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

2001; Elsevier BV; Volume: 276; Issue: 47 Linguagem: Inglês

10.1074/jbc.m105880200

ISSN

1083-351X

Autores

Karen Tan, Shing Leng Chan, Kuan Onn Tan, Victor C. Yu,

Tópico(s)

Ubiquitin and proteasome pathways

Resumo

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, F1Aα, has been described. Although there is little evidence to link F1Aα to sex determination, F1Aα 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 Ca2+/calmodulin (CaM)-dependent protein kinase phosphatase (rCaMKPase). hFEM-2 and FEM-2, but not PP2Cα, 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 F1Aα 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. 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, F1Aα, has been described. Although there is little evidence to link F1Aα to sex determination, F1Aα 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 Ca2+/calmodulin (CaM)-dependent protein kinase phosphatase (rCaMKPase). hFEM-2 and FEM-2, but not PP2Cα, 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 F1Aα 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. programmed cell death polymerase chain reaction glutathione S-transferase open reading frame Ca2+/calmodulin rat Ca2+/calmodulin-dependent kinase phosphatase Ca2+/calmodulin-dependent kinase II enhanced green fluorescent protein poly(ADP-ribose) polymerase polyacrylamide gel electrophoresis bovine serum albumin cytomegalovirus hemagglutinin phosphate-buffered saline fluorescein isothiocyanate phenylmethylsulfonyl fluoride Apoptosis, or programmed cell death (PCD),1 plays important roles in tissue homeostasis and development in essentially all multicellular organisms (1Metzstein M.M. Stanfield G.M. Horvitz H.R. Trends Genet. 1998; 14: 410-416Abstract Full Text Full Text PDF PubMed Scopus (389) Google Scholar, 2Vaux D.L. Korsmeyer S.J. Cell. 1999; 96: 245-254Abstract Full Text Full Text PDF PubMed Scopus (1361) Google Scholar). Genetic analyses of the PCD pathway in Caenorhabditis elegans have successfully identified key regulatory genes that define the core machinery of cell death. Orthologues of the apoptosis genes of C. elegans have subsequently been identified in other organisms including mammals (3Hengartner M.O. Nature. 1998; 391: 440-442Crossref Scopus (111) Google Scholar,4Horvitz H.R. Cancer Res. 1999; 59: 1701s-1706sPubMed Google Scholar), suggesting that the molecular strategies that regulate the fundamental aspects of this important biological process are likely to be conserved across species. The molecular strategies that are involved in sex determination among species, however, are thought to be quite diverse (5Schafer A.J. Goodfellow P.N. Bioessays. 1996; 18: 955-963Crossref PubMed Scopus (48) Google Scholar, 6Marin I. Baker B.S. Science. 1998; 281: 1990-1994Crossref PubMed Scopus (244) Google Scholar). Indeed, genes in the sex-determining pathway are known to be rapidly diverged even between two closely related species, C. elegans and Caenorhabditis briggsae (7Hansen D. Pilgrim D. Genetics. 1998; 149: 1353-1362Crossref PubMed Google Scholar, 8Hansen D. Pilgrim D. Mech. Dev. 1999; 83: 3-15Crossref PubMed Scopus (21) Google Scholar). In C. elegans, many genes have been identified that affect sexual fate. One of the key steps in the sex determination pathway in C. elegans is regulated by three fem genes, fem-1,fem-2, and fem-3 (8Hansen D. Pilgrim D. Mech. Dev. 1999; 83: 3-15Crossref PubMed Scopus (21) Google Scholar, 9Kimble J. Edgar L. Hirsh D. Dev. Biol. 1984; 105: 234-239Crossref PubMed Scopus (102) Google Scholar). Loss-of-function mutations in any one of the fem genes prevent all aspects of male development and transform both males and hermaphrodites into females (9Kimble J. Edgar L. Hirsh D. Dev. Biol. 1984; 105: 234-239Crossref PubMed Scopus (102) Google Scholar, 10Doniach T. Hodgkin J. Dev. Biol. 1984; 106: 223-235Crossref PubMed Scopus (143) Google Scholar). fem-1 encodes a protein with ankyrin repeats (11Spence A.M. Coulson A. Hodgkin J. Cell. 1990; 60: 981-990Abstract Full Text PDF PubMed Scopus (132) Google Scholar), and fem-2 encodes a serine/threonine protein phosphatase of the PP2C type (12Pilgrim D. McGregor A. Jackle P. Johnson T. Hansen D. Mol. Biol. Cell. 1995; 6: 1159-1171Crossref PubMed Scopus (64) Google Scholar), which interacts directly with FEM-3 (13Chin-Sang I.D. Spence A.M. Genes Dev. 1996; 10: 2314-2325Crossref PubMed Scopus (71) Google Scholar, 14Ahringer J. Rosenquist T.A. Lawson D.N. Kimble J. EMBO J. 1992; 11: 2303-2310Crossref PubMed Scopus (81) Google Scholar), a protein without any recognizable functional motif. F1Aα has recently been identified as a mammalian Fas death domain-interacting protein in a yeast two-hybrid screen (15Chan S.L. Tan K.O. Zhang L. Yee K.S. Ronca F. Chan M.Y. Yu V.C. J. Biol. Chem. 1999; 274: 32461-32468Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Overexpression of F1Aα induces apoptosis in mammalian cells, and F1Aα is a caspase substrate (15Chan S.L. Tan K.O. Zhang L. Yee K.S. Ronca F. Chan M.Y. Yu V.C. J. Biol. Chem. 1999; 274: 32461-32468Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Interestingly, F1Aα was found to be highly homologous throughout its entire protein sequence, to the C. elegans sex determination protein FEM-1 (15Chan S.L. Tan K.O. Zhang L. Yee K.S. Ronca F. Chan M.Y. Yu V.C. J. Biol. Chem. 1999; 274: 32461-32468Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 16Chan S.L. Yee K.S. Tan K.M. Yu V.C. J. Biol. Chem. 2000; 275: 17925-17928Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). The degree of similarity between FEM-1 and F1Aα (30% amino acid identity) is comparable with that between several of the functionally conserved components of PCD in nematodes and mammals, e.g.the amino acid identity between CED-9 and Bcl-2 is 23% (17Hengartner M.O. Horvitz H.R. Cell. 1994; 76: 665-676Abstract Full Text PDF PubMed Scopus (1044) Google Scholar), and that between CED-3 and caspase-3 is 34% (18Xue D. Shaham S. Horvitz H.R. Genes Dev. 1996; 10: 1073-1083Crossref PubMed Scopus (280) Google Scholar). Despite their exclusive roles in masculinizing somatic tissues in males and regulating the production of male germ cells (8Hansen D. Pilgrim D. Mech. Dev. 1999; 83: 3-15Crossref PubMed Scopus (21) Google Scholar, 9Kimble J. Edgar L. Hirsh D. Dev. Biol. 1984; 105: 234-239Crossref PubMed Scopus (102) Google Scholar), FEM-1 and FEM-2 proteins are expressed throughout development in all somatic tissues at equivalent levels in both sexes (12Pilgrim D. McGregor A. Jackle P. Johnson T. Hansen D. Mol. Biol. Cell. 1995; 6: 1159-1171Crossref PubMed Scopus (64) Google Scholar, 19Gaudet J. VanderElst I. Spence A.M. Mol. Biol. Cell. 1996; 7: 1107-1121Crossref PubMed Scopus (43) Google Scholar). This observation raises the question as to whether these proteins would have additional functions other than sex determination. Although there is no evidence to link F1Aα to sex determination function, FEM-1 and its mammalian homologue, F1Aα, were found to induce apoptosis when overexpressed in mammalian cells (15Chan S.L. Tan K.O. Zhang L. Yee K.S. Ronca F. Chan M.Y. Yu V.C. J. Biol. Chem. 1999; 274: 32461-32468Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 16Chan S.L. Yee K.S. Tan K.M. Yu V.C. J. Biol. Chem. 2000; 275: 17925-17928Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). In vitro experiments demonstrated that FEM-1 is cleaved by the C. elegans caspase, CED-3, demonstrating a striking parallel to its mammalian homologue, F1Aα (16Chan S.L. Yee K.S. Tan K.M. Yu V.C. J. Biol. Chem. 2000; 275: 17925-17928Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). It is possible that fem-1, in contrast to many other sex-determining genes in C. elegans, was conserved during evolution because of its function in apoptosis. The functional conservation between FEM-1 and F1Aα in mediating apoptosis in mammalian cells raises an intriguing possibility that perhaps not only FEM-1 but a subset of genes in the C. elegans sex determination pathway may also be conserved if they have a role to play in apoptosis signaling. To test our hypothesis, we initiated a search for putative mammalian homologues of fem genes. A search in the GenBank™ data base revealed a human cDNA that encodes a full-length protein, which we named hFEM-2, that exhibits extensive amino acid sequence similarity to FEM-2 (28% amino acid identity). hFEM-2 exhibited enzymatic characteristics of the PP2C type similar to those for FEM-2. Like FEM-2, hFEM-2 associates with FEM-3, whereas a related PP2C phosphatase, PP2Cα (20Mann D.J. Campbell D.G. McGowan C.H. Cohen P.T. Biochim. Biophys. Acta. 1992; 1130: 100-104Crossref PubMed Scopus (60) Google Scholar), does not. The putative human homologue of fem-2 shares 79% amino acid identity with the rat Ca2+/calmodulin-dependent protein kinase phosphatase (rCaMKPase) that was recently cloned from rat brain (21Kitani T. Ishida A. Okuno S. Takeuchi M. Kameshita I. Fujisawa H. J Biochem. (Tokyo). 1999; 125: 1022-1028Crossref PubMed Scopus (51) Google Scholar). CaM-dependent protein kinases are multifunctional protein kinases that control a variety of cellular functions including apoptosis (22Nairn A.C. Picciotto M.R. Semin. Cancer Biol. 1994; 5: 295-303Crossref PubMed Scopus (21) Google Scholar, 23Fujisawa H. J. Biochem. (Tokyo). 2001; 129: 193-199Crossref PubMed Scopus (70) Google Scholar, 24Soderling T.R. Curr. Opin. Neurobiol. 2000; 10: 375-380Crossref PubMed Scopus (201) Google Scholar, 25Soderling T.R. Trends Biochem. 1999; 24: 232-237Abstract Full Text Full Text PDF PubMed Scopus (441) Google Scholar, 26Cohen O. Feinstein E. Kimchi A. EMBO J. 1997; 16: 998-1008Crossref PubMed Scopus (377) Google Scholar, 27Wright S.C. Schellenberger U. Ji L. Wang H. Larrick J.W. FASEB J. 1997; 11: 843-849Crossref PubMed Scopus (81) Google Scholar). CaM kinase II is activated through autophosphorylation, whereas CaM kinase I and IV are activated through phosphorylation by upstream Ca2+/calmodulin-dependent protein kinase kinases (23Fujisawa H. J. Biochem. (Tokyo). 2001; 129: 193-199Crossref PubMed Scopus (70) Google Scholar). CaM kinase phosphatase was initially purified from rat brain using a synthetic peptide corresponding to the autophosphorylation site of CaM kinase II (28Ishida A. Kameshita I. Fujisawa H. J. Biol. Chem. 1998; 273: 1904-1910Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). The rCaMKPase dephosphorylated and deactivated CaM kinase II activated by autophosphorylation (28Ishida A. Kameshita I. Fujisawa H. J. Biol. Chem. 1998; 273: 1904-1910Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar), and was later shown to be able to dephosphorylate CaM kinases I and IV activated upon phosphorylation by CaM kinase kinases (28Ishida A. Kameshita I. Fujisawa H. J. Biol. Chem. 1998; 273: 1904-1910Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 29Ishida A. Okuno S. Kitani T. Kameshita I. Fujisawa H. Biochem. Biophys. Res. Commun. 1998; 253: 159-163Crossref PubMed Scopus (39) Google Scholar). We show that FEM-2, hFEM-2, and rCaMKPase mediate caspase-dependent cell death when overexpressed in mammalian cells. We also demonstrate that FEM-2 and its human homologue can dephosphorylate autophosphorylated CaM kinase II efficiently in vitro. Hence, FEM-2 and its mammalian homologues are evolutionarily conserved CaM kinase phosphatases that may play a role in apoptosis signaling. Mono- and polyclonal antibodies against the Myc epitope (9E10, A14), monoclonal antibody against the HA epitope (F7), and polyclonal antibody against poly(ADP-ribose) polymerase (PARP) (A20) were obtained from Santa Cruz Biotechnology. 293T human embryo kidney cells, NIH3T3 fibroblast cells, and HeLa cells were originally from the American Type Culture Collection (ATCC). tumor necrosis factor-sensitive MCF7 breast carcinoma cells were maintained as described previously (15Chan S.L. Tan K.O. Zhang L. Yee K.S. Ronca F. Chan M.Y. Yu V.C. J. Biol. Chem. 1999; 274: 32461-32468Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 16Chan S.L. Yee K.S. Tan K.M. Yu V.C. J. Biol. Chem. 2000; 275: 17925-17928Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). Ca2+/calmodulin-dependent kinase II (CaM kinase II) was obtained from Calbiochem. Plasmids containing the cDNAs for fem-1, fem-2, and fem-3 were kindly provided by Dr. Andrew Spence (University of Toronto, Toronto, Ontario, Canada). The cDNA for rat Ca2+/calmodulin kinase phosphatase was kindly provided by Dr. Hitoshi Fujisawa (Asahikawa Medical College, Asahikawa, Hokkaido, Japan). cDNA fragments for hFEM-2 and hPP2Cα (20Mann D.J. Campbell D.G. McGowan C.H. Cohen P.T. Biochim. Biophys. Acta. 1992; 1130: 100-104Crossref PubMed Scopus (60) Google Scholar) were obtained by polymerase chain reaction (PCR) amplification from human spleen cDNA (CLONTECH) with the Expand™ high fidelity PCR system (Roche Molecular Biochemicals) with primers incorporated with appropriate restriction sites and inserted into the pXJ40 mammalian expression vector driven by the CMV promoter (30Yee K.S. Yu V.C. J. Biol. Chem. 1998; 273: 5366-5374Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). The constructs were sequenced to ensure that no PCR error was introduced. All epitope tags are at the NH2 termini. Point mutations were introduced using the Transformer™ site-directed mutagenesis kit (CLONTECH). Human multiple tissue Northern blots (CLONTECH) were hybridized with a32P-labeled probe corresponding to the NH2-terminal 295-base pair coding region of hFEM-2 using ExpressHyb™ hybridization solution (CLONTECH) according to the instructions of the manufacturer. MCF-7 cells were transfected at 70% confluence on sterile glass coverslips using LipofectAMINE™ with 2 μg of Myc-hFEM-2, and 16 h later cells were harvested and washed gently with PBS. Cells were then fixed with methanol for 5 min at −20 °C, washed twice with PBS, and permeabilized with 0.2% Triton X-100 in PBS for 10 min, followed by four washes with PBS. Cells were then blocked with 10% fetal bovine serum in PBS for 10 min and incubated with mouse monoclonal anti-Myc antibody for 1 h, followed by washing three times with PBS containing 0.1% Triton X-100. Cells were then incubated with anti-mouse FITC-conjugated antibody for 1 h and washed three times with PBS containing 0.1% Triton X-100. Cells were then incubated with propidium iodide for 10 min to stain the nuclei and washed three times with PBS. Cells were mounted in anti-fading agent (Molecular Probes), placed on a glass slide, and viewed using a confocal microscope. Co-immunoprecipitation analyses were performed as described (15Chan S.L. Tan K.O. Zhang L. Yee K.S. Ronca F. Chan M.Y. Yu V.C. J. Biol. Chem. 1999; 274: 32461-32468Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 16Chan S.L. Yee K.S. Tan K.M. Yu V.C. J. Biol. Chem. 2000; 275: 17925-17928Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). Briefly, 293T cells seeded on a 100-mm plate at 80% confluence were transfected with 5 μg each of expression plasmids driven by the CMV promoter (pXJ40) (15Chan S.L. Tan K.O. Zhang L. Yee K.S. Ronca F. Chan M.Y. Yu V.C. J. Biol. Chem. 1999; 274: 32461-32468Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 16Chan S.L. Yee K.S. Tan K.M. Yu V.C. J. Biol. Chem. 2000; 275: 17925-17928Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar) encoding the indicated NH2-terminal HA- and Myc-tagged proteins using LipofectAMINE™ (Life Technologies, Inc.). 16 h after transfection, the cells were harvested and lysed in 1 ml of lysis buffer (50 mm HEPES, pH 7.6, 250 mm NaCl, 0.1% Nonidet P-40, 5 mm EDTA, 1 mm PMSF, 50 μg/ml aprotinin, and 10 μg/ml leupeptin). An aliquot (1%) of the cell lysates (1 ml) was fractionated on SDS-PAGE for visualization of the expression of proteins. The remaining cell lysates were subjected to immunoprecipitation using 1 μg of polyclonal anti-Myc antibody. 20 μl of 1:1 slurry of protein A-agarose was added after 1 h and incubated for another 1 h at 4 °C. The agarose beads were washed once in 1 ml of lysis buffer, two times in 1 ml of lysis buffer containing 500 mm NaCl, and once in 1 ml of lysis buffer before fractionation on SDS-PAGE followed by Western blotting analyses. Sequence encoding hFEM-2 was excised from pXJ40 vector and cloned in-frame into the glutathione S-transferase (GST) fusion protein bacterial expression vector pGEX-TK4E. The plasmids were transformed into the Escherichia coli strain BL21. GST and GST fusion protein were prepared by standard methods (30Yee K.S. Yu V.C. J. Biol. Chem. 1998; 273: 5366-5374Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar), and the recombinant proteins were immobilized on glutathione-agarose beads. Labeled proteins were prepared by in vitro transcription/translation of pXJ-HA-constructs using the TNT T7-coupled reticulocyte lysate system (Promega) in the presence of [35S]methionine. The integrity of the 35S-labeled proteins was verified by SDS-PAGE. For in vitro protein interaction, equal amounts of total 35S -labeled lysate (7 × 105 cpm of trichloroacetic acid-precipitable counts) were diluted into 0.2 ml of GST binding buffer (20 mm Tris, pH 7.5, 1 mmEDTA, 150 mm NaCl, 0.2% Nonidet P-40) and incubated for 1 h with the various GST fusion proteins immobilized on the beads (∼2 μg). Samples were subsequently washed six times with binding buffer and boiled for 3 min in loading buffer before fractionation on 10% SDS-PAGE. Bound proteins were visualized by autoradiography. 293T cells seeded on 150-mm plates at 80% confluence were transfected with 10 μg of expression plasmids driven by the CMV promoter (pXJ40) (15Chan S.L. Tan K.O. Zhang L. Yee K.S. Ronca F. Chan M.Y. Yu V.C. J. Biol. Chem. 1999; 274: 32461-32468Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 16Chan S.L. Yee K.S. Tan K.M. Yu V.C. J. Biol. Chem. 2000; 275: 17925-17928Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar) encoding the indicated NH2-terminal Myc-tagged proteins using LipofectAMINE™ (Life Technologies, Inc.). 24 h after transfection, cells were harvested and lysed in 1 ml of storage buffer (50 mm Tris, pH 7.6, 150 mm NaCl, 0.1 mm EGTA, 0.1% β-mercaptoethanol, 0.1% Triton X-100, 1 mm PMSF, 50 μg/ml aprotinin, and 10 μg/ml leupeptin). Lysates were subjected to immunoprecipitation using 3 μg of polyclonal anti-Myc antibodies. 20 μl of 1:1 slurry of protein A-agarose was added after 1 h and incubated for another 1 h at 4 °C. The agarose beads were washed three times with storage buffer and resuspended in 50 μl of storage buffer before incubating with the phosphothreonine peptide substrate, RRA(pT)VA, and PP2C buffer (50 mm imidazole, pH 7.2, 0.2 mm EGTA, 20 mm MgSO4, 0.02% β-mercaptoethanol, 0.1 mg/ml BSA) at 30 °C for 1 h. In experiments that determine cation requirement, MgSO4 is replaced with the sulfates of the indicated cations. Free phosphate generated was then quantitated according to the instructions of the Serine/Threonine Phosphatase Assay System™ (Promega). Autophosphorylation of CaMKII (Calbiochem) was performed as described (28Ishida A. Kameshita I. Fujisawa H. J. Biol. Chem. 1998; 273: 1904-1910Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). Briefly, CaMKII (10 μg/ml) was autophosphorylated at 30 °C for 1 min in a reaction mixture containing 40 mm Hepes-NaOH, pH 8.0, 5 mm Mg(CH3CO2)2, 0.1 mm EGTA, 5 μm calmodulin, 0.4 mmCaCl2, 0.02% Tween 20, and 0.33 μm[γ-32P]ATP (6000 Ci/mmol). Excess EDTA (12.3 mm) and BSA (1 mg/ml) were added to stop the reaction, and the reaction mixture was applied to a P30 microspin column (Bio-Rad) buffered with 50 mm Tris, pH 7.5, 0.2 m NaCl, 0.05% Tween 20, and 1 mm dithiothreitol. The eluate from the spin column was collected and stored at −80 °C. Dephosphorylation of CaMKII was performed as described (28Ishida A. Kameshita I. Fujisawa H. J. Biol. Chem. 1998; 273: 1904-1910Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). Dephosphorylation of [32P]CaMKII was carried out at 30 °C for 20 min in a 30-μl reaction mixture containing ∼2 μg of GST, GST-rCaMKPase, GST-hFEM-2, GST-FEM-2, or GST-PP2Cα, or immunoprecipitates of Myc-hFEM-2, Myc-hFEM-2, or Myc vector, in 50 mm Tris-HCl, pH 7.5, 100 mm KCl, 2 mm MgCl2, 2 mm MnCl2, 0.1 mm EGTA, 0.01% Tween 20, 10 μg/ml poly(Lys), and 28,000 cpm autophosphorylated [32P]CaMKII. After preincubation for 30 s at 30 °C, the reaction was started by adding the phosphoprotein. Where indicated, 1 mm orthovanadate or 25 mm EDTA was added. After incubation for 20 min, the reaction was terminated by mixing with SDS-PAGE sample buffer. The mixture was boiled for 2 min and centrifuged for 2 min, and an aliquot of the supernatant was run on a 10% SDS-PAGE. The gel was stained with Coomassie Blue to ensure equal loading of protein, following which the gel was dried and visualized by autoradiography. HeLa cells were seeded onto glass coverslips at 70% confluence and transfected with pEGFP, pEGFP-FEM-2, or pEGFP-PP2Cα using LipofectAMINE™. 24 h after transfection, the cells were fixed, rinsed with PBS, and then incubated for 2 min with Hoechst 33342 dye (Molecular Probes Inc.) to enable nuclear staining. The cells were subsequently fixed and then visualized using a Zeiss Axioplan microscope. Apoptosis assays were performed as described (15Chan S.L. Tan K.O. Zhang L. Yee K.S. Ronca F. Chan M.Y. Yu V.C. J. Biol. Chem. 1999; 274: 32461-32468Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 16Chan S.L. Yee K.S. Tan K.M. Yu V.C. J. Biol. Chem. 2000; 275: 17925-17928Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). Briefly, HeLa, MCF7, or NIH3T3 cells were transiently co-transfected with 2 μg each of the expression plasmids or vector and 0.5 μg of pCMV-β-galactosidase. Vector plasmid was supplemented to bring the total amount of plasmids for each transfection to 5 μg. Transfections were carried out with LipofectAMINE™ for 6 h, followed by change of medium, and ZVAD added to the fresh medium at this point where indicated. 24 h later, the cells were fixed with 2% formaldehyde and 0.2% glutaraldehyde and stained with 5-bromo-4-chloro-3-indolyl β-d-galactopyranoside (X-gal) solution at 37 °C. Cells were visualized by phase-contrast microscopy and blue (β-galactosidase-positive) cells were scored for apoptotic morphology. The data (mean ± S.D.) shown are percentage of round blue cells as a function of total number of blue cells counted (∼400–500 cells/sample) from three to five randomly chosen fields. For detection of PARP cleavage, HeLa cells cultured on 100-mm dishes were transiently transfected with the various pXJ-Myc constructs (10 μg). Whole cell extracts were prepared by lysing the cells in 0.2 ml of lysis buffer (50 mm HEPES, pH 7.6, 250 mm NaCl, 0.1% Nonidet P-40, 5 mmEDTA, 1 mm PMSF, 50 μg/ml aprotinin, and 10 μg/ml leupeptin). The extracts were fractionated on SDS-PAGE followed by Western blotting analyses using PARP-specific antibody (A20). To identify mammalian homologues of fem-2 and fem-3, we searched the GenBank™ data base for cDNAs encoding proteins with homology to these genes. No gene with significant similarity to fem-3 was found. However, the amino acid sequence predicted by analysis of an open reading frame (ORF) of a cDNA clone, kiaa0015 (GenBank™ accession no. D13640), derived from randomly sampled cDNA clones prepared from human immature myeloid cell line KG-1 (31Nomura N. Miyajima N. Sazuka T. Tanaka A. Kawarabayasi Y. Sato S. Nagase T. Seki N. Ishikawa K. Tabata S. DNA Res. 1994; 1: 27-56Crossref PubMed Scopus (272) Google Scholar), showed significant homology to FEM-2. The kiaa0015 clone contained a single long ORF encoding a protein of 454 amino acids (Fig.1 A). The 5′ and 3′ regions adjacent to the ORF contain stop codons in all three reading frames preceding the predicted translation start site and subsequent to the end of the ORF, suggesting that it encodes a full-length protein. Using the 5′ and 3′ sequence information derived from this clone, several independent cDNA clones encoding kiaa0015 product were generated by PCR from double-stranded cDNAs prepared from human spleen (CLONTECH). Three of the independent clones were sequenced completely in both directions, and the deduced amino acid sequences encoded by the clones were found to be identical to the sequence encoded by kiaa0015. We named this protein hFEM-2. hFEM-2 shares relatively high amino acid sequence homology with FEM-2, with a relatively long amino-terminal extension flanking the catalytic domain. Over the entire length of the protein, the amino acid identity between hFEM-2 and FEM-2 is 28%. The degree of similarity is not uniform over the entire length of the protein. In the catalytic domain, the two proteins are 30% identical, whereas in the amino-terminal region, the proteins are 18% identical. The catalytic domains among different members (α, β, γ) (20Mann D.J. Campbell D.G. McGowan C.H. Cohen P.T. Biochim. Biophys. Acta. 1992; 1130: 100-104Crossref PubMed Scopus (60) Google Scholar, 32Marley A.E. Kline A. Crabtree G. Sullivan J.E. Beri R.K. FEBS Lett. 1998; 431: 121-124Crossref PubMed Scopus (23) Google Scholar, 33Travis S.M. Welsh M.J. FEBS Lett. 1997; 412: 415-419Crossref PubMed Scopus (47) Google Scholar) of PP2C family generally share high degree of sequence similarity (34Cohen P. Annu. Rev. Biochem. 1989; 58: 453-508Crossref PubMed Scopus (2146) Google Scholar); however, the catalytic domain of FEM-2 is more similar in sequence to that of hFEM-2 than to other mammalian PP2Cs. The expression profile of hFEM-2 in human tissues was assessed by Northern blot analysis. Two transcripts, with estimated length of 6.5 and 3 kb, were detected in all adult tissues studied (Fig.1 B). The level of hFEM-2 mRNA message was variable from tissue to tissue, with the highest expression in testis (Fig.1 B). The subcellular localization of hFEM-2 was assessed in MCF-7 cells using immunocytochemistry. Myc-tagged hFEM-2 was expressed in MCF-7 cells, and the expression of the protein was detected using FITC-conjugated Myc antibody, whereas propidium iodide was used to stain the nuclei. hFEM-2 was consistently found diffusely distributed in the cytosol with no notable expression in the nucleus (Fig.1 C), suggesting that hFEM-2 is a cytosolic protein. As FEM-2 is known to exhibit PP2C phosphatase activity (13Chin-Sang I.D. Spence A.M. Genes Dev. 1996; 10: 2314-2325Crossref PubMed Scopus (71) Google Scholar), we proceeded to examine the phosphatase activity of hFEM-2 in vitro. PP2C is the main enzyme subtype of the PPM family of serine-threonine phosphatases, and some of its members include mammalian PP2Cα (20Mann D.J. Campbell D.G. McGowan C.H. Cohen P.T. Biochim. Biophys. Acta. 1992; 1130: 100-104Crossref PubMed Scopus (60) Google Scholar), PP2Cβ (32Marley A.E. Kline A. Crabtree G. Sullivan J.E. Beri R.K. FEBS Lett. 1998; 431: 121-124Crossref PubMed Scopus (23) Google Scholar), and PP2Cγ (33Travis S.M. Welsh M.J. FEBS Lett. 1997; 412: 415-419Crossref PubMed Scopus (47) Google Scholar). In contrast to other protein phosphatases, the dephosphorylation activity of PP2C absolutely requires the metal cations, Mn2+ or Mg2+, but its activity is not sensitive to the tumor promoter okadaic acid and other inhibitors of the PPP family (34Cohen P. Annu. Rev. Biochem. 1989; 58: 453-508Crossref PubMed Scopus (2146) Google Scholar). Full-length NH2-terminal Myc-tagged hFEM-2, FEM-2, or hPP2Cα were transiently overexpressed in 293T cells. The Myc-tagged proteins were immunoprecipitated using anti-Myc antibody, and the immunoprecipitates were assayed for phosphatase activity in vitro. The phosphatase activity was assayed by dephosphorylating the phosphothreonine peptide RRA(pT)VA as described under "Experimental Procedures." Immunoprecipitates of hFEM-2 protein from transiently transfected 293T cells exhibited phosphatase activity to an extent similar to that of FEM-2 and PP2

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