Worm Breeder's Gazette 14(1): 31 (October 1, 1995)
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.
Department of Biological Sciences, University of Alberta, Edmonton, Alberta
As discussed previously, sequencing of fem-2 has revealed its similarity to protein phosphatases type 2C (PP2C) from various different organisms (WBG 13 no. 4, p.44, Pilgrim et al., in press) This suggested that FEM-2's role in determining sex is carried out, at least in part, through dephosphorylation. The regions of homology between FEM-2 and the other protein phosphatases are limited to only six motifs in the central portion of the predicted protein. It is formally possible that the regions of homology do not all correspond to domains specifically responsible for phosphatase activity, but also for some other function such as regularoty protein binding. In order to test this we isolated FEM-2 protein fused to GST (Glutathione S-transferase) and tested it in vitro for phosphatase activity. We used 32P labeled casein as a substrate which is commonly used to measure the phosphatase activity of PP2C enzymes. GST-FEM-2 demonstrated significant phosphatase activity as shown by a high level of released 32P as compared to negative controls. This indicates not only that FEM-2 has phosphatase activity but also that, at least in the conditions used, FEM-2 does not require any other C. elegans proteins for activity. PTC1 is a FEM-2 homologue from S. cerevisiae that has previously been shown to have phosphatase activity under the conditions that are characteristic of PP2C enzymes (Maeda et al.,1993). The predicted proteins are homologous in the six motifs described previously yet differ greatly in other aspects of their primary structure such as the much larger amino terminus in FEM-2 (see below). Yeast strains that contain a null mutation of ptc1 have no obvious phenotype when grown at 30 degrees but grow very poorly at 37 degrees Constitutive expression of full length fem-2 cDNA using an ADH1 promoter in a ptc1 mutant yeast strain is able to rescue the temperature sensitive phenotype. This further suggests that FEM-2 does in fact have phosphatase activity and that it is quite conserved since it is able to functionally replace the yeast homologue. Since PTC1 is lacking the long amino terminus that is present in FEM-2, this could suggest that its function is not directly involved in dephosphorylation but rather some other function such as specifying the target of dephosphorylation or the binding of a protein involved in regulation of the activity. The important next step is to determine the actual target of FEM-2 phosphatase activity and how this is responsible for FEM-2¹s role in sex determination.