Worm Breeder's Gazette 14(1): 30 (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.

Protein-protein interaction between FEM-3 and FEM-2, a Mg2+-Dependent Protein Phosphatase

Ian Chin Sang, Andrew Spence

Dept. of Medical Genetics, University of Toronto, 1 King's College Circle,

Toronto, Canada

      Male development in C. elegans requires the activities of the three
fem genes.  Current models of sex determination suggest that in X0
animals, they promote male cell fates in somatic tissues by negatively
regulating tra-1, and that in XX animals, they are prevented from
interfering with tra-1 activity by the transmembrane protein encoded by
the tra-2 locus, TRA-2A.  We presented evidence at the last C. elegans
meeting that a direct interaction between the C-terminal domain of TRA-2A
and FEM-3 is important for negatively regulating the masculinizing
activity of the fem genes. [See abstract by Spence et al.]  On the
hypothesis that when liberated from the influence of TRA-2A, FEM-3 engages
in new specific interactions to bring about male development, we used the
yeast two-hybrid system to screen for other proteins that interact with
      We used a "bait" protein consisting of FEM-3 fused to the DNA-
binding domain of GAL4 to screen a library of C. elegans cDNAs fused to
the GAL4 transcriptional activation domain.  The library was constructed
and generously provided by R. Barstead, and the fem-3 cDNA was a gift from
J. Kimble.  A screen of 2.3 million yeast transformants yielded three
cDNAs encoding proteins which interacted specifically with the FEM-3 bait.
One encoded a C-terminal fragment of TRA-2A, as expected from our earlier
observations.  The other two mapped to the immediate vicinity of fem-2 on
the genomic physical map.  Expression of the larger of the two from the
heat-shock promoter partially rescued male somatic development in X0
animals homozygous for the putative null mutation fem-2(e2105).  Both
cDNAs can encode a member of the Type 2C protein serine/threonine
phosphatase family identical in sequence to that predicted from the
sequence of fem-2, and provided before publication, by D. Pilgrim and
      Coimmunoprecipitation of FEM-2 and myc epitope-tagged FEM-3
following their synthesis in rabbit reticulocyte lysates confirmed their
ability to associate.  Bacterially expressed GST-FEM-2 fusion protein was
also able to bind radiolabelled FEM-3 from in vitro translation reactions
in batch affinity experiments.  We have used the latter assay in a
deletion analysis to locate the region of FEM-2 responsible for binding
FEM-3, and our results to date suggest that a region of about 190 amino
acids near the N-terminus contains sequences required for FEM-3 binding.
      Bacterially expressed GST-FEM-2 exhibits phosphatase activity in
vitro with 32P-labelled casein as substrate.  The activity is completely
magnesium-dependent, confirming that FEM-2 is a Type 2C protein
phosphatase.  A report that other Type 2C phosphatases require Mg2+ for
substrate binding led us to test the magnesium dependence of the
interaction between FEM-2 and FEM-3.  The interaction does not require
magnesium, but we cannot exclude the possibility that FEM-3 may
nevertheless be a substrate for the FEM-2 phosphatase.  We have made
several point mutations which alter conserved residues in FEM-2 and
abolish its phosphatase activity in vitro without affecting its ability to
bind FEM-3.  We are currently testing alleles carrying these mutations for
their ability to rescue the phenotype of fem-2 mutants.
      We propose that a direct interaction between FEM-3 and FEM-2 protein
phosphatase is required for male development in C. elegans.  We cannot
discriminate among several plausible hypotheses concerning the biochemical
consequences of the interaction.  FEM-3 may become the target of an
(unknown) inhibitory kinase upon binding to TRA-2A and require
dephosphorylation by FEM-2 for its activation.  Alternatively, FEM-3 may
regulate the localization, activity or substrate specificity of the FEM-2