A single C.elegans gene,
pkc-2, encodes Ca2+ & diacylglycerol (DAG) activated protein kinase C. PKC-2 null animals are athermotactic and unable to respond to chemoattractants that stimulate receptors in AWA and AWC neurons. We are using a combination of proteomics and molecular genetics to characterize upstream activators, substrates and downstream effectors of PKC-2. Total protein was isolated from PKC-2 null animals and transgenic C. elegans expressing PKC-2 at ~40X the wild type level (designated 40X PKC-2). Protein samples from L4 animals were labeled with Cy3 or Cy5 dyes, mixed and analyzed by 2-D difference gel electrophoresis (DIGE). Fluorescent proteins were mapped and quantified using DeCyder software. In gel comparison of proteins from 40X PKC-2 and null worms revealed several proteins with shifted mobilities, indicating alterations in phosphorylation or other post-translational modifications. Changes in protein isoelectric point or molecular weight may be caused by direct, PKC-2-catalyzed phosphorylation or PKC-2 mediated regulation of intermediary signaling proteins. Proteins excised from the 2D-gel were trypsinized and analyzed by mass spectrometry. An initial set of 3 proteins that were shifted in 40X PKC-2 animals was characterized. Peroxiredoxin (
prdx-2), translationally controlled tumor protein (
tct-1) and elongation factor (EF)-1 beta/delta 2 (Y41E3.10) were identified as potential PKC-2 effectors. EF-1 beta/delta is a guanine nucleotide exchange factor that regulates the binding of aminoacyl tRNA to the ribosomes. Proteins involved in regulation of translation are often involved in learning and the creation of memory. Translationally controlled tumor proteins are involved in Ca2+binding, apoptosis and microtubule stabilization. A
tct-1(tm 2743) deletion mutant is lethal. Peroxiredoxins (PRDXs) detoxify reactive oxygen species and regulate peroxide-mediated signaling cascades. A deletion mutant in
prdx-2(
gk169) displays an athermotactic phenotype (animals wander randomly over a temperature gradient instead of locating their cultivation temperature) that closely parallels the behavior of PKC-2 null worms.
prdx-2(
gk169) is being tested for its ability to suppress the cryophilic phenotype of 40X PKC-2 nematodes. The potential link between a Ca2+, DAG-regulated PKC (PKC-2), a Ca2+-binding protein, a mediator of stress signaling (PRDX-2) and a specific physiological output (thermotaxis) indicates that the proteomics-molecular genetics approach is a promising route for elucidating in vivo regulation and functions of signaling proteins. DIGE analysis has also been extended to phorbol ester (mimics DAG) treated worms to increase the probability of identifying direct PKC2 targets.