We found that TTX-4, a homolog of nPKC ("novel" PKC)-epsilon, is essential for sensation of environmental signals such as temperature, volatile and soluble compounds, and osmolarity (Okochi et al., this meeting). Biochemical analyses mostly based on mammalian cell cultures have revealed that nPKC species are activated only by diacylglycerol (DAG) or other lipids such as phosphatidylinositol-3,4,5-trisphosphate (PIP 3 ) or phosphatidylserine without requirement of calcium ion which is necessary for cPKC ("conventional" PKC) activation. Little evidence has been reported, though, to prove in vivo activation of nPKC by any of those lipids. Moreover, in vivo substrates of nPKC, whose activities are regulated by nPKC-dependent phosphorylation and required for transducing specific signal from nPKC, are poorly understood. To address these questions, we are undertaking reverse and molecular genetic approaches to identify upstream and downstream molecules of the TTX-4 nPKC-epsilon. We identified C. elegans homologs of candidate genes for nPKC pathway from the genome database, and have been making GFP reporter constructs of those genes. If the candidates are expressed in the same cells as those expressing ttx-4
, we will screen TMP/UV library for knockout mutants (Inada et al., this meeting). (I) Upstream candidates: DAG is a strong candidate molecule as an activator of TTX-4. DAG can be generated through hydrolysis of phosphatidylinositol-4,5-bisphosphate (PIP 2 ) by phospholipase C (PLC) or through dephosphorylation of phosphatidic acid (PA) by PA phosphatase (PAP). Thus, those enzymes are candidates for upstream gene products of TTX-4. We are currently analyzing expression pattern of these genes. (II) Downstream candidates and binding proteins: Several proteins have been reported to be phosphorylated by nPKCs in mammals. Biological significance of those phosphorylations however remain questionable. We are currently analyzing a couple of homologs for these proteins, beta'-COP (F38E11.5) and adducin (F57F5.4). Interestingly, the adducin (F57F5.4) is located adjacent to ttx-4
(F57F5.5) on the physical map. We are also screening yeast two hybrid library using wild type or kinase negative TTX-4 catalytic domain as a bait.