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.

Protein Kinase C of C. elegans

Toshihiro Sassa and Johji Miwa

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 
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.