Differential Phosphorylation of c-Jun and JunD in Response to the Epidermal Growth Factor Is Determined by the Structure of MAPK Targeting Sequences

Abstract

MAPK phosphorylation of various substrates is mediated by the presence of docking sites, including the D domain and the DEF motif. Depending on the number and sequences of these domains, substrates are phosphorylated by specific subsets of MAPKs. For example, a D domain targets JNK to c-Jun, whereas a DEF motif is required for ERK phosphorylation of c-Fos. JunD, in contrast, contains both D and DEF domains. Here we show that these motifs mediate JunD phosphorylation in response to either ERK or JNK activation. An intact D domain is required for phosphorylation and activation of JunD by both subtypes of MAPK. The DEF motif acts together with the D domain to elicit efficient phosphorylation of JunD in response to the epidermal growth factor (EGF) but has no function on JunD phosphorylation and activation by JNK signaling. Furthermore, we show that conversion of a c-Jun sequence to a canonical DEF domain, as it is present in JunD, elicits c-Jun activation in response to EGF. Our results suggest that evolution of a particular modular system of MAPK targeting sequences has determined a differential response of JunD and c-Jun to ERK activation. MAPK phosphorylation of various substrates is mediated by the presence of docking sites, including the D domain and the DEF motif. Depending on the number and sequences of these domains, substrates are phosphorylated by specific subsets of MAPKs. For example, a D domain targets JNK to c-Jun, whereas a DEF motif is required for ERK phosphorylation of c-Fos. JunD, in contrast, contains both D and DEF domains. Here we show that these motifs mediate JunD phosphorylation in response to either ERK or JNK activation. An intact D domain is required for phosphorylation and activation of JunD by both subtypes of MAPK. The DEF motif acts together with the D domain to elicit efficient phosphorylation of JunD in response to the epidermal growth factor (EGF) but has no function on JunD phosphorylation and activation by JNK signaling. Furthermore, we show that conversion of a c-Jun sequence to a canonical DEF domain, as it is present in JunD, elicits c-Jun activation in response to EGF. Our results suggest that evolution of a particular modular system of MAPK targeting sequences has determined a differential response of JunD and c-Jun to ERK activation. The AP-1 transcription factor activates the transcription of several genes in response to a variety of external stimuli, including growth factors, tumor promoters, and cytokines. Active AP-1 is a dimeric protein complex whose components are the products of the Jun/ATF and Fos/Fra gene families (1Angel P. Karin M. Biochim. Biophys. Acta. 1991; 107: 129-157Google Scholar, 2Karin M. Liu Z. Zandi E. Curr. Opin. Cell Biol. 1997; 9: 240-246Crossref PubMed Scopus (2324) Google Scholar). The three Jun proteins, c-Jun, JunB, and JunD, share a high degree of sequence homology; however, they display distinct transactivation properties and have different effects on cell proliferation (3Mechta-Grigoriou F. Gerald D. Yaniv M. Oncogene. 2001; 20: 2378-2389Crossref PubMed Scopus (279) Google Scholar). Compared with c-Jun, JunD plays an opposite role in cell growth regulation and transformation; whereas c-Jun behaves as a positive regulator of cell growth and may cause cell transformation, JunD antagonizes both of these effects in immortalized fibroblast (4Pfarr C.M. Mechta F. Spyrou G. Lallemand D. Carillo S. Yaniv M. Cell. 1994; 76: 747-760Abstract Full Text PDF PubMed Scopus (287) Google Scholar, 5Weitzman J.B. Fiette L. Matsuo K. Yaniv M. Mol. Cell. 2000; 6: 1109-1119Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). However, JunD -/- primary fibroblasts present an increased sensitivity to p53-dependent apoptosis induced by UV or tumor necrosis factor-α (5Weitzman J.B. Fiette L. Matsuo K. Yaniv M. Mol. Cell. 2000; 6: 1109-1119Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). Recently, it has been shown that the JNK 1The abbreviations used are: JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; MEK, MAPK/MEK kinase; MKK7, MAPK kinase 7; EGF, epidermal growth factor; HEK 293, human embryonic kidney 293; HA, hemagglutinin A; wt, wild-type./JunD pathway can collaborate with NF-κB to protect embryonic fibroblasts from tumor necrosis factor-α-induced cell death (6Lamb A.J. Ventura J.J. Hess P. Flavell R.A. Davis R.J. Mol. Cell. 2003; 11: 1479-1489Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). A similar scenario has been also reported for c-Jun; however, the molecular mechanisms through which c-Jun and JunD influence cell-survival seem to be different (7Wisdom R. Johnson S.R. Moore C. EMBO J. 1999; 18: 188-197Crossref PubMed Scopus (533) Google Scholar, 8Eferi R. Ricci R. Kenner L. Zenz R. David J.P. Rath M. Wagner E.F. Cell. 2003; 112: 181-192Abstract Full Text Full Text PDF PubMed Scopus (408) Google Scholar). The N-terminal transactivation domains of c-Jun and JunD are more divergent than other functional domains (9Musti A.M. Treier M. Peverali F.A. Bohmann D. Biol. Chem. 1996; 377: 619-624PubMed Google Scholar), suggesting that this difference may account for their diverse biological effects. The N terminus of JunD contains three MAPK phospho-acceptor sites (serine 90, serine 100, and threonine 117), the local sequence contexts of which are essentially identical to those present in the analogous region of c-Jun (serine 63, serine 73, and threonine 91). In the case of c-Jun, it has been well established that phosphorylation of these sites correlates with biological activity (2Karin M. Liu Z. Zandi E. Curr. Opin. Cell Biol. 1997; 9: 240-246Crossref PubMed Scopus (2324) Google Scholar, 10Shaulian E. Karin M. Oncogene. 2001; 20: 2390-2400Crossref PubMed Scopus (1394) Google Scholar). Phosphorylation of c-Jun is predominantly responsive to JNK activation (10Shaulian E. Karin M. Oncogene. 2001; 20: 2390-2400Crossref PubMed Scopus (1394) Google Scholar, 11Davis R.J. Cell. 2000; 103: 239-252Abstract Full Text Full Text PDF PubMed Scopus (3666) Google Scholar); however, in certain cell types serine 63 and serine 73 can be phosphorylated by sustained activation of ERK signaling (12Leppa S. Saffrich R. Ansorge W. Bohmann D. EMBO J. 1999; 17: 4404-4413Crossref Scopus (291) Google Scholar). Conversely the N-terminal domain of JunD is rapidly phosphorylated upon the transient activation of ERK1/2 (13Gallo A. Cuozzo C. Esposito I. Maggiolini M. Bonofiglio D. Vivacqua A. Garramone M. Weiss C. Bohmann D. Musti A.M. Oncogene. 2002; 21: 6434-6445Crossref PubMed Scopus (78) Google Scholar). In addition, JunD can be phosphorylated in vitro by active JNK and activated by the JNK pathway in vivo (14Yazgan A. Pfarr C.M. J. Biol. Chem. 2002; 33: 29710-29718Abstract Full Text Full Text PDF Scopus (45) Google Scholar), but less efficiently than c-Jun (15Kallunki T. Deng T. Hibi M. Karin M. Cell. 1996; 87: 929-939Abstract Full Text Full Text PDF PubMed Scopus (408) Google Scholar, 16Gupta S. Barrett T. Whitmarsh A.J. Cavanagh J. Sluss H.K. Derijard B. Davis R.J. EMBO J. 1996; 15: 2760-2770Crossref PubMed Scopus (1183) Google Scholar). Taken together, these studies suggest that c-Jun and JunD each display a different extent and kinetics of phosphorylation in response to either the JNK or ERK subtype of MAPK. Accumulating evidence indicates that substrate specificity for MAPKs is determined not only by the sequence in the vicinity of the phospho-acceptor sites but also by distinct targeting domains, also referred to as docking sites, which enhance the efficiency of phosphorylation by mediating the interaction of the kinases with their substrate (17Jacobs D. Glossip D. Xing H. Muslin A.J. Kornfeld K. Genes Dev. 1999; 13: 163-175Crossref PubMed Scopus (443) Google Scholar, 18Douglas A. Fantz D.J. Glossip D. Kornfeld K. J. Biol. Chem. 2001; 29: 27256-27265Google Scholar). The first docking site, here referred to as the D domain, has been identified within the c-Jun δ domain, and it was shown to be required for targeting JNK1/2 to c-Jun (19Derijard B. Hibi M. Wu I.H. Barrett T. Su B. Deng T. Karin M. Davis R.J. Cell. 1994; 76: 1025-1037Abstract Full Text PDF PubMed Scopus (2957) Google Scholar, 20Kallunki T. Tsigelny S.B. Sluss I. Derijard H.K. Moore B. Davis R.J. Karin M. Genes Dev. 1994; 8: 2996-3007Crossref PubMed Scopus (595) Google Scholar). Further studies have revealed that analogous D domains exist in other MAPK-responsive transcription factors such as Elk-1 and SAP-1 (21Yang S.H. Yates P.R. Whitmarsh A.J. Davis R.J. Sharrocks A.D. Mol. Cell. Biol. 1998; 18: 710-720Crossref PubMed Scopus (234) Google Scholar, 22Holland P.M. Cooper J.A. Curr. Biol. 1999; 9: R329-R331Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 23Galanis A. Yang S.H. Sharrocks A.D. J. Biol. Chem. 2001; 276: 965-973Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar) and can specify substrate phosphorylation by one or two different subtypes of MAPK (23Galanis A. Yang S.H. Sharrocks A.D. J. Biol. Chem. 2001; 276: 965-973Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 24Yang S.H. Whitmarsh A.J. Davis R.J. Sharrocks A.D. EMBO J. 1998; 17: 1740-1749Crossref PubMed Scopus (268) Google Scholar, 25Barsyte-Lovejoy D. Galanis A. Sharrocks A.D. J. Biol. Chem. 2002; 12: 9896-9903Abstract Full Text Full Text PDF Scopus (71) Google Scholar). A second motif, referred to either as the FXF or DEF motif, has been found in certain substrates and shown to function together with the D domain to increase the affinity for ERK and the kinetics of ERK phosphorylation (18Douglas A. Fantz D.J. Glossip D. Kornfeld K. J. Biol. Chem. 2001; 29: 27256-27265Google Scholar). However, the DEF domain can function independently from the D domain but requires a sustained signal to specify ERK phosphorylation. This appears to be the case with c-Fos (26Murphy L.O. Smith S. Chen R.H. Fingar D.C. Blenis J. Nat. Cell Biol. 2002; 8: 556-564Crossref Scopus (767) Google Scholar). c-Jun and JunD display divergent configurations of the D and DEF domains, the former presenting only a D box whereas the latter contains both an N-terminal D motif and a C-terminal DEF domain (Fig. 1A), suggesting that this difference could account for the distinct responses of the two Jun proteins to ERK signaling. Consistent with this hypothesis, Drosophila Jun is a target of the Drosophila ERK type of MAP kinase called Rolled (27Peverali F.A. Isaksson A. Papavassiliou A.G. Plastina P. Staszewski L.M. Mlodzik M. Bohmann D. EMBO J. 1996; 15: 3943-3950Crossref PubMed Scopus (49) Google Scholar) and contains both D and DEF putative domains (Fig. 1A). In the present study we have analyzed the role of putative MAPK targeting motifs present in JunD in regulating JunD phosphorylation and transcriptional activity. Also, we have investigated whether the introduction of a canonical DEF motif in c-Jun might rescue c-Jun activation in response to EGF. Cell Culture and Transfection—Human embryonic kidney cell line HEK 293 cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum (Invitrogen). Mouse fibroblast cell line NIH-3T3 cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% calf serum (Invitrogen). All media were supplemented with 2 mm glutamine, 100 IU/ml penicillin-G, and 100 μg/ml streptomycin (Invitrogen). HEK 293 and NIH-3T3 cells were seeded at 8 × 105 cells per 100-mm plate and transfected with the indicated plasmids DNA on the following day by LipofectAMINE as recommended by the manufacturer (Invitrogen) Transfected cells were harvested 36-48 h after transfection. Plasmids—All DNA manipulations were performed essentially as described (28Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). The expression vectors for the JunD-HA, c-Jun-HA, ERK2-MEK1-LA, and MKK7 plasmids have been described (9Musti A.M. Treier M. Peverali F.A. Bohmann D. Biol. Chem. 1996; 377: 619-624PubMed Google Scholar, 30Tournier C. Whitmarsh A.J. Cavanagh J. Barrett T. Davis R.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 14: Scopus Google Scholar, E. Curr. Biol. 1998; 8: Full Text Full Text PDF PubMed Google Scholar). The expression plasmids for the and proteins have been described (13Gallo A. Cuozzo C. Esposito I. Maggiolini M. Bonofiglio D. Vivacqua A. Garramone M. Weiss C. Bohmann D. Musti A.M. Oncogene. 2002; 21: 6434-6445Crossref PubMed Scopus (78) Google Scholar). The expression for was by a DNA to a of human c-Jun a expression with a DNA domain of c-Jun and JunD as well as and were by a system proteins were with the and a expression in HEK 293 were performed the as recommended by the manufacturer were for and activity was with a to the of the activity was to the by the from HEK 293 cells were in a mm mm mm and were in for and the proteins were on 10% and by as described (9Musti A.M. Treier M. Peverali F.A. Bohmann D. Biol. Chem. 1996; 377: 619-624PubMed Google Scholar). proteins were by a and were by an that JunD it is phosphorylated at serine 100 and c-Jun is phosphorylated at serine ERK1/2 was by a was a ERK1/2 and JNK1/2 kinases were by and were used to the of the were by a by Phosphorylation of JunD by D and DEF contains two domains with sequence to the ERK docking sites referred to as the D domain, and referred to as the DEF motif (Fig. 1A). have investigated whether the D and DEF domains present in JunD to ERK phosphorylation. this we have JunD in which the motif of the D domain or the motif were either or as indicated in the of these on JunD phosphorylation in response to the transient activation of ERK signaling by EGF. JunD proteins were in HEK 293 and JunD phosphorylation was by an that JunD only it is phosphorylated at serine 100 (13Gallo A. Cuozzo C. Esposito I. Maggiolini M. Bonofiglio D. Vivacqua A. Garramone M. Weiss C. Bohmann D. Musti A.M. Oncogene. 2002; 21: 6434-6445Crossref PubMed Scopus (78) Google Scholar). indicated by the sensitivity of phosphorylation to the A.J. R.A. F. R.A. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar) with efficiently and rapidly JunD phosphorylation in an In contrast, phosphorylation of JunD was in the which a D The which the canonical DEF motif, was whereas phosphorylation was in the in which both the D and the DEF motifs are results that the D domain is for phosphorylation of JunD and that it can phosphorylation of JunD independently of the DEF motif. In contrast, the DEF motif can only function in the presence of an intact D domain but is required for efficient phosphorylation of JunD in response to EGF. in the D and DEF motifs of JunD or no on the of JunD to ERK which can be found in the of this However, we found that c-Jun is also to with ERK suggesting that may to the of ERK by the and for an of JunD also investigated the role of the D and DEF domains on in vivo phosphorylation of JunD in response to JNK signaling. or the various in the D and DEF domains were in HEK 293 and the JNK pathway was induced by a of ERK were performed in the presence of the shown in phosphorylation of the JunD serine 100 was in the JunD an D domain or in the both the D and DEF In contrast, of the DEF motif not phosphorylation of serine 100, as by with and results that the D domain is to mediate in vivo phosphorylation of JunD by the DEF motif is not required for JunD phosphorylation by the JNK Furthermore, of in vivo by that the of a D domain on JunD phosphorylation may be from docking JNK found in the of this these results the that are of JunD D and DEF we investigated the role of the D and DEF domains on transcriptional activation by JunD in response to EGF. transcription was in vivo by a system in HEK 293 The protein contains the N-terminal region of JunD to the DNA domain of (13Gallo A. Cuozzo C. Esposito I. Maggiolini M. Bonofiglio D. Vivacqua A. Garramone M. Weiss C. Bohmann D. Musti A.M. Oncogene. 2002; 21: 6434-6445Crossref PubMed Scopus (78) Google Scholar). have of a protein the indicated in activation following was for each of the proteins as well as for a of serine 90, serine 100, and threonine shown in to of activity but no on the that the of JunD transcriptional activity was mediated by the phosphorylation of MAPK only in the D domain or in both the D and DEF domain of of the DEF motif to the of on activity. results well with the of the on phosphorylation of JunD (Fig. and that the D domain is and for JunD activity in response to however, an intact DEF domain is required for JunD activation. have shown that of a active of the kinase protein E. Curr. Biol. 1998; 8: Full Text Full Text PDF PubMed Google Scholar), the activity of but has no on (13Gallo A. Cuozzo C. Esposito I. Maggiolini M. Bonofiglio D. Vivacqua A. Garramone M. Weiss C. Bohmann D. Musti A.M. Oncogene. 2002; 21: 6434-6445Crossref PubMed Scopus (78) Google Scholar). we transcriptional activation of the various in response to of increased the activity of but no on expression no on or (Fig. of the DEF domain only a on activity induced by ERK2-MEK1-LA, suggesting that the DEF motif is less in presence of sustained ERK activation. A DEF to c-Jun in to results shown in 2 and that the D domain and the DEF motif of JunD function to mediate phosphorylation and activation of JunD in response to in an c-Jun phosphorylation and transcriptional activity are not responsive to transient activation of ERK signaling (2Karin M. Liu Z. Zandi E. Curr. Opin. Cell Biol. 1997; 9: 240-246Crossref PubMed Scopus (2324) Google Scholar, F. Gerald D. Yaniv M. Oncogene. 2001; 20: 2378-2389Crossref PubMed Scopus (279) Google Scholar). A of JunD and c-Jun sequences the MAPK sites that c-Jun from JunD in the region the latter contains the DEF motif (Fig. 1A). The sequence present in JunD contains the and in and The sequence present in c-Jun the in which has been shown to be for ERK phosphorylation of either proteins or c-Fos (18Douglas A. Fantz D.J. Glossip D. Kornfeld K. J. Biol. Chem. 2001; 29: 27256-27265Google Scholar, L.O. Smith S. Chen R.H. Fingar D.C. Blenis J. Nat. Cell Biol. 2002; 8: 556-564Crossref Scopus (767) Google Scholar). This to whether this could account for the differential activation of c-Jun and JunD proteins in response to ERK signaling. this hypothesis, we the activity of a protein with a the sequence of c-Jun was to as it is in the DEF motif of JunD (Fig. also a the was a protein the MAPK phospho-acceptor sites to (Fig. The various proteins were in HEK 293 cells together with a and their in response to were shown in no on the activity of or but induced the activity of that the of a canonical DEF domain c-Jun responsive to in a the that the of the protein was we activity in response to JNK signaling by the MKK7 kinase C. Whitmarsh A.J. Cavanagh J. Barrett T. Davis R.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 14: Scopus Google Scholar). of MKK7 induced the activity of (Fig. However, MKK7 a on suggesting that conversion of the motif in the domain a on c-Jun activation by we the phosphorylation of the c-Jun or protein in response to transient activation of ERK signaling. or JunD proteins were in and the phosphorylation of each in response to serum was by an that either c-Jun or JunD only they are phosphorylated at serine 73 or serine 100, phosphorylation of JunD was rapidly induced in response to serum (Fig. In contrast, of serum an on the of c-Jun phosphorylation (Fig. However, phosphorylation of the was induced after of the with the results shown in these that introduction of the DEF motif c-Jun to be responsive to transient activation ERK signaling. AP-1 activity by MAPK that mediate phosphorylation of distinct substrates (2Karin M. Liu Z. Zandi E. Curr. Opin. Cell Biol. 1997; 9: 240-246Crossref PubMed Scopus (2324) Google Scholar, 10Shaulian E. Karin M. Oncogene. 2001; 20: 2390-2400Crossref PubMed Scopus (1394) Google Scholar). Depending on the of serum and growth factor have been shown to AP-1 by distinct activation of ERK, as by EGF, to phosphorylation of transcription factors of the complex factor such as Elk-1 of the gene (10Shaulian E. Karin M. Oncogene. 2001; 20: 2390-2400Crossref PubMed Scopus (1394) Google Scholar). have shown that AP-1 regulation by ERK signaling may also at the of phosphorylation and activation of JunD (13Gallo A. Cuozzo C. Esposito I. Maggiolini M. Bonofiglio D. Vivacqua A. Garramone M. Weiss C. Bohmann D. Musti A.M. Oncogene. 2002; 21: 6434-6445Crossref PubMed Scopus (78) Google Scholar). The present study that activation of JunD in response to is mediated by MAPK targeting motifs present in the N-terminal region of The of the (Fig. on JunD phosphorylation indicates that the phosphorylation of JunD is mediated by ERK activation. Furthermore, the of JunD phosphorylation in response to the activation kinetics of ERK than of JNK as shown by the of the phosphorylation of ERK1/2 and JNK1/2 (Fig. The functional phosphorylation and JunD activation is by the that transcription was induced by only the three MAPK phospho-acceptor sites, present in the transactivation domain, were intact (Fig. substrates of ERK have been shown to docking referred to as the D domain and the DEF motif, on their number and the affinity for ERK (17Jacobs D. Glossip D. Xing H. Muslin A.J. Kornfeld K. Genes Dev. 1999; 13: 163-175Crossref PubMed Scopus (443) Google Scholar, 18Douglas A. Fantz D.J. Glossip D. Kornfeld K. J. Biol. Chem. 2001; 29: 27256-27265Google Scholar). ERK target proteins both such as Elk-1 and have been shown to have affinity for ERK than other substrates only a DEF or a D motif, such as proteins P.M. Cooper J.A. Curr. Biol. 1999; 9: R329-R331Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). that JunD contains both the D domain and the DEF motif the MAPK phospho-acceptor sites in a that is of the of Elk-1 and SAP-1 (Fig. 1A). sequence for MAPK docking sites have been determined for both the D and the DEF motifs (17Jacobs D. Glossip D. Xing H. Muslin A.J. Kornfeld K. Genes Dev. 1999; 13: 163-175Crossref PubMed Scopus (443) Google Scholar, 18Douglas A. Fantz D.J. Glossip D. Kornfeld K. J. Biol. Chem. 2001; 29: 27256-27265Google Scholar). of JunD of these sequences has to that the D domain JunD phosphorylation by both ERK and JNK and the D domain was to JunD activation by JNK signaling and both the D and DEF motifs were required for efficient phosphorylation and activation of JunD in response to transient activation of ERK, as by (Fig. and the other the DEF domain not to be for JunD activation by sustained activation of ERK signaling (Fig. suggest that the D domain is to specify both JNK and ERK however, the presence of the D and DEF by JunD affinity for ERK, JunD to be activated in response to EGF. Consistent with this c-Fos and c-Jun, the DEF or the D domain (Fig. 1A), are phosphorylated by extracellular that cause sustained activation of ERK signaling (12Leppa S. Saffrich R. Ansorge W. Bohmann D. EMBO J. 1999; 17: 4404-4413Crossref Scopus (291) Google Scholar, L.O. Smith S. Chen R.H. Fingar D.C. Blenis J. Nat. Cell Biol. 2002; 8: 556-564Crossref Scopus (767) Google Scholar), but not in response to (10Shaulian E. Karin M. Oncogene. 2001; 20: 2390-2400Crossref PubMed Scopus (1394) Google Scholar, S. Saffrich R. Ansorge W. Bohmann D. EMBO J. 1999; 17: 4404-4413Crossref Scopus (291) Google Scholar). In addition, conversion of the sequence of c-Jun the motif c-Jun an transcription factor (Fig. of evidence that introduction of a DEF motif increased ERK phosphorylation of the DEF domain could rescue transcription only the MAPK sites were intact (Fig. introduction of the DEF domain c-Jun phosphorylation by transient activation of ERK signaling. Depending on the cell transient or sustained activation of the pathway has been with specific biological such as or cell (26Murphy L.O. Smith S. Chen R.H. Fingar D.C. Blenis J. Nat. Cell Biol. 2002; 8: 556-564Crossref Scopus (767) Google Scholar, Cell. Full Text PDF PubMed Scopus Google Scholar, T. Cell. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). Our results that evolution of a distinct of ERK targeting sites has c-Jun and JunD in their to the of (Fig. to this Drosophila a target of both and (27Peverali F.A. Isaksson A. Papavassiliou A.G. Plastina P. Staszewski L.M. Mlodzik M. Bohmann D. EMBO J. 1996; 15: 3943-3950Crossref PubMed Scopus (49) Google Scholar, J. Staszewski L.M. Mlodzik M. Bohmann D. Genes Dev. 1997; 13: Scopus Google Scholar), contains putative D and DEF motifs (Fig. 1A). In study that of ERK activation are the of c-Jun and JunD activation. In a differential regulation by ERK signaling may account for the distinct through which c-Jun and JunD cell proliferation and R.J. Davis for the MKK7 and M. for the are to for and A to D. Bohmann for on the also and for their of the R. and D. for with