Worm Breeder's Gazette 11(4): 37

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.

Programmed Cell Death May Be Regulated by Phosphorylation

Junying Yuan, Nancy Tsung and Bob Horvitz

The genes ced-3 and ced-4 are essential for the 131 programmed cell 
deaths that occur during normal C.  elegans development.  Mosaic 
analysis has shown that the actions of both ced-3 and ced-4 are cell 
autonomous.  We have previously cloned and sequenced ced-4, and found 
that this gene encodes a protein with two possible calcium-binding 
domains.  We have now initiated molecular studies of ced-3.We 
identified a Bristol-specific Tc1 polymorphism closely linked to ced-3,
cloned this polymorphism and, in collaboration with John Sulston and 
Alan Coulson, defined a 200 kb contig.  By mapping RFLPs between the 
Bristol and Bergerac strains, we localized ced-3 to a region of about 
2-3 cosmids.  We microinjected cosmids from this region along with the 
unc-31(+) cosmid C14G10 into ced-1; ced-3 animals 
and observed rescue of the ced-3 phenotype using cosmid C48D1.  We 
identified a 7 kb subclone of C48D1 with ced-3(+) activity and showed 
that a slightly smaller 6 kb subclone lacks this activity.  This 7 kb 
clone detects a 2.8 kb RNA on a Northern blot.  This transcript is 
expressed primarily during embryogenesis, consistent with the 
observation that most programmed cell deaths are embryonic.  This 
transcript is very abundant, with a level in eggs comparable to that 
of actin I RNA.  The level of this transcript is not altered in eggs 
derived from ced-4 animals.
We have isolated a number of cDNAs that correspond to this 
transcript, and have sequenced one strand of a 2.5 kb cDNA, pJ87.  
This cDNA contains a single long open reading frame that can be 
translated into a protein of 518 amino acids.  However, the 5' region 
of this cDNA consists of a sequence of 24 consecutive T's, which most 
likely is a cloning artifact.  A search of the Genbank protein 
sequence database (release 63.0) revealed no sequence highly similar 
to that of ced-3.  However, the region near the amino terminus of the 
putative ced-3 protein is very striking: it has 34 serines within a 
stretch of 120 amino acids.  Serines are common targets for serine and 
threonine protein kinases.  Furthermore, many of these serines are 
flanked by basic residues, as are a variety of the phosphorylation 
sites of protein kinase C and cAMP-dependent protein kinases.  We have 
made peptides corresponding to three serine-rich regions of ced-3.  
Preliminary results indicated that at least one of these peptides can 
be phosphorylated by cAMP-dependent protein kinase.  The high level of 
expression of the ced-3 transcript suggests that ced-3 expression 
might not be specific to dying cells.  We suggest that ced-3 function 
might be regulated by phosphorylation, with either phosphorylation or 
dephosphorylation activating ced-3 and consequently causing cell death.