Worm Breeder's Gazette 10(2): 33

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.

Paramyosin Phosphorylation

Lawrence A. Schriefer and Robert H. Waterston

Paramyosin from C.  elegans, although the product of a single gene, 
displays several species on isoelectric focusing gels.  In unc-82(
e1220) and unc-82(1323) mutants the most acidic species are missing 
with increased amounts in the more basic spots.  Because the unc-82 
mutations exhibit unanticipated interactions with unc-15 alleles (
Waterston, et al.  1980) and because unc-82 mutants contain paramyosin 
aggregates, we were particularly intrigued when, in the process of 
looking for the target of the unc-22 kinase, we found evidence for 
paramyosin phosphorylation (not by unc-22 however).
Our in vitro method for phosphorylation studies uses the insoluble 
pellet derived from extensive washing of French press broken worms 
with low salt buffer.  Under these conditions, thick filaments are 
broken, but their components remain insoluble, and short range protein-
protein associations should be maintained.  The labelling of 
paramyosin in these pellets implies that the kinase activity is also 
insoluble in low salt, and could even be part of the thick filaments.
To test whether these in vitro findings have any relevance to the in 
vivo situation, we purified paramyosin from worms without using 
phosphates in the buffers and we took precautions against phosphate 
contamination.  Our initial phosphate assays of pure paramyosin 
indicates there are 1-2 moles of phosphate per mole of paramyosin, 
suggesting there may be more than one site of phosphorylation.
To learn more about the possible site(s) of phosphorylation we have 
pursued two lines of exploration.  In vitro labeled paramyosin was 
acid hydrolyzed and amino acids analyzed by TLC.  Label from the 
paramyosin migrated with phosphoserine, not phosphothreonine or 
phosphotyrosine.  We have also begun to localize the labelled serines 
within paramyosin.  NTCB cleaves at cysteine residues and yields 3 
fragments from paramyosin.  The smallest fragment of 15,000 Da is 
labelled and Hiro Kagawa's unpublished sequencing data indicates that 
this includes the N-terminus of paramyosin.  CNBr digestions also 
yield a labelled fragment of a size consistent with an origin from the 
N-terminus.  Unfortunately, similar sized peptides can arise from 
elsewhere in the polypeptide.  To localize the site(s) more precisely, 
we have digested labelled paramyosin with endoproteinase-Lys-C (ELC).  
ELC cuts at lysine and should yield 68 fragments.  We are currently 
analyzing 2 labelled HPLC fractions.
The in vitro significance of paramyosin phosphorylation is unclear.  
Paramyosin phosphorylation has also been observed in vitro in 
molluscan muscle.  Hopefully, analysis of unc-82 mutants or other 
muscle mutants might reveal the function of this phenomenon.