Ubiquitinylation Is Not an Absolute Requirement for Degradation of c-Jun Protein by the 26 S Proteasome

Degradation of rapidly turned over cellular proteins is commonly thought to be energy dependent, to require tagging of protein substrates by multi-ubiquitin chains, and to involve the 26 S proteasome, which is the major neutral proteolytic activity in both the cytosol and the nucleus. The c-Jun oncoprotein is very unstable in vivo. Using cell-free degradation assays, we show that ubiquitinylation, along with other types of tagging, is not an absolute prerequisite for ATP-dependent degradation of c-Jun by the 26 S proteasome. This indicates that a protein may bear intrinsic structural determinants allowing its selective recognition and breakdown by the 26 S proteasome. Moreover, taken together with observations by different groups, our data point to the notion of the existence of multiple degradation pathways operating on c-Jun. Degradation of rapidly turned over cellular proteins is commonly thought to be energy dependent, to require tagging of protein substrates by multi-ubiquitin chains, and to involve the 26 S proteasome, which is the major neutral proteolytic activity in both the cytosol and the nucleus. The c-Jun oncoprotein is very unstable in vivo. Using cell-free degradation assays, we show that ubiquitinylation, along with other types of tagging, is not an absolute prerequisite for ATP-dependent degradation of c-Jun by the 26 S proteasome. This indicates that a protein may bear intrinsic structural determinants allowing its selective recognition and breakdown by the 26 S proteasome. Moreover, taken together with observations by different groups, our data point to the notion of the existence of multiple degradation pathways operating on c-Jun. c-Jun protein is a transcription factor belonging to the AP-1 family (for a complete review, see Ref. 1Angel P. Herrlich P. The FOS and JUN Families of Transcription Factors. CRC Press, Boca Raton, FL1994Google Scholar). It is a basic domain-leucine zipper protein that must homodimerize or heterodimer-ize with other partners such as c-Fos to recognize specific DNA-binding motifs known as TRE, for 12-O-tetradecanoyulphorbol-13-acetate-responsive element or AP-1 binding sites. Several lines of evidence points to imply this protein as a positive regulator of cell proliferation. For example, (i) it is rapidly and transiently induced in quiescent embryonic fibroblasts stimulated by growth factors (2Quantin B. Breatnach R. Nature. 1988; 334: 538-540Crossref PubMed Scopus (185) Google Scholar, 3Ryseck R.P. Hirai S.-H. Yaniv M. Bravo R. Nature. 1988; 334: 535-538Crossref PubMed Scopus (385) Google Scholar), (ii) microinjection of anti-c-Jun antibodies inhibits growth of fibroblasts (4Kovary K. Bravo R. Mol. Cell. Biol. 1991; 11: 4466-4472Crossref PubMed Scopus (394) Google Scholar), (iii) disruption of c-jun gene by homologous recombination entails retarded cell growth in culture (5Johnson R.S. Lingen B.V. Papaionnou V.E. Spiegelman B.M. Genes & Dev. 1993; 7: 1309-1317Crossref PubMed Scopus (345) Google Scholar), and (iv) its overexpression confers a transformed phenotype to chicken embryo fibroblasts (6Bos T.J. Monteclaro F.S. Mitsunobu F. Ball A.R. Chang C.H.W. Nishimura T. Vogt P. K Genes & Dev. 1990; 4: 1677-1687Crossref PubMed Scopus (161) Google Scholar, 7Morgan I.M. Asano M. Havarstein L.S. Ishikawa H. Hiiragi T. Ito Y. Vogt P.K. Oncogene. 1993; 8: 1135-1140PubMed Google Scholar). Indeed, c-jun gene has originally been identified in the acutely transforming ASV17 chicken retrovirus (8Maki Y. Bos T.J. Davis C. Starbuck M. Vogt P.K. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 2848-2852Crossref PubMed Scopus (353) Google Scholar). In this case, alterations of the protein, and particularly deletion of a 27-amino acid motif termed the 8 domain, confers increased transforming potential (8Maki Y. Bos T.J. Davis C. Starbuck M. Vogt P.K. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 2848-2852Crossref PubMed Scopus (353) Google Scholar). c-jun is also involved in the control of certain differentiation pathways. As a matter of fact, its knock-out is responsible for embryonic death most likely linked to abnormal hepatogenesis (9Hilberg F. Aguzzi A. Howells N. Wagner E.F. Nature. 1993; 365: 179-181Crossref PubMed Scopus (468) Google Scholar). Finally, c-jun expression may be instrumental for triggering apoptosis (10Coletta F. Polentarutti N. Sironi M. Mantovani A. J. Biol. Chem. 1992; 267: 18278-18283Abstract Full Text PDF PubMed Google Scholar). c-Jun is a short-lived protein with an approximate half-life of 90 min in fibroblast cultures (11Lamph W.W. Wamsley P. Sassone-Corsi P. Verma I.M. Nature. 1988; 334: 629-631Crossref PubMed Scopus (501) Google Scholar, 12Treier M. Staszewski L.M. Bohmann D. Cell. 1994; 78: 787-798Abstract Full Text PDF PubMed Scopus (847) Google Scholar). Interestingly, at least two catabolic pathways seem to operate on this protein. First, two lines of evidence support the idea that calpains, which are abundant cytoplasmic calcium-dependent cystein proteases, can initiate degradation of c-Jun along with that of c-Fos: (i) both proteins have been shown to constitute actual substrates for these proteases in vitro either under purified form or in cytoplasmic extracts (13Hirai S.-L. Kawasaki H. Yaniv M. Susuki K FEBS Lett. 1991; 287: 57-61Crossref PubMed Scopus (149) Google Scholar, 14Watt F. Molloy P.L. Nucleic Acids Res. 1993; 21: 5092-5100Crossref PubMed Scopus (93) Google Scholar, 15Carillo S. Pariat M. Steff A.-M. Roux P. Etienne-Julan M. Lorca T. Piechaczyk M. Oncogene. 1994; 9: 1679-1689PubMed Google Scholar) and (ii) specific modulation of calpain activity in vivo modifies c-Jun- and c-Fos-dependent AP-1 transcription complex activity in a transient co-transfection assay (13Hirai S.-L. Kawasaki H. Yaniv M. Susuki K FEBS Lett. 1991; 287: 57-61Crossref PubMed Scopus (149) Google Scholar). In this situation, it must, however, be emphasized that other proteolytic systems must take over the action of calpains since the latter cleave their substrates only to a limited extent. Moreover, this pathway presumably concerns only cytoplasmic degradation of c-Jun and c-Fos because of the restricted intracellular distribution of calpains. Second, it has recently been demonstrated that c-Jun can be ubiquitinylated in vivo (12Treier M. Staszewski L.M. Bohmann D. Cell. 1994; 78: 787-798Abstract Full Text PDF PubMed Scopus (847) Google Scholar). However, although the correlation between the capability to be ubiquitinylated and rapid turnover is highly suggestive, the final demonstration that ubiquitinylation is actually responsible for the triggering of c-Jun protein breakdown is still missing. Characterization of the involved enzymatic activities and reconstitution of the proteolytic machinery is necessary for full understanding of the process by which c-Jun is broken down. As a first step toward this aim, we have developed in vitro assays using recombinant rat c-Jun and either rat liver extracts or purified proteasome, the latter being the major soluble neutral intracellular proteolytic activity that has been characterized so far and which is putatively responsible for degradation of most cytoplasmic and nuclear proteins (for a review, see Refs. 16Rivett J.A. Biochem. J. 1993; 291: 1-10Crossref PubMed Scopus (383) Google Scholar, 17Peters J.-M. Trends Biochem. Sci. 1994; 19: 377-382Abstract Full Text PDF PubMed Scopus (300) Google Scholar, 18Ciechanover A. Cell. 1994; 79: 13-21Abstract Full Text PDF PubMed Scopus (1602) Google Scholar). We report here that ubiquitinylation is not an absolute requirement for in vitro degradation of c-Jun by the 26 S proteasome. This points to the notion that c-Jun, and possibly other proteins, does not necessarily require tagging or co-factors of any sort for degradation by the 26 S proteasome but possesses intrinsic structural motifs allowing direct recognition and subsequent breakdown by the latter (see “Discussion”). Moreover, this also suggests the existence of an additional putative degradation pathway operating on c-Jun. For preparation of F2 extracts, rat livers were homogenized in 1 volume of 50 mM Tris-HCl, pH 7.5, 2 mM dithiothreitol (DTT), 1The abbreviations used are: DTT, dithiothreitol; PBS, phosphate-buffered saline. 0.1 mM soybean trypsin inhibitor, 0.1 mM phenylmethylsulfonyl fluoride using a Waring blender. Nuclei and tissue debris were eliminated through two steps of centrifugation at 4 °C (6,000 × g for 30 min and 70,000 × g for 2 h). Crude extracts were incubated in the presence of 20 mM deoxyglucose and 2 mM dinitrophenol for 1 h at room temperature to deplete ATP. DEAE-cellulose chromatography and concentration of proteins were carried out as described in Hershko et al. (19Hershko A. Heller H. Elias S. Ciechanover A. J. Biol. Chem. 1983; 258: 8206-8214Abstract Full Text PDF PubMed Google Scholar). F2 extracts (20 mg/ml) were aliquoted and kept at –20 °C in 50 mM Tris-HCl, pH 7.5, 2 mM DTT, 20% glycerol until use. Their activity was stable over months. Purification of 20 and 26 S proteasome was conducted as described by Sawada et al. (20Sawada H. Muto K. Fujimuro M. Akaishi T. Sawada M.T. Yokosawa H. FEBS Lett. 1993; 335: 207-212Crossref PubMed Scopus (29) Google Scholar) through successive chromatography steps using Mono Q and Superose 6 columns (20Sawada H. Muto K. Fujimuro M. Akaishi T. Sawada M.T. Yokosawa H. FEBS Lett. 1993; 335: 207-212Crossref PubMed Scopus (29) Google Scholar). Purified particles were kept at-20 “C in 20 mM Tris-HCl, pH 7.5, 2 mM ATP, and 50% glycerol. 26 S proteasome was used within a few days after purification because of rapid loss of activity. On the contrary, 20 S proteasome was stable at least for weeks. Proteins were fractionated through 15% Polyacrylamide gels containing SDS according to Laemmli (21Laemmli U.K. Nature. 1990; 227: 680-685Crossref Scopus (207537) Google Scholar) using the mini-Protean II system from Bio-Rad. Proteins were electrotransferred onto nitrocellulose membranes (0.2-μm pores; Schleicher and Schuell) for 45 min at 0.8 mA/cm2 using the semidry transfer apparatus from LKB. Membranes were blocked by incubation for 1 h at room temperature in 10% skimmed milk into PBS (0.15 M NaCl, 0.01 M sodium phosphate pH 7). Anti-protein antibodies were added for 2 h at room temperature at a concentration of 0.1 μg/ml. Membranes were washed 3 times for 10 min in PBS. A 1:1000 dilution of a peroxidase-conjugated sheep anti-mouse or anti-rabbit IgG (Amersham Corp.) as a second antibody was then added and incubated for 2 h at room temperature. Membranes were washed 3 times in PBS, 0.1% Tween 20 and 3 times with PBS. Immunodetected proteins were revealed using the ECL chemoluminescent system from DuPont NEN according to the specification of the supplier. c-Jun was detected using the SC45 anti-c-Jun from Santa-Cruz. Ubiquitin was detected using the Z148 rabbit antiserum from Dakko. Rabbit anti-p53 antibodies were a gift from Dr. L. Debüssche (Rhone-Poulenc-Rohrer, France). Anti-E1 antibodies were obtained by immunization of rabbits with E1 enzyme purified from Xenopus oocytes. Anti-whole proteasome and proteasome subunits were gifts from Drs. K. and K. were using protein as described in and D. Scholar). In a of 10 mg/ml) were added to the volume of F2 was carried out for h at °C under of protein in 20 mM Tris-HCl, pH binding of were and the was to for at 4 was eliminated through a centrifugation step × g for The process was and extracts were to 20% glycerol being aliquoted and until use. Purified and rabbit are from The recombinant protein was a gift from Dr. L. It as been from a using the antibody as described in Ref. Cell. 1992; Full Text PDF PubMed Scopus Google c-Jun C. D. T. & 1990; Google Scholar) was purified from as of were induced for 3 h in the presence of 1 mM in 3 of 50 mM Tris-HCl, pH 1 mM mM NaCl, 0.1 mM phenylmethylsulfonyl and on for 20 4 of and of were and the was incubated for 30 min at which the of c-Jun, were × g for in 10 of 50 mM Tris-HCl, pH 10 mM mM NaCl, 0.1 mM phenylmethylsulfonyl fluoride and washed under for min at room temperature. The was and the was in 3 of mM Tris-HCl, pH mM NaCl, 0.1 mM phenylmethylsulfonyl 8 M for 1 h at The was for 1 h at × g at room and the was either or used for c-Jun, which of proteins of proteins as by was purified to by through Polyacrylamide gels using the apparatus from Bio-Rad. The described is an of that of Chang et al. L.M. P. J. Biol. Chem. Full Text PDF PubMed Google Scholar) and c-Jun, in a volume of 50 of mM Tris-HCl, pH 0.1% SDS was to using at room temperature. The was then through a and for h 50 of 50 mM Tris-HCl, pH 50 mM DTT, 2 mM M in to c-Jun. was obtained through of of 50 mM Tris-HCl, pH mM NaCl, 2 mM DTT, 2 mM 0.1% at The was to 50% and c-Jun was kept at –20 °C at a concentration of until use. of was using the as described in Hershko et al. (19Hershko A. Heller H. Elias S. Ciechanover A. J. Biol. Chem. 1983; 258: 8206-8214Abstract Full Text PDF PubMed Google Scholar). were incubated for 30 min at °C in a volume of 50 in the presence of 20 mM Tris-HCl, pH 7.5, 2 mM ATP, mM 10 mM 1 2 mM DTT, of and 2 protein were through a 15% Polyacrylamide and then Degradation were at “C in a volume of in the presence of 20 mM Tris-HCl, pH 7.5, of recombinant rat c-Jun other and of proteins from the different extracts were obtained with and mM and the were were at points and for 10 min in Laemmli Proteins were fractionated through 15% Polyacrylamide gels and electrotransferred onto was carried out as described The of proteins multiple steps A. Cell. 1994; 79: 13-21Abstract Full Text PDF PubMed Scopus (1602) Google Scholar, A. Ciechanover A. Biochem. 1992; PubMed Scopus Google Scholar, S. 1992; PubMed Scopus Google Scholar). is first by an proteins then the transfer of from E1 to protein substrates to specific ubiquitinylation is necessary for the rapid breakdown of c-Jun, ubiquitinylation and rat cell-free degradation assays were of protein extracts was carried out as In a first a protein was purified by chromatography from a rat liver according to Hershko et al. (19Hershko A. Heller H. Elias S. Ciechanover A. J. Biol. Chem. 1983; 258: 8206-8214Abstract Full Text PDF PubMed Google Scholar). two points are being First, was from the to of in its is eliminated the chromatography Second, F2 still as as but not of and together with major soluble proteolytic the of ubiquitinylation in degradation assays, either of two additional were to In case, was eliminated using an for both and In the other case, the ubiquitinylation pathway was by E1 that and were eliminated from F2 and However, to the system used are in types of extracts and may in F2 and The of the of degradation however, out on incubation of F2 extracts for 2 h under degradation (see not to any in the of on the other in a carried out in the presence of we detected of as by does not ubiquitinylation, as by of of to proteins of the in to both F2 and F2 with a which by the are shown to be for ubiquitinylation For degradation rat c-Jun C. D. T. & 1990; Google Scholar), Cell. 1992; Full Text PDF PubMed Scopus Google Scholar), and rabbit were added in to the different extracts in the or in the of ATP, and of the were at different points for of c-Jun was both by its to homodimerize and to to AP-1 motifs and to transcription in an in vitro assay and by of since this revealed a to both its for structural determinants and its limited action on c-Jun (13Hirai S.-L. Kawasaki H. Yaniv M. Susuki K FEBS Lett. 1991; 287: 57-61Crossref PubMed Scopus (149) Google Scholar, 14Watt F. Molloy P.L. Nucleic Acids Res. 1993; 21: 5092-5100Crossref PubMed Scopus (93) Google Scholar, 15Carillo S. Pariat M. Steff A.-M. Roux P. Etienne-Julan M. Lorca T. Piechaczyk M. Oncogene. 1994; 9: 1679-1689PubMed Google Scholar). c-Jun turned out to be at a in extracts in an ATP-dependent On the contrary, and in with the observations that antibodies are very stable although is very unstable and through the the enzyme specific for ubiquitinylation of is from F2 A. D. M. B. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar). Interestingly, the of of to F2 not c-Jun Moreover, calpains, as are of c-Jun (13Hirai S.-L. Kawasaki H. Yaniv M. Susuki K FEBS Lett. 1991; 287: 57-61Crossref PubMed Scopus (149) Google Scholar, 14Watt F. Molloy P.L. Nucleic Acids Res. 1993; 21: 5092-5100Crossref PubMed Scopus (93) Google Scholar, 15Carillo S. Pariat M. Steff A.-M. Roux P. Etienne-Julan M. Lorca T. Piechaczyk M. Oncogene. 1994; 9: 1679-1689PubMed Google Scholar), not in degradation since a highly specific M. H. K. M. T. M. J. Biol. Chem. Full Text PDF Google Scholar) from which is the of calpains, does not c-Jun. selective degradation of c-Jun in F2 rat liver extracts is on ubiquitinylation stimulated by the presence of The 26 S S is the major soluble neutral proteolytic activity in the proteolytic the 20 S proteasome, is in in to the 26 S for proteins J.A. Biochem. J. 1993; 291: 1-10Crossref PubMed Scopus (383) Google Scholar, 17Peters J.-M. Trends Biochem. Sci. 1994; 19: 377-382Abstract Full Text PDF PubMed Scopus (300) Google Scholar, 18Ciechanover A. Cell. 1994; 79: 13-21Abstract Full Text PDF PubMed Scopus (1602) Google Scholar). the proteasome is responsible for the ATP-dependent degradation of c-Jun in rat liver extracts, F2 was using an antiserum shown for such a Y. S. T. S.-L. K. K. A. Nature. 1992; PubMed Scopus Google Scholar). was as (i) proteasome proteolytic activity was shown to be using the which is to be a for the proteasome K. T. A. A. A. M. K. T. J. Biol. 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As a support to the of the and revealed a to both types of in the presence of and (i) of not c-Jun, out by calpain (ii) 10% of c-Jun is any of ATP, on of in the the 26 S proteasome is in the presence of 2 (iii) c-Jun is to of 20 S proteasome in an but selective since and under the and (iv) c-Jun degradation is still selective and ATP-dependent at a concentration of 26 S proteasome of purified 26 S not The proteolytic pathway is thought to for the degradation of the of short-lived and abnormal cellular proteins A. Cell. 1994; 79: 13-21Abstract Full Text PDF PubMed Scopus (1602) Google Scholar, A. Ciechanover A. Biochem. 1992; PubMed Scopus Google Scholar, S. 1992; PubMed Scopus Google Scholar). It has also been in for subsequent of by major complex M.T. C. Nature. 1993; PubMed Scopus Google Scholar, C. L. K. R. L. D. Cell. 1994; 78: Full Text PDF PubMed Scopus Google Scholar) and protein as in the of the protein T. Cell. 1994; 78: Full Text PDF PubMed Scopus Google Scholar). 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We report here that c-Jun protein can be in a selective and ATP-dependent the proteasome in rat liver extracts since and protein with data not are not under the this to the very few characterized cellular substrates of the proteasome. Moreover, we also show that tagging by is not an absolute requirement for recognition and degradation of c-Jun by the 26 S proteasome. which is of the most rapidly turned over proteins, is the only other of such a Using the as a cell-free degradation et al. Y. Ciechanover A. C. J. Biol. Chem. Full Text PDF PubMed Google Scholar) have shown that breakdown is ATP-dependent but using both different cell extracts and purified proteasome, et al. Y. S. T. S.-L. K. K. A. Nature. 1992; PubMed Scopus Google Scholar) have identified the 26 S proteasome as the responsible for It is that breakdown of does not require tagging by triggering of is of with a specific with specific is for degradation of c-Jun by purified 26 S proteasome. our c-Jun is the first demonstration that a cellular protein can bear intrinsic for selective recognition and ATP-dependent degradation by the 26 S proteasome. of does not that co-factors may be involved in the of c-Jun breakdown in vivo. c-Jun with to protein since proteolytic systems have been to operate on First, as in the in vitro and in vivo lines of evidence that c-Jun degradation can be in the by calpains (13Hirai S.-L. Kawasaki H. Yaniv M. Susuki K FEBS Lett. 1991; 287: 57-61Crossref PubMed Scopus (149) Google Scholar, 14Watt F. Molloy P.L. Nucleic Acids Res. 1993; 21: 5092-5100Crossref PubMed Scopus (93) Google Scholar, 15Carillo S. Pariat M. Steff A.-M. Roux P. Etienne-Julan M. Lorca T. Piechaczyk M. Oncogene. 1994; 9: 1679-1689PubMed Google Scholar). However, since of c-Jun by calpains is it is likely that other proteolytic activities are involved in the of by our point to the of the proteasome. Second, et al. (12Treier M. Staszewski L.M. Bohmann D. Cell. 1994; 78: 787-798Abstract Full Text PDF PubMed Scopus (847) Google Scholar) have recently shown that (i) a of c-Jun is ubiquitinylated in (ii) a 27-amino acid in the of c-Jun and which is from the protein, is for the of at multiple on the protein, and (iii) the capability to be ubiquitinylated with rapid turnover since is or However, the demonstration that degradation of c-Jun is actually by of has not been and the of the proteasome has not been It be out that the is instrumental for rapid degradation of c-Jun through we report here that c-Jun can be by the 26 S proteasome in the of ubiquitinylation, at least in to is to and pathways actually in vivo for c-Jun breakdown and or under different or in different cell our the that an unstable protein can be according to different pathways has not been It however, that the has been described at the since two of the have been shown to their under different cell culture Genes & Dev. PubMed Scopus Google Scholar). the of the of the different proteolytic involved in c-Jun degradation in vivo however, be As a matter fact, is known the of calpains in vivo 1991; PubMed Scopus Google Scholar), on and the 26 S proteasome have according to its cellular intracellular on the other can that certain c-Jun in an of Purification and of the different 26 S be necessary for this We Dr. K. and K. for the gift of proteasome and We also Dr. L. Debüssche for the gift of recombinant and Drs. and U. for of the