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.

FEM-2 Shows Phosphatase Activity in vitro and in vivo

Dave Hansen, Dave Pilgrim

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.