Male development in C. elegans requires the activities of the fem genes,
fem-1,
fem-2, and
fem-3. The prevailing model of sex determination suggests that in the somatic tissues of XO animals, the FEM proteins negatively regulate TRA-1A, a sequence-specific DNA-binding protein that would otherwise cause female development. In the germline of both XX and XO animals, the FEM proteins regulate other targets to promote spermatogenesis. Greater understanding of the mechanism of sex determination requires knowledge of the interactions in which the FEM proteins engage and the regulatory mechanisms that they employ. The sequence of FEM-3 is unique and therefore offers no clues as to its biochemical activity. To address the question of how FEM-3 fulfills its role in sex determination, we employed the yeast two-hybrid system to screen for clones encoding FEM-3 - interacting proteins. Here we report the isolation of
fem-2 cDNA clones on the basis of an interaction between FEM-2 and FEM-3. We used two assays to demonstrate that FEM-2 and FEM-3 associate in vitro. In the first, we used a monoclonal antibody against an epitope tag in FEM-3 to coimmunoprecipitate both proteins from in vitro translation reactions. In the second, we showed that glutathione Sepharose beads coated with bacterially expressed GST-FEM-2 specifically bound radiolabelled FEM-3 produced by in vitro translation in reticulocyte lysates. GST-coated beads did not bind FEM-3. Deletion analysis of FEM-2 suggests that a region of about 190 amino acids contains sequences required for FEM-3 binding. As reported by D. Pilgrim and colleagues (1), FEM-2 is similar in sequence to protein serine/threonine phosphatases of Type 2C (PP2C). We showed that GST-FEM-2 indeed exhibits casein phosphatase activity in vitro, and that its activity is magnesium-dependent, as is typical of PP2C. Substitution of a highly conserved arginine residue abolishes the casein phosphatase activity of FEM-2 but does not affect its FEM-3 - binding activity, suggesting that the mutant protein is not grossly abnormal in structure. When expressed from the heat shock promoter in transgenic
fem-2 m-z- XO animals, FEM-2 lacking casein phosphatase activity is much less active in rescuing male development than wild-type FEM-2. The mutant and wild-type proteins exhibit similar levels of expression and stability. Our observations suggest that FEM-2 must be an active protein phosphatase to fulfill its normal role in sex determination. We conclude that the mechanism of sex determination in C. elegans involves protein phosphorylation, and that FEM-2 antagonizes the feminizing effect of a kinase that remains to be identified. We propose that interaction between FEM-2 and FEM-3 is necessary for normal male development, and we are currently testing the requirement for the interaction. Several possibilities exist as to the functional consequences of the FEM-2-FEM-3 interaction. For example, FEM-3 might regulate the localization or substrate specificity of FEM-2. (1) Pilgrim, D., et al. (1995). Mol. Biol. Cell 6: 1159-1171.