Ubiquitylation of MEKK1 Inhibits Its Phosphorylation of MKK1 and MKK4 and Activation of the ERK1/2 and JNK Pathways
2003; Elsevier BV; Volume: 278; Issue: 3 Linguagem: Inglês
10.1074/jbc.c200616200
ISSN1083-351X
AutoresJames Witowsky, Gary L. Johnson,
Tópico(s)PI3K/AKT/mTOR signaling in cancer
ResumoMEKK1 is a MAPK kinase kinase that is activated in response to stimuli that alter the cytoskeleton and cell shape. MEKK1 phosphorylates and activates MKK1 and MKK4, leading to ERK1/2 and JNK activation. MEKK1 has a plant homeobox domain (PHD) that has been shown to have E3 ligase activity. (Lu, Z., Xu, S., Joazeiro, C., Cobb, M. H., and Hunter, T. (2002) Mol. Cell 9, 945–956). MEKK1 kinase activity is required for ubiquitylation of MEKK1. MEKK1 ubiquitylation is inhibited by mutation of cysteine 441 to alanine (C441A) within the PHD. The functional consequence of MEKK1 ubiquitylation is the inhibition of MEKK1 catalyzed phosphorylation of MKK1 and MKK4 resulting in inhibition of ERK1/2 and JNK activation. The C441A mutation within the PHD of MEKK1 prevents ubiquitylation and preserves the ability of MEKK1 to catalyze MKK1 and MKK4 phosphorylation. MEKK1 ubiquitylation represents a mechanism for inhibiting the ability of a protein kinase to phosphorylate substrates and regulate downstream signaling pathways. MEKK1 is a MAPK kinase kinase that is activated in response to stimuli that alter the cytoskeleton and cell shape. MEKK1 phosphorylates and activates MKK1 and MKK4, leading to ERK1/2 and JNK activation. MEKK1 has a plant homeobox domain (PHD) that has been shown to have E3 ligase activity. (Lu, Z., Xu, S., Joazeiro, C., Cobb, M. H., and Hunter, T. (2002) Mol. Cell 9, 945–956). MEKK1 kinase activity is required for ubiquitylation of MEKK1. MEKK1 ubiquitylation is inhibited by mutation of cysteine 441 to alanine (C441A) within the PHD. The functional consequence of MEKK1 ubiquitylation is the inhibition of MEKK1 catalyzed phosphorylation of MKK1 and MKK4 resulting in inhibition of ERK1/2 and JNK activation. The C441A mutation within the PHD of MEKK1 prevents ubiquitylation and preserves the ability of MEKK1 to catalyze MKK1 and MKK4 phosphorylation. MEKK1 ubiquitylation represents a mechanism for inhibiting the ability of a protein kinase to phosphorylate substrates and regulate downstream signaling pathways. ubiquitin-activating enzyme ubiquitin-conjugating enzyme ubiquitin ligase cysteine 441 to alanine mutation extracellular signal-regulated kinase glutathioneS-transferase hemagglutinin epitope tag homology to E6 COOHterminus: His6, hexahistidine c-Jun NH2-terminal kinase mitogen-activated protein kinase mitogen-activated protein kinase kinase kinase 1 mitogen-activated protein kinase kinase 1 mitogen-activated protein kinase kinase 4 planthomeobox domain reallyinteresting new gene ubiquitin interaction motif nickel-nitrilotriacetic acid Ubiquitin is a highly conserved 76-amino acid globular protein that was identified as the first protein to act as a covalent modifier. Ubiquitylation is the process of attachment of ubiquitin to a target protein and is a multistep process that involves the action of at least three classes of enzyme: the ubiquitin-activating enzyme (E1),1 a ubiquitin-conjugating enzyme (E2), and a ubiquitin ligase (E3), which assists in substrate recognition and the transfer of ubiquitin to the target protein. There are two main types of E3 enzyme, those that contain a homology to E6 Cterminus (HECT) domain and those that have areally interesting newgene (RING finger or RING finger-like) domain (1Glickman M.H. Ciechanover A. Physiol. Rev. 2002; 82: 373-428Crossref PubMed Scopus (3304) Google Scholar). The plant homeobox domain (PHD) is a RING finger-like domain defined by a series of cysteine and histidine residues with a characteristic spacing that mediates coordination of two zinc ions in a cross-brace structure (2Pascual J. Martinez-Yamout M. Dyson H.J. Wright P.E. J. Mol. Biol. 2000; 304: 723-729Crossref PubMed Scopus (125) Google Scholar). Disruption of a metal coordinating residue within the RING finger or PHD has been shown to inactivate the ligase properties of the enzyme (3Hashizume R. Fukuda M. Maeda I. Nishikawa H. Oyake D. Yabuki Y. Ogata H. Ohta T. J. Biol. Chem. 2001; 276: 14537-14540Abstract Full Text Full Text PDF PubMed Scopus (542) Google Scholar).Classically, ubiquitin is involved in the homeostasis of cellular proteins by removing unnecessary, deleterious, or misfolded proteins primarily through the ubiquitin-proteasome degradation pathway (4Jennissen H.P. Eur. J. Biochem. 1995; 231: 1-30Crossref PubMed Scopus (112) Google Scholar). Ubiquitin has been shown to function in many cellular processes including cell cycle progression, apoptosis, cell differentiation, and DNA repair (5Naujokat C. Hoffmann S. Lab. Invest. 2002; 82: 965-980Crossref PubMed Scopus (218) Google Scholar). Recently, ubiquitylation of specific proteins has been shown to have functions independent of proteasome-mediated degradation (6Marx J. Science. 2002; 297: 1792-1794Crossref PubMed Scopus (40) Google Scholar). For example, vesicular sorting and TRAF6 organization of the TAK1 signal transduction complex has been shown to involve ubiquitylation of proteins for the control of protein-protein interactions (7Katzmann D.J. Babst M. Emr S.D. Cell. 2001; 106: 145-155Abstract Full Text Full Text PDF PubMed Scopus (1110) Google Scholar, 8Deng L. Wang C. Spencer E. Yang L. Braun A. You J. Slaughter C. Pickart C. Chen Z.J. Cell. 2000; 103: 351-361Abstract Full Text Full Text PDF PubMed Scopus (1485) Google Scholar, 9Wang C. Deng L. Hong M. Akkaraju G.R. Inoue J. Chen Z.J. Nature. 2001; 412: 346-351Crossref PubMed Scopus (1612) Google Scholar).MEKK1 is a 196-kDa serine/threonine MAPK kinase kinase that can regulate both the extracellular-regulated kinase (ERK1/2) and the c-Jun NH2-terminal kinase (JNK) pathways in response to specific stimuli (10Yujiri T. Sather S. Fanger G.R. Johnson G.L. Science. 1998; 282: 1911-1914Crossref PubMed Scopus (280) Google Scholar). We and others have shown that MEKK1 is activated by a variety of stimuli that alter cell shape and is required for normal cell motility (11Yujiri T. Ware M. Widmann C. Oyer R. Russell D. Chan E. Zaitsu Y. Clarke P. Tyler K. Oka Y. Fanger G.R. Henson P. Johnson G.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7272-7277Crossref PubMed Scopus (207) Google Scholar, 12Xia Y. Makris C., Su, B., Li, E. Yang J. Nemerow G.R. Karin M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5243-5248Crossref PubMed Scopus (231) Google Scholar). MEKK1 is a complex protein containing multiple identified domains that may serve to regulate function. The E3 activity of the PHD of MEKK1 was recently described by Lu et al.(13Lu Z., Xu, S. Joazeiro C. Cobb M.H. Hunter T. Mol. Cell. 2002; 9: 945-956Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar). Here we demonstrate that full-length MEKK1 requires an intact PHD for its own ubiquitylation, and this ubiquitylation impairs its ability to phosphorylate MKK1 and MKK4.DISCUSSIONMEKK1 is unique among MAPK kinase kinases in that it encodes a PHD in its NH2 terminus. The MEKK1 PHD has E3 ligase activity (13Lu Z., Xu, S. Joazeiro C. Cobb M.H. Hunter T. Mol. Cell. 2002; 9: 945-956Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar) that is dependent on the kinase activity of the full-length 196-kDa MEKK1 protein. Lu et al. (13Lu Z., Xu, S. Joazeiro C. Cobb M.H. Hunter T. Mol. Cell. 2002; 9: 945-956Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar) demonstrated that a GST fusion of the MEKK1 PHD had E3 ligase activity in vitro. Our work demonstrates that a functional PHD is required for MEKK1 poly/multiubiquitylation. Thus, activation of MEKK1 not only stimulates its ability to phosphorylate and activate MKK1 and MKK4 in the ERK1/2 and JNK pathways but also leads to the ubiquitylation of MEKK1 itself. This ubiquitylation consequently inhibits MEKK1-catalyzed phosphorylation of MKK1 and MKK4, thereby down-regulating the MEKK1 activation of the ERK1/2 and JNK pathways. The inhibition of MEKK1 phosphorylation activity is independent of proteasome-dependent degradation of MEKK1. In fact, ubiquitylated MEKK1 seems to be a stable protein in cells. Although it is difficult to quantitate because of the poly/multiubiquitylation of MEKK1 and the consequent laddering in SDS-PAGE, it appears that ubiquitylated MEKK1 actually accumulates in cells and is not degraded significantly by the proteasome. We propose that the ubiquitylation-dependent uncoupling of MEKK1-catalyzed phosphorylation of MKK1 and MKK4 represents a novel function for protein ubiquitylation.It appears that the C441A MEKK1 protein has greater activity toward MKK1 and MKK4 than wild type MEKK1, based on the relative protein levels in the immunoprecipitations. This is consistent with the PHD having a negative regulatory function that correlates with its E3 ligase activity. Certainly, other proteins that interact with MEKK1 may be ubiquitylated by the PHD E3 ligase activity of MEKK1. The E3 ligase function of MEKK1 is likely to down-regulate MEKK1 activation of ERK1/2 and JNK and also ubiquitylate other proteins to regulate their function and/or targeting to the proteasome for degradation.How could MEKK1 ubiquitylation inhibit MKK1 and MKK4 phosphorylation? The ubiquitin modification of MEKK1 may simply block binding of MKK1 and MKK4. Adjacent to the COOH-terminal kinase domain are two UIMs that could interact with ubiquitins covalently bound to MEKK1 and induce a steric hindrance for substrate interaction. The fact that a full-length 196-kDa MEKK1 is required for MEKK1 ubiquitylation suggests that an intramolecular interaction is required for regulation of the MEKK1 E3 ligase activity. It should be noted that our studies were done using transfection. We must still define the role of ubiquitylation of endogenous MEKK1 in cells.The rapidly expanding functions for ubiquitin modification of proteins indicates that ubiquitin is used to regulate protein targeting, scaffolding, and activity. In regards to positive regulation of signal transduction, the polyubiquitylation of TRAF6 is involved in the organization of p38 and NFκB signaling complexes (8Deng L. Wang C. Spencer E. Yang L. Braun A. You J. Slaughter C. Pickart C. Chen Z.J. Cell. 2000; 103: 351-361Abstract Full Text Full Text PDF PubMed Scopus (1485) Google Scholar, 9Wang C. Deng L. Hong M. Akkaraju G.R. Inoue J. Chen Z.J. Nature. 2001; 412: 346-351Crossref PubMed Scopus (1612) Google Scholar). In contrast, activation of the E3 ligase activity of c-Cbl by Src kinase phosphorylation of c-Cbl (17Yokouchi M. Kondo T. Sanjay A. Houghton A. Yoshimura A. Komiya S. Zhang H. Baron R. J. Biol. Chem. 2001; 276: 35185-35193Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar, 18Kassenbrock C.K. Hunter S. Garl P. Johnson G.L. Anderson S.M. J. Biol. Chem. 2002; 277: 24967-24975Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar) results in the ubiquitylation of Src and the receptors for EGF (19Levkowitz G. Waterman H. Zamir E. Kam Z. Oved S. Langdon W.Y. Beguinot L. Geiger B. Yarden Y. Genes Dev. 1998; 12: 3663-3674Crossref PubMed Scopus (714) Google Scholar, 20Yokouchi M. Kondo T. Houghton A. Bartkiewicz M. Horne W.C. Zhang H. Yoshimura A. Baron R. J. Biol. Chem. 1999; 274: 31707-31712Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar, 21Levkowitz G. Waterman H. Ettenberg S.A. Katz M. Tsygankov A.Y. Alroy I. Lavi S. Iwai K. Reiss Y. Ciechanover A. Lipkowitz S. Yarden Y. Mol. Cell. 1999; 4: 1029-1040Abstract Full Text Full Text PDF PubMed Scopus (829) Google Scholar) and PDGF (22Rosenkranz S. Ikuno Y. Leong F.L. Klinghoffer R.A. Miyake S. Band H. Kazlauskas A. J. Biol. Chem. 2000; 275: 9620-9627Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Thus, c-Cbl down-regulates tyrosine kinase signaling. It has also been shown in yeast that pheromone activation of the mating pathway involving the MAPK, Fus3, stimulates a feedback ubiquitylation and degradation of Ste11, a MAPK kinase kinase (23Esch R.K. Errede B. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 9160-9165Crossref PubMed Scopus (48) Google Scholar). Our results demonstrate that the PHD of MEKK1 is required for inhibition of ERK1/2 and JNK activation by MEKK1. The C441A MEKK1 mutant protein stimulates the ERK1/2 and JNK pathways, and this activation is not down-regulated as is seen with wild type MEKK1, showing the requirement of the PHD of MEKK1 for ubiquitin-dependent inhibition of MEKK1 substrate phosphorylation. The regulation of Ste11 and MEKK1, both MAPK kinase kinases, by ubiquitylation appears different. Ste11 is degraded and MEKK1 substrate phosphorylation is inhibited when each is modified with ubiquitin. The function of the MEKK1 PHD is a previously unrecognized mechanism for controlling the activity of a MAPK kinase kinase and the turn-off of MAPK signaling in cells. Ubiquitin is a highly conserved 76-amino acid globular protein that was identified as the first protein to act as a covalent modifier. Ubiquitylation is the process of attachment of ubiquitin to a target protein and is a multistep process that involves the action of at least three classes of enzyme: the ubiquitin-activating enzyme (E1),1 a ubiquitin-conjugating enzyme (E2), and a ubiquitin ligase (E3), which assists in substrate recognition and the transfer of ubiquitin to the target protein. There are two main types of E3 enzyme, those that contain a homology to E6 Cterminus (HECT) domain and those that have areally interesting newgene (RING finger or RING finger-like) domain (1Glickman M.H. Ciechanover A. Physiol. Rev. 2002; 82: 373-428Crossref PubMed Scopus (3304) Google Scholar). The plant homeobox domain (PHD) is a RING finger-like domain defined by a series of cysteine and histidine residues with a characteristic spacing that mediates coordination of two zinc ions in a cross-brace structure (2Pascual J. Martinez-Yamout M. Dyson H.J. Wright P.E. J. Mol. Biol. 2000; 304: 723-729Crossref PubMed Scopus (125) Google Scholar). Disruption of a metal coordinating residue within the RING finger or PHD has been shown to inactivate the ligase properties of the enzyme (3Hashizume R. Fukuda M. Maeda I. Nishikawa H. Oyake D. Yabuki Y. Ogata H. Ohta T. J. Biol. Chem. 2001; 276: 14537-14540Abstract Full Text Full Text PDF PubMed Scopus (542) Google Scholar). Classically, ubiquitin is involved in the homeostasis of cellular proteins by removing unnecessary, deleterious, or misfolded proteins primarily through the ubiquitin-proteasome degradation pathway (4Jennissen H.P. Eur. J. Biochem. 1995; 231: 1-30Crossref PubMed Scopus (112) Google Scholar). Ubiquitin has been shown to function in many cellular processes including cell cycle progression, apoptosis, cell differentiation, and DNA repair (5Naujokat C. Hoffmann S. Lab. Invest. 2002; 82: 965-980Crossref PubMed Scopus (218) Google Scholar). Recently, ubiquitylation of specific proteins has been shown to have functions independent of proteasome-mediated degradation (6Marx J. Science. 2002; 297: 1792-1794Crossref PubMed Scopus (40) Google Scholar). For example, vesicular sorting and TRAF6 organization of the TAK1 signal transduction complex has been shown to involve ubiquitylation of proteins for the control of protein-protein interactions (7Katzmann D.J. Babst M. Emr S.D. Cell. 2001; 106: 145-155Abstract Full Text Full Text PDF PubMed Scopus (1110) Google Scholar, 8Deng L. Wang C. Spencer E. Yang L. Braun A. You J. Slaughter C. Pickart C. Chen Z.J. Cell. 2000; 103: 351-361Abstract Full Text Full Text PDF PubMed Scopus (1485) Google Scholar, 9Wang C. Deng L. Hong M. Akkaraju G.R. Inoue J. Chen Z.J. Nature. 2001; 412: 346-351Crossref PubMed Scopus (1612) Google Scholar). MEKK1 is a 196-kDa serine/threonine MAPK kinase kinase that can regulate both the extracellular-regulated kinase (ERK1/2) and the c-Jun NH2-terminal kinase (JNK) pathways in response to specific stimuli (10Yujiri T. Sather S. Fanger G.R. Johnson G.L. Science. 1998; 282: 1911-1914Crossref PubMed Scopus (280) Google Scholar). We and others have shown that MEKK1 is activated by a variety of stimuli that alter cell shape and is required for normal cell motility (11Yujiri T. Ware M. Widmann C. Oyer R. Russell D. Chan E. Zaitsu Y. Clarke P. Tyler K. Oka Y. Fanger G.R. Henson P. Johnson G.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7272-7277Crossref PubMed Scopus (207) Google Scholar, 12Xia Y. Makris C., Su, B., Li, E. Yang J. Nemerow G.R. Karin M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5243-5248Crossref PubMed Scopus (231) Google Scholar). MEKK1 is a complex protein containing multiple identified domains that may serve to regulate function. The E3 activity of the PHD of MEKK1 was recently described by Lu et al.(13Lu Z., Xu, S. Joazeiro C. Cobb M.H. Hunter T. Mol. Cell. 2002; 9: 945-956Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar). Here we demonstrate that full-length MEKK1 requires an intact PHD for its own ubiquitylation, and this ubiquitylation impairs its ability to phosphorylate MKK1 and MKK4. DISCUSSIONMEKK1 is unique among MAPK kinase kinases in that it encodes a PHD in its NH2 terminus. The MEKK1 PHD has E3 ligase activity (13Lu Z., Xu, S. Joazeiro C. Cobb M.H. Hunter T. Mol. Cell. 2002; 9: 945-956Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar) that is dependent on the kinase activity of the full-length 196-kDa MEKK1 protein. Lu et al. (13Lu Z., Xu, S. Joazeiro C. Cobb M.H. Hunter T. Mol. Cell. 2002; 9: 945-956Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar) demonstrated that a GST fusion of the MEKK1 PHD had E3 ligase activity in vitro. Our work demonstrates that a functional PHD is required for MEKK1 poly/multiubiquitylation. Thus, activation of MEKK1 not only stimulates its ability to phosphorylate and activate MKK1 and MKK4 in the ERK1/2 and JNK pathways but also leads to the ubiquitylation of MEKK1 itself. This ubiquitylation consequently inhibits MEKK1-catalyzed phosphorylation of MKK1 and MKK4, thereby down-regulating the MEKK1 activation of the ERK1/2 and JNK pathways. The inhibition of MEKK1 phosphorylation activity is independent of proteasome-dependent degradation of MEKK1. In fact, ubiquitylated MEKK1 seems to be a stable protein in cells. Although it is difficult to quantitate because of the poly/multiubiquitylation of MEKK1 and the consequent laddering in SDS-PAGE, it appears that ubiquitylated MEKK1 actually accumulates in cells and is not degraded significantly by the proteasome. We propose that the ubiquitylation-dependent uncoupling of MEKK1-catalyzed phosphorylation of MKK1 and MKK4 represents a novel function for protein ubiquitylation.It appears that the C441A MEKK1 protein has greater activity toward MKK1 and MKK4 than wild type MEKK1, based on the relative protein levels in the immunoprecipitations. This is consistent with the PHD having a negative regulatory function that correlates with its E3 ligase activity. Certainly, other proteins that interact with MEKK1 may be ubiquitylated by the PHD E3 ligase activity of MEKK1. The E3 ligase function of MEKK1 is likely to down-regulate MEKK1 activation of ERK1/2 and JNK and also ubiquitylate other proteins to regulate their function and/or targeting to the proteasome for degradation.How could MEKK1 ubiquitylation inhibit MKK1 and MKK4 phosphorylation? The ubiquitin modification of MEKK1 may simply block binding of MKK1 and MKK4. Adjacent to the COOH-terminal kinase domain are two UIMs that could interact with ubiquitins covalently bound to MEKK1 and induce a steric hindrance for substrate interaction. The fact that a full-length 196-kDa MEKK1 is required for MEKK1 ubiquitylation suggests that an intramolecular interaction is required for regulation of the MEKK1 E3 ligase activity. It should be noted that our studies were done using transfection. We must still define the role of ubiquitylation of endogenous MEKK1 in cells.The rapidly expanding functions for ubiquitin modification of proteins indicates that ubiquitin is used to regulate protein targeting, scaffolding, and activity. In regards to positive regulation of signal transduction, the polyubiquitylation of TRAF6 is involved in the organization of p38 and NFκB signaling complexes (8Deng L. Wang C. Spencer E. Yang L. Braun A. You J. Slaughter C. Pickart C. Chen Z.J. Cell. 2000; 103: 351-361Abstract Full Text Full Text PDF PubMed Scopus (1485) Google Scholar, 9Wang C. Deng L. Hong M. Akkaraju G.R. Inoue J. Chen Z.J. Nature. 2001; 412: 346-351Crossref PubMed Scopus (1612) Google Scholar). In contrast, activation of the E3 ligase activity of c-Cbl by Src kinase phosphorylation of c-Cbl (17Yokouchi M. Kondo T. Sanjay A. Houghton A. Yoshimura A. Komiya S. Zhang H. Baron R. J. Biol. Chem. 2001; 276: 35185-35193Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar, 18Kassenbrock C.K. Hunter S. Garl P. Johnson G.L. Anderson S.M. J. Biol. Chem. 2002; 277: 24967-24975Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar) results in the ubiquitylation of Src and the receptors for EGF (19Levkowitz G. Waterman H. Zamir E. Kam Z. Oved S. Langdon W.Y. Beguinot L. Geiger B. Yarden Y. Genes Dev. 1998; 12: 3663-3674Crossref PubMed Scopus (714) Google Scholar, 20Yokouchi M. Kondo T. Houghton A. Bartkiewicz M. Horne W.C. Zhang H. Yoshimura A. Baron R. J. Biol. Chem. 1999; 274: 31707-31712Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar, 21Levkowitz G. Waterman H. Ettenberg S.A. Katz M. Tsygankov A.Y. Alroy I. Lavi S. Iwai K. Reiss Y. Ciechanover A. Lipkowitz S. Yarden Y. Mol. Cell. 1999; 4: 1029-1040Abstract Full Text Full Text PDF PubMed Scopus (829) Google Scholar) and PDGF (22Rosenkranz S. Ikuno Y. Leong F.L. Klinghoffer R.A. Miyake S. Band H. Kazlauskas A. J. Biol. Chem. 2000; 275: 9620-9627Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Thus, c-Cbl down-regulates tyrosine kinase signaling. It has also been shown in yeast that pheromone activation of the mating pathway involving the MAPK, Fus3, stimulates a feedback ubiquitylation and degradation of Ste11, a MAPK kinase kinase (23Esch R.K. Errede B. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 9160-9165Crossref PubMed Scopus (48) Google Scholar). Our results demonstrate that the PHD of MEKK1 is required for inhibition of ERK1/2 and JNK activation by MEKK1. The C441A MEKK1 mutant protein stimulates the ERK1/2 and JNK pathways, and this activation is not down-regulated as is seen with wild type MEKK1, showing the requirement of the PHD of MEKK1 for ubiquitin-dependent inhibition of MEKK1 substrate phosphorylation. The regulation of Ste11 and MEKK1, both MAPK kinase kinases, by ubiquitylation appears different. Ste11 is degraded and MEKK1 substrate phosphorylation is inhibited when each is modified with ubiquitin. The function of the MEKK1 PHD is a previously unrecognized mechanism for controlling the activity of a MAPK kinase kinase and the turn-off of MAPK signaling in cells. MEKK1 is unique among MAPK kinase kinases in that it encodes a PHD in its NH2 terminus. The MEKK1 PHD has E3 ligase activity (13Lu Z., Xu, S. Joazeiro C. Cobb M.H. Hunter T. Mol. Cell. 2002; 9: 945-956Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar) that is dependent on the kinase activity of the full-length 196-kDa MEKK1 protein. Lu et al. (13Lu Z., Xu, S. Joazeiro C. Cobb M.H. Hunter T. Mol. Cell. 2002; 9: 945-956Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar) demonstrated that a GST fusion of the MEKK1 PHD had E3 ligase activity in vitro. Our work demonstrates that a functional PHD is required for MEKK1 poly/multiubiquitylation. Thus, activation of MEKK1 not only stimulates its ability to phosphorylate and activate MKK1 and MKK4 in the ERK1/2 and JNK pathways but also leads to the ubiquitylation of MEKK1 itself. This ubiquitylation consequently inhibits MEKK1-catalyzed phosphorylation of MKK1 and MKK4, thereby down-regulating the MEKK1 activation of the ERK1/2 and JNK pathways. The inhibition of MEKK1 phosphorylation activity is independent of proteasome-dependent degradation of MEKK1. In fact, ubiquitylated MEKK1 seems to be a stable protein in cells. Although it is difficult to quantitate because of the poly/multiubiquitylation of MEKK1 and the consequent laddering in SDS-PAGE, it appears that ubiquitylated MEKK1 actually accumulates in cells and is not degraded significantly by the proteasome. We propose that the ubiquitylation-dependent uncoupling of MEKK1-catalyzed phosphorylation of MKK1 and MKK4 represents a novel function for protein ubiquitylation. It appears that the C441A MEKK1 protein has greater activity toward MKK1 and MKK4 than wild type MEKK1, based on the relative protein levels in the immunoprecipitations. This is consistent with the PHD having a negative regulatory function that correlates with its E3 ligase activity. Certainly, other proteins that interact with MEKK1 may be ubiquitylated by the PHD E3 ligase activity of MEKK1. The E3 ligase function of MEKK1 is likely to down-regulate MEKK1 activation of ERK1/2 and JNK and also ubiquitylate other proteins to regulate their function and/or targeting to the proteasome for degradation. How could MEKK1 ubiquitylation inhibit MKK1 and MKK4 phosphorylation? The ubiquitin modification of MEKK1 may simply block binding of MKK1 and MKK4. Adjacent to the COOH-terminal kinase domain are two UIMs that could interact with ubiquitins covalently bound to MEKK1 and induce a steric hindrance for substrate interaction. The fact that a full-length 196-kDa MEKK1 is required for MEKK1 ubiquitylation suggests that an intramolecular interaction is required for regulation of the MEKK1 E3 ligase activity. It should be noted that our studies were done using transfection. We must still define the role of ubiquitylation of endogenous MEKK1 in cells. The rapidly expanding functions for ubiquitin modification of proteins indicates that ubiquitin is used to regulate protein targeting, scaffolding, and activity. In regards to positive regulation of signal transduction, the polyubiquitylation of TRAF6 is involved in the organization of p38 and NFκB signaling complexes (8Deng L. Wang C. Spencer E. Yang L. Braun A. You J. Slaughter C. Pickart C. Chen Z.J. Cell. 2000; 103: 351-361Abstract Full Text Full Text PDF PubMed Scopus (1485) Google Scholar, 9Wang C. Deng L. Hong M. Akkaraju G.R. Inoue J. Chen Z.J. Nature. 2001; 412: 346-351Crossref PubMed Scopus (1612) Google Scholar). In contrast, activation of the E3 ligase activity of c-Cbl by Src kinase phosphorylation of c-Cbl (17Yokouchi M. Kondo T. Sanjay A. Houghton A. Yoshimura A. Komiya S. Zhang H. Baron R. J. Biol. Chem. 2001; 276: 35185-35193Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar, 18Kassenbrock C.K. Hunter S. Garl P. Johnson G.L. Anderson S.M. J. Biol. Chem. 2002; 277: 24967-24975Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar) results in the ubiquitylation of Src and the receptors for EGF (19Levkowitz G. Waterman H. Zamir E. Kam Z. Oved S. Langdon W.Y. Beguinot L. Geiger B. Yarden Y. Genes Dev. 1998; 12: 3663-3674Crossref PubMed Scopus (714) Google Scholar, 20Yokouchi M. Kondo T. Houghton A. Bartkiewicz M. Horne W.C. Zhang H. Yoshimura A. Baron R. J. Biol. Chem. 1999; 274: 31707-31712Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar, 21Levkowitz G. Waterman H. Ettenberg S.A. Katz M. Tsygankov A.Y. Alroy I. Lavi S. Iwai K. Reiss Y. Ciechanover A. Lipkowitz S. Yarden Y. Mol. Cell. 1999; 4: 1029-1040Abstract Full Text Full Text PDF PubMed Scopus (829) Google Scholar) and PDGF (22Rosenkranz S. Ikuno Y. Leong F.L. Klinghoffer R.A. Miyake S. Band H. Kazlauskas A. J. Biol. Chem. 2000; 275: 9620-9627Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Thus, c-Cbl down-regulates tyrosine kinase signaling. It has also been shown in yeast that pheromone activation of the mating pathway involving the MAPK, Fus3, stimulates a feedback ubiquitylation and degradation of Ste11, a MAPK kinase kinase (23Esch R.K. Errede B. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 9160-9165Crossref PubMed Scopus (48) Google Scholar). Our results demonstrate that the PHD of MEKK1 is required for inhibition of ERK1/2 and JNK activation by MEKK1. The C441A MEKK1 mutant protein stimulates the ERK1/2 and JNK pathways, and this activation is not down-regulated as is seen with wild type MEKK1, showing the requirement of the PHD of MEKK1 for ubiquitin-dependent inhibition of MEKK1 substrate phosphorylation. The regulation of Ste11 and MEKK1, both MAPK kinase kinases, by ubiquitylation appears different. Ste11 is degraded and MEKK1 substrate phosphorylation is inhibited when each is modified with ubiquitin. The function of the MEKK1 PHD is a previously unrecognized mechanism for controlling the activity of a MAPK kinase kinase and the turn-off of MAPK signaling in cells.
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