FBW2 Targets GCMa to the Ubiquitin-Proteasome Degradation System

The GCM proteins GCMa/1 and GCMb/2 are novel zinc-containing transcription factors critical for glial cell differentiation in fly and for placental as well as parathyroid gland development in mouse. Previous pulse-chase experiments have demonstrated differential protein stabilities of GCM proteins with half-lives from ∼30 min to 2 h (Tuerk, E. E., Schreiber, J., and Wegner, M. (2000) J. Biol. Chem. 275, 4774–4782). However, little is known about the machinery that controls GCM protein degradation. Here, we report the identification of an SCF complex as the GCM ubiquitin-protein isopeptide ligase (E3) that regulates human GCMa (hGCMa) degradation. We found that SKP1 and CUL1, two key components of the SCF complex, associate with hGCMa in vivo. We further identify the human F-box protein FBW2 (hFBW2) as the substrate recognition subunit in the SCF E3 complex for hGCMa. We show that hFBW2 interacts with hGCMa in a phosphorylation-dependent manner and promotes hGCMa ubiquitination. Supporting a critical role for hFBW2 in hGCMa degradation, knockdown of hFBW2 expression by RNA interference leads to a reduction in hGCMa ubiquitination and a concomitant increase in hGCMa protein stability. Our study identifies the SCFhFBW2 E3 complex as the key machinery that targets hGCMa to the ubiquitin-proteasome degradation system. The GCM proteins GCMa/1 and GCMb/2 are novel zinc-containing transcription factors critical for glial cell differentiation in fly and for placental as well as parathyroid gland development in mouse. Previous pulse-chase experiments have demonstrated differential protein stabilities of GCM proteins with half-lives from ∼30 min to 2 h (Tuerk, E. E., Schreiber, J., and Wegner, M. (2000) J. Biol. Chem. 275, 4774–4782). However, little is known about the machinery that controls GCM protein degradation. Here, we report the identification of an SCF complex as the GCM ubiquitin-protein isopeptide ligase (E3) that regulates human GCMa (hGCMa) degradation. We found that SKP1 and CUL1, two key components of the SCF complex, associate with hGCMa in vivo. We further identify the human F-box protein FBW2 (hFBW2) as the substrate recognition subunit in the SCF E3 complex for hGCMa. We show that hFBW2 interacts with hGCMa in a phosphorylation-dependent manner and promotes hGCMa ubiquitination. Supporting a critical role for hFBW2 in hGCMa degradation, knockdown of hFBW2 expression by RNA interference leads to a reduction in hGCMa ubiquitination and a concomitant increase in hGCMa protein stability. Our study identifies the SCFhFBW2 E3 complex as the key machinery that targets hGCMa to the ubiquitin-proteasome degradation system. The gcm1 (glial cell missing 1) gene was first identified in a Drosophila melanogaster mutant line that produces additional neurons at the expense of glial cells. Conversely, ectopic expression of gcm1 leads to glial differentiation at the expense of other cell types (1Hosoya T. Takizawa K. Nitta K. Hotta Y. Cell. 1995; 82: 1025-1036Abstract Full Text PDF PubMed Scopus (365) Google Scholar, 2Jones B.W. Fetter R.D. Tear G. Goodman C.S. Cell. 1995; 82: 1013-1023Abstract Full Text PDF PubMed Scopus (399) Google Scholar). In D. melanogaster, GCM1 mediates the differentiation of lateral (but not midline) glial cells and the proliferation of plasmatocyte precursors (1Hosoya T. Takizawa K. Nitta K. Hotta Y. Cell. 1995; 82: 1025-1036Abstract Full Text PDF PubMed Scopus (365) Google Scholar, 2Jones B.W. Fetter R.D. Tear G. Goodman C.S. Cell. 1995; 82: 1013-1023Abstract Full Text PDF PubMed Scopus (399) Google Scholar). Recently, another gcm gene in D. melanogaster, gcm2, was identified and shown to have biological functions similar to those of gcm1 (3Alfonso T.B. Jones B.W. Dev. Biol. 2002; 248: 369-383Crossref PubMed Scopus (136) Google Scholar, 4Kammerer M. Giangrande A. EMBO J. 2001; 20: 4664-4673Crossref PubMed Scopus (59) Google Scholar). Two homologs of Drosophila gcm1 named GCMa and GCMb have also been isolated from mouse and human (5Kanemura Y. Hiraga S. Arita N. Ohnishi T. Izumoto S. Mori K. Matsumura H. Yamasaki M. Fushiki S. Yoshimine T. FEBS Lett. 1999; 442: 151-156Crossref PubMed Scopus (49) Google Scholar, 6Kim J. Jones B.W. Zock C. Chen Z. Wang H. Goodman C.S. Anderson D.J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12364-12369Crossref PubMed Scopus (171) Google Scholar). Unlike the Drosophila gcm genes, the mammalian GCM genes are not primarily expressed in the nervous system. Mouse GCMa is highly expressed in labyrinthine trophoblast cells (7Basyuk E. Cross J.C. Corbin J. Nakayama H. Hunter P. Nait-Oumesmar B. Lazzarini R.A. Dev. Dyn. 1999; 214: 303-311Crossref PubMed Scopus (129) Google Scholar). The GCMa knockout mouse is embryonic lethal, with a failure to form a labyrinth layer and no fusion of trophoblast cells to syncytiotrophoblasts (8Anson-Cartwright L. Dawson K. Holmyard D. Fisher S.J. Lazzarini R.A. Cross J.C. Nat. Genet. 2000; 25: 311-314Crossref PubMed Scopus (344) Google Scholar, 9Schreiber J. Riethmacher-Sonnenberg E. Riethmacher D. Tuerk E.E. Enderich J. Bosl M.R. Wegner M. Mol. Cell. Biol. 2000; 20: 2466-2474Crossref PubMed Scopus (165) Google Scholar). In contrast, mouse GCMb is expressed in parathyroid cells, and the GCMb knockout mouse fails to develop a parathyroid gland (10Gunther T. Chen Z.F. Kim J. Priemel M. Rueger J.M. Amling M. Moseley J.M. Martin T.J. Anderson D.J. Karsenty G. Nature. 2000; 406: 199-203Crossref PubMed Scopus (318) Google Scholar). Therefore, although Drosophila gcm and mammalian GCM genes are evolutionarily conserved at the nucleotide and protein levels, they appear to execute different biological functions during development. GCM proteins form a novel family of transcription factors that all share sequence homology in the N-terminal region that constitutes the DNA-binding domain called the GCM motif, which has a preferred binding sequence of 5′-(A/G)CCC(T/G)CAT-3′ or its complement (11Akiyama Y. Hosoya T. Poole A.M. Hotta Y. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14912-14916Crossref PubMed Scopus (175) Google Scholar, 12Schreiber J. Enderich J. Wegner M. Nucleic Acids Res. 1998; 26: 2337-2343Crossref PubMed Scopus (64) Google Scholar). Moreover, two zinc ions have been found to be tightly coordinated by cysteine and histidine residues and to be required for the DNA-binding activity in the GCM motif (13Cohen S.X. Moulin M. Hashemolhosseini S. Kilian K. Wegner M. Muller C.W. EMBO J. 2003; 22: 1835-1845Crossref PubMed Scopus (74) Google Scholar). Sequence homology is less preserved outside the GCM motif; a transactivation domain has been identified in the C terminus of GCM proteins (11Akiyama Y. Hosoya T. Poole A.M. Hotta Y. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14912-14916Crossref PubMed Scopus (175) Google Scholar, 14Schreiber J. Sock E. Wegner M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4739-4744Crossref PubMed Scopus (102) Google Scholar). Target genes of fly GCM1 include gcm1 and several glial cells-specific genes such as pointed, repo, tramtrack, and prospero (15Van De Bor V. Giangrande A. Curr. Opin. Genet. Dev. 2002; 12: 465-472Crossref PubMed Scopus (40) Google Scholar). Target genes of human (h) 1The abbreviations used are: h, human; E2, ubiquitin carrier protein; E3, ubiquitin-protein isopeptide ligase; RNAi, RNA interference; HA, hemagglutinin; mAb, monoclonal antibody; Ub, ubiquitin; λ-PPase, λ-protein phosphatase; GST, glutathione S-transferase; siRNA, small interfering RNA. GCMa have been reported recently. For instance, the promoter region of a placental aromatase gene that is responsible for estrogen biosynthesis has been found to contain GCM-binding sites that are recognized by GCMa (16Yamada K. Ogawa H. Honda S. Harada N. Okazaki T. J. Biol. Chem. 1999; 274: 32279-32286Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). In addition, we have demonstrated that hGCMa regulates expression of the syncytin gene, which encodes a fusogenic membrane protein mediating the fusion of trophoblast cells (17Yu C. Shen K. Lin M. Chen P. Lin C. Chang G.-D. Chen H. J. Biol. Chem. 2002; 277: 50062-50068Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar). Therefore, hGCMa appears to be a key factor in the formation of the human syncytiotrophoblast layer. GCM proteins contain PEST sequences, which are frequently found in labile proteins. Previous pulse-chase experiments have shown that GCM proteins have short half-lives from ∼30 min to 2 h (18Tuerk E.E. Schreiber J. Wegner M. J. Biol. Chem. 2000; 275: 4774-4782Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Because GCM proteins are labile and because little is known about the machinery that controls GCM protein degradation, in this study, we investigate the role of the ubiquitin-proteasome degradation system in GCM protein turnover. Ubiquitin-mediated protein degradation plays an important role in controlling numerous processes, including cell cycle progression, signal transduction, transcriptional regulation, receptor down-regulation, and endocytosis. Ubiquitin is a polypeptide of 76 amino acids, and its primary sequence is highly conserved from yeast to mammals. Ubiquitin conjugation of target proteins requires a cascade of reactions. First, ubiquitin is activated by the ubiquitin-activating enzyme in an ATP-dependent process. Second, activated ubiquitin is transferred to the ubiquitin-conjugating enzyme (ubiquitin carrier protein (E2)). Finally, E2 transfers ubiquitin to a substrate protein by itself or in cooperation with ubiquitin-protein isopeptide ligase (E3) by formation of an amide isopeptide bond between the carboxyl group of the C-terminal glycine residue of ubiquitin and the ϵ-amino group of an internal lysine residue of the substrate protein (19Pickart C.M. Annu. Rev. Biochem. 2001; 70: 503-533Crossref PubMed Scopus (2944) Google Scholar). The monoubiquitinated substrate protein becomes polyubiquitinated via the same cascade of reactions. In eukaryotes, polyubiquitinated proteins are targeted to the 26 S proteasome, which consists of a proteolytic core particle, the 20 S proteasome, and two 19 S regulatory complexes (20Pickart C.M. Cohen R.E. Nat. Rev. Mol. Cell Biol. 2004; 5: 177-187Crossref PubMed Scopus (609) Google Scholar). In the ubiquitin-proteasome degradation system, E3 controls substrate specificity and transfers ubiquitin to lysine residues of the substrate protein. E3 ligases have been classified into HECT (homologous to E6-AP Cterminus) domain E3 ligases, single-subunit RING finger E3 ligases, and multisubunit RING finger E3 ligases. In the ubiquitination process, HECT E3 ligases form thioester catalytic intermediates with ubiquitin, whereas RING finger E3 ligases facilitate direct transfer of ubiquitin from E2 to substrate, probably without formation of covalent intermediates. The SCF (SKP1/cullin/F-box protein) complex belongs to the multisubunit RING finger E3 ligases, which are composed of CUL1, SKP1, RBX1, and an F-box protein. CUL1 functions as a scaffold and interacts with SKP1 and RBX1 at its N and C termini, respectively. RBX1 is a RING finger protein that recruits E2 to the E3 complex. SKP1 interacts with the F-box domain of an F-box protein, which mediates substrate recognition via another interaction domain (21Cardozo T. M. Nat. Rev. Mol. Cell Biol. 2004; 5: PubMed Scopus Google Scholar). In this study, we that hGCMa be and by the 26 S Moreover, the human F-box protein FBW2 (hFBW2) is identified to with hGCMa in a phosphorylation-dependent manner and to ubiquitination of hGCMa. The interaction between hGCMa and hFBW2 and the critical role of hFBW2 in degradation of hGCMa are further by domain and RNA interference Our study identifies the SCFhFBW2 E3 complex as the key machinery that targets hGCMa to the ubiquitin-proteasome degradation system. GCMa expression hGCMa a at its C was by the of hGCMa into The expression was by hGCMa into in this and by J. of The of CUL1 was from and into to The the F-box proteins and from a human placental or expressed sequence and into a expression of the at the C The and mutant expression by first the DNA-binding domain into and the of hGCMa or its by the Cell and cell line was from and at in with and The cell line was from and at in of with and the same study the of hGCMa protein cells in with by at for h and with or for another The protein of was by mouse monoclonal For the in a and at for The with mouse a as by R.E. J. PubMed Google Scholar). study the of in cells, cells in and with for 2 h to with or without for the proteins by with In study ubiquitination of cells with and the ubiquitin expression for h and with or without for another h at for of proteins by as by H. A. J. A. EMBO J. 1997; PubMed Scopus Google Scholar). study the interaction of hGCMa and components of the SCF complex, cells with and in 20 and a h as for and the to with for min at with the complexes by with was for cells with and by with and with an F-box protein was with cells with and or mAb, and the complexes by mouse and in study of hGCMa in cells with for h and with with for was with as of was by the complexes with of λ-protein at for The was by and study the of of hGCMa the interaction of hGCMa and cells with and or for in 20 and a or without the 20 and and with of λ-PPase, by with and with For in proteins from the cells with or without of and with of glutathione or in at for 2 the complexes by the of hGCMa and the domain of hGCMa that interacts with cells with and or its mutant was mAb, and the complexes by the domain of hFBW2 that interacts with hFBW2 and its into the expression fusion proteins expressed in cells and glutathione to the cell was by cells in C and experiments by of cell and of or the fusion protein in C glutathione at for the by by of small interfering RNA for the and by a and the of the and sequences, and into the The and expression for and hFBW2 expression and expression a sequence no homology to human gene was used as a study the of gene of hFBW2 the ubiquitination of cells with different of by A. for and as in the to and with of hGCMa was by and by In addition, a was by and by study the of gene of hFBW2 the protein of cells with or h cells with for 2 h and with for the for with and by and The in the of the hGCMa protein is by the 26 S degradation system, cells with and with or without including and shown in an of and not the protein of with the However, an of and the 26 S proteasome, the protein of an of the 26 S proteasome, also the protein of that the protein of hGCMa be by the 26 S degradation system in vivo. Because GCMa is primarily expressed in we hGCMa is also by the 26 S degradation system in placental cells. this the human placental cell line was used to via a expression system. cells with and for different of in the or of shown in a of between and 2 h in cells in the of However, the protein for h in the of that the GCMa protein is by the 26 S degradation system in placental cells. Because the 26 S mediates the degradation of we hGCMa is in vivo. this cells with and and with or without proteins in the cell from cells and by The of in the proteins was further by shown in the protein of was which is to a of protein degradation. In contrast, the protein of in the of was In the of GCMa no signal for was in cells with as However, for in the and group and further in the of that hGCMa be in vivo. Drosophila GCM1 and mouse GCMb also was with in the of not that GCM proteins be and by the 26 S of the F-box with the SCF E3 complex is in the degradation of transcription we an SCF E3 complex is required for the degradation of hGCMa. We first hGCMa interacts with components of the SCF complex by cells with and in shown in the protein was in the complex. a was cells with and interaction and was also that hGCMa with the SCF E3 complex in vivo. Because the substrate specificity for an SCF E3 complex its F-box protein, we identified the F-box protein that hGCMa. We the for several human F-box proteins known to be expressed in including and and for interaction with hGCMa in We a interaction between hGCMa and not or or not that hFBW2 is the F-box protein that hGCMa. between hGCMa and further of hGCMa is required for interaction with hFBW2 because several well F-box proteins such as and in a phosphorylation-dependent manner H. A. J. A. EMBO J. 1997; PubMed Scopus Google Scholar, A. A. M. S. 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We of hGCMa is required for interaction with this cells first with and or the cell from cells was in the or of and further with λ-PPase, by shown in a interaction between hGCMa and hFBW2 (but not was in the of this interaction was the was in the of that of hGCMa is required for interaction with experiments to further the phosphorylation-dependent interaction between hGCMa and was first from cells and with or without λ-PPase, by with or a interaction was between and However, this interaction was the protein was with interaction was between and or not with that hFBW2 the form of the hGCMa protein. of the of hGCMa and the interaction of hGCMa and the domain of expression the DNA-binding domain with or its with into cells. between and or its by shown in an interaction was between hFBW2 and hGCMa as well as the C-terminal amino and However, the N-terminal amino and not with Therefore, the domain of hGCMa was identified as region which also the GCM the domain of fusion proteins of hFBW2 and hFBW2 from E. and with cell for The hFBW2 polypeptide consists of an F-box and The F-box motif interacts with SKP1 of the SCF complex, whereas the are to interaction with protein shown in the N-terminal region of the hFBW2 polypeptide the F-box not with However, the hFBW2 N-terminal amino and with the the binding activity for that amino C-terminal to the substrate that the domain of hFBW2 is region which the FBW2 the of we found that hGCMa interacts with that hFBW2 of the SCFhFBW2 E3 complex is in the ubiquitination of hGCMa for degradation. this we used to the protein of hFBW2 in and ubiquitination of hGCMa was the protein of hFBW2 was We first for Two expression and from the hFBW2 sequence for to expression of and hFBW2 in cells. shown in (but not or an was highly in the protein of In addition, and for to the hFBW2 protein in cells with shown in the protein of hFBW2 was by not by or We ubiquitination of hGCMa is expression of the hFBW2 gene is cells with or with or with and by shown in of the of was by the of not was to the of in the proteins. the of was by the of not that hFBW2 is in the ubiquitination of hGCMa in vivo. We further the protein of hGCMa is expression of the hFBW2 gene is experiments with hGCMa in cells with or The of in cells with and was between and 2 h, whereas was as as h in cells with and that hGCMa is with knockdown of hFBW2 gene GCMa is primarily expressed in and plays a key role in mediating trophoblast cell In this study, we first that hGCMa be and by the 26 S degradation system by that a 26 S increase the of the hGCMa protein and its polyubiquitinated form in vivo. we demonstrated that an SCF complex is in the of hGCMa because hGCMa with two key components of the complex and in vivo. We further several human F-box proteins expressed in for with hGCMa and identified hFBW2 as the F-box protein for hGCMa in and in hGCMa is the first identified substrate protein of proteins a conserved domain the F-box have been identified in the human M. Biol. 2000; Google Scholar, C. S.J. Curr. Biol. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, J. T. S.J. M. 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