Physiological functions, effectors and upstream regulators of Ca 2+ and diacylglycerol (DAG) activated, classical protein kinase C isoforms (cPKCs) are only partially understood. Either depletion or over-expression of PKC-2, the only cPKC in C. elegans, disrupts thermotaxis.
pkc-2 null (
pkc-2(
ok328)),
tax-4(
p678),
tax-2(
p671),
pkc-2(
ok328);
tax-2(
p671) and
pkc-2(
ok328);
tax-4(
p678) mutants share an athermotactic (AT) phenotype. Mutant animals lose the ability to detect or localize at their previous cultivation temperature upon transfer to a thermal gradient. Instead, they move randomly, suggesting that TAX-2, TAX-4 and PKC-2 function together in a signaling pathway that couples changes in temperature to directed movement.
tax-4(
p678) and
tax-2(
p671) suppress cryophilic behavior induced by 12-fold overexpression of PKC-2 (
pkc-2(12X) ). Increased substrate phosphorylation was evident in transgenic animals (
pkc-2(12x)) that sustain a supraphysiological PKC-2 concentration. However, phosphorylation of PKC substrates was suppressed below the basal WT level in TAX-2 or TAX-4 deficient animals (
tax-4(
p678),
tax-2(
p671),
tax-4(
p678);
pkc-2(12x) and
tax-2(
p671);
pkc-2(12x)) expressing either normal or highly elevated amounts of PKC-2. A DAG mimetic, PMA, elicits a cryophilic phenotype in WT and TAX-2 deficient animals, whereas
pkc-2 null,
tax-2(
p671);
pkc-2(
ok328) and
tax-4(
p678) worms are athermotactic. Thus, epistasis analysis places PKC-2 downstream from TAX-2. TAX-2 and TAX-4 form a multimeric, cGMP-regulated cation channel that transports Ca2+. TAX-2 modulates the activity of TAX-4, which directly mediates cation influx. TAX-4, TAX-2 and PKC-2 are co-expressed in many neurons, including AFD, the predominant thermosensor, and other neurons in thermotaxis circuitry. The results suggest that Ca2+ entry mediated by the TAX-2/TAX-4 channel is critical for activation of PKC-2 and subsequent downstream signaling in the thermotaxis response. Expression of normal amounts of WT PKC-2 in the
pkc-2 null background rescues the thermotaxis defect. A transgene encoding mutant PKC-2, in which the C2 (Ca2+ binding) domain is disrupted, failed to rescue either thermotaxis or substrate phosphorylation. This verifies that Ca2+ is essential for PKC-2 activation in thermotaxis and shows DAG binding via a C1 domain is insufficient for in vivo PKC-2 activation. TAX-4 may be a PKC-2 effector. A recombinant TAX-4-His-fusion protein was phosphorylated to high stoichiometry, invitro by PKC. Phosphorylated Ser/Thr in TAX-4 will be identified and expression of transgenes encoding phospho-mimetic and non-phosphorylatable TAX-4 mutants will be used to assess the physiological relevance of the PKC-2 target sites.