PKC-2 is the only classical protein kinase C (cPKC) in C.elegans that is activated by both diacylglycerol (DAG) and Ca2+. In mammals 3 genes encode cPKCs. PKCs phosphorylate specific Ser/Thr amino acid residues on their targets, however the function and specific substrates of individual isoforms is not known. PKC-2 null,
pkc-2(
ok328) nematodes have an athermotactic (AT) phenotype, in which worms migrate randomly to temperatures other than their cultivation temperature, while PKC-2 over expressing worms,
pkc-2(40xs), have a cryophilic phenotype (worms migrate to a colder temperature than their cultivation temperature). Proteomic analysis of total protein from
pkc-2(
ok328) and (
pkc-2(40xs) worms using 2-D difference gel electrophoresis (DIGE) revealed proteins with altered mobility's, indicating different levels of phosphorylation or other post-translational modifications. A 20 kDa protein that carried increased negative charge (consistent with increased phosphorylation) in
pkc-2(40x) worms was identified as PRDX-2. PRDX-2 is a 2-Cys peroxiredoxin that catalyzes peroxide reduction to detoxify reactive oxygen species and diminish peroxide generated by metabolism and actions of growth factors and cytokines. A PRDX-2 deletion mutant,
prdx-2(
gk169), is also AT and suppresses the cryophilic phenotype of
pkc-2(40x) animals. This suppressor of elevated PKC-2 activity could be a substrate. Purified recombinant PRDX-2 was phosphorylated to high stoichiometry by PKC in vitro. We are identifying phosphorylated Ser/Thr in PRDX-2 and will express phospho-mimetic and non-phosphorylatable PRDX-2 mutants in vivo to reveal their physiological relevance.
prdx-2(
gk169),nematodes are sensitive to peroxide treatment and have a shorter life span compared to that of wild type (WT) worms (Monika Olahova et al., P.N.A.S.,105, 19839 (2008)). We have examined the life span of PKC-2 nulls and found they also have a reduced life span and in addition are sensitive to peroxide treatment.
pkc-2(
ok328) have a shorter life span, compared to WT nematodes, that is similar to the life span of
prdx-2(
gk179). In addition
pkc-2(
ok328) are more sensitive to hydrogen peroxide, compared to WT worms, that is similar to the sensitivity for peroxide treatment of
prdx-2(
gk179). The involvement of PKC-2 in the aging response will be defined. The longevity and stress response of PKC-2 over expressing and
pkc-2(
ok328);
prdx-2(
gk179) doubly mutant worms is being analyzed.