The Caenorhabditis elegansSex-determining Protein FEM-2 and Its Human Homologue, hFEM-2, Are Ca2+/Calmodulin-dependent Protein Kinase Phosphatases That Promote Apoptosis

Abstract

In Caenorhabditis elegans, fem-1, fem-2, and fem-3 play pivotal roles in sex determination. Recently, a mammalian homologue of the C. elegans sex-determining protein FEM-1, F1A␣, has been described. Although there is little evidence to link F1A␣ to sex determination, F1A␣ and FEM-1 both promote apoptosis in mammalian cells. Here we report the identification and characterization of a human homologue of the C. elegans sex-determining protein FEM-2, hFEM-2. Similar to FEM-2, hFEM-2 exhibited PP2C phosphatase activity and associated with FEM-3. hFEM-2 shows striking similarity (79% amino acid identity) to rat Ca 2؉ /calmodulin (CaM)-dependent protein kinase phosphatase (rCaMKPase). hFEM-2 and FEM-2, but not PP2C␣, were demonstrated to dephosphorylate CaM kinase II efficiently in vitro, suggesting that hFEM-2 and FEM-2 are specific phosphatases for CaM kinase. Furthermore, hFEM-2 and FEM-2 associated with F1A␣ and FEM-1 respectively. Overexpression of hFEM-2, FEM-2, or rCaMKPase all mediated apoptosis in mammalian cells. The catalytically active, but not the inactive, forms of hFEM-2 induced caspase-dependent apoptosis, which was blocked by Bcl-XL or a dominant negative mutant of caspase-9. Taken together, our data suggest that hFEM-2 and rCaMKPase are mammalian homologues of FEM-2 and they are evolutionarily conserved CaM kinase phosphatases that may have a role in apoptosis signaling.

Regulation of the Multifunctional Ca2+/Calmodulin-dependent Protein Kinase II by the PP2C Phosphatase PPM1F in Fibroblasts

“…The N-terminal region of PPM1F (amino acids 1-145) has been suggested to be an inhibitory domain of hFEM-2 (30). Contrary to the results reported for hFEM-2, a deletion made to the N terminus of PPM1F, such as that generated in the ⌬1-148 mutant, resulted in a catalytically inactive protein even though stable protein was made.…”

“…Because this mutant was able to bind CaMKII, the deletion of this amino acid sequence did not appear to fully disrupt the protein structure. Our result with the N-terminal deletion mutant implies that the 157-454 mutant of hFEM-2 is most likely an inactive phosphatase mutant; therefore, the apoptotic effect associated with its overexpression is not a result of its catalytic function as proposed by Tan et al (30).…”

“…Since then, two groups of investigators have independently isolated the same cDNA from different sources and have named it hFEM-2 (30) and POPX2 (31), depending on its identified function. Both groups recognized what we had found earlier during the course of our investigation, 2 the human sequence shares 80% identity with the rat Ca 2ϩ /calmodulin-dependent protein kinase phosphatase (rCaMKPase) (32); however, neither group fully investigated this possible function of the gene, other than to demonstrate that a partially purified hFEM-2 could dephosphorylate CaMKII in vitro (30). The rCaMKPase has characteristics similar to members of the PP2C family, including a dependence on divalent cations for activity and insensitivity to okadaic acid (OA), and has been shown to dephosphorylate as well as deactivate autophosphorylated CaMKII in vitro (33).…”

“…By taking this into consideration, an N-terminal deletion mutant Myc⅐PPM1F⌬1-148 was made by using a convenient EcoRI restriction site. This mutant corresponds to a similar deletion made in hFEM-2 that was shown to enhance the apoptotic effect associated with its overexpression in HeLa cells (30). C-terminal deletion mutants Myc⅐PPM1F⌬419 -454 and Myc⅐PPM1F⌬422-454 were generated by PCR cloning and were included in this analysis based on the absence or presence of the phosphatase activity of these two proteins, respectively, toward 32 P-AutoCaMtide3 (see Table I).…”

p21-Activated Kinase 2 in Neutrophils Can Be Regulated by Phosphorylation at Multiple Sites and by a Variety of Protein Phosphatases

“…7). FEM-2 is a possible candidate for this Mg 2ϩ /Mn 2ϩ -dependent phosphatase because it is activated by Mg 2ϩ , catalyzes dephosphorylation of the AL of Pak, is partially inhibited by F Ϫ and insensitive to orthorvanadate and calyculin A (29,36,39). Chronic activation of Pak can promote viral replication, metastasis, and cellular transformation (e.g., Refs.…”