Worm Breeder's Gazette 11(4): 49
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
Ca++-activated phospholipid dependent protein kinase (protein kinase C; PKC), discovered by Y. Nishizuka, is a serine/threonine kinase and plays a crucial role in cellular signal transduction. This enzyme is activated by diacylglycerol, a product of receptor stimulated inositol lipid breakdown. Also, it is activated by tumor promoters, such as 12- 0-tetradecanoylphorbol-13-acetate (TPA). The action of TPA is pleiotropic both in vivo and in vitro. Miwa and Tabuse found that the nematode stops growing and displays strongly disturbed undulatory movement when cultured in the presence of TPA. Genetic analysis of mutants that grow and move undisturbed in the presence of TPA identified the gene tpa-1, which is apparently involved in the TPA action on the nematode. Molecular cloning analysis of the tpa-1 gene has shown that its DNA sequence is highly homologous to those of mammalian PKCs. However, it has not yet been tested whether PKC molecules exist in the nematode. Since we have now detected the PKC activity in the nematode, we describe some of the nematode PKC properties. PKC activity was measured by incorporation of [32P] into histone III- S from gamma-[32P] ATP, and [3H]-PDBu binding was measured by the cold acetone filtration method. After grinding the nematode under liquid nitrogen, the homogenate was centrifuged at 100,000 X g for 60 min. The supernatant was applied to a DEAE cellulose column and eluted by a 0.025 to 1.0M linear NaCl gradient. We observed a single peak of protein kinase activity, which was enhanced in the presence of phosphatidylserine, Ca++ ion, and TPA. Also, this peak produced the highest [3H]-PDBu binding activity, providing further evidence that it contained PKC. We purified the fractions of this peak to near homogeneity by two sequential column chromatographies, polylysine agarose column and phosphatidylserine affinity column. SDS- polyacrylamide gel electrophoresis showed a single band corresponding to the molecular mass of 79,000, and the specific activity was 1.8 micro mole/min/mg, similar to those of mammalian PKCs. We have yet to know whether this PKC is the same as the tpa-1 gene product.