-4 or
tax-2 , which are loss of function mutants that encode defective subunits of a cGMP gated Ca 2+ channel, exhibit an athermotactic phenotype (V Komatsu H, Mori I, Rhee J-S, Akaike N, Ohshima Y, Neuron 17: 707-718 1996) in the absence or presence of over expression of PKC-2. TAX-2 and/or TAX-4 may be substrates of PKC2 , regulated by a PKC2 effector or generate Ca 2+ that could activate PKC2. N and C terminal portions of TAX-4 and TAX-2, generated as His 6 -fusion proteins in E.coli, were not phosphorylated by PKC2 in vitro. Thus it is more likely that the TAX-2/TAX-4 channel imports Ca 2+ that activates PKC2. Analysis of functions of PKC2 exons (and corresponding protein domains) is under investigation. Large segments of genomic DNA upstream from C and B promoters were deleted from the 30kbp
kin-11 gene. The modified DNA was sufficient to drive normal PKC2 expression and rescue the athermotactic phenotype of null worms. Like mammalian PKC beta, PKC2 uses alternative splicing of the final and penultimate exons to generate potentially functionally divergent isoforms. Expression of PKC2 cDNA containing only the penultimate exon is capable of rescuing the athermotactic phenotype in
kin-11 -/- worms. Its ability to rescue the chemotactic response of PKC2 null worms is being tested. We are currently utilizing genetic and proteomic analysis to identify PKC2 substrates.