FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2092762008> ?p ?o ?g. }

• W2092762008 endingPage "32168" @default.
• W2092762008 startingPage "32163" @default.
• W2092762008 abstract "Regulatory proteins are often ubiquitinated, depending on their phosphorylation status as well as on their association with ancillary proteins that serve as adapters of the ubiquitination machinery. We previously demonstrated that c-Jun is targeted for ubiquitination by its association with inactive c-Jun NH2-terminal kinase (JNK). Phosphorylation by activated JNK protects c-Jun from ubiquitination, thus by prolonging its half-life. In the study reported here, we determined the ability of JNK to target ubiquitination of its other substrates (Elk1 and activating transcription factor 2 (ATF2)) and associated proteins (ATF2 and JunB). We demonstrate that phosphorylation by JNK protects ATF2, but not Elk1, from JNK-targeted ubiquitination. We also show that association of inactive JNK with JunB or ATF2 is necessary to target them for ubiquitination. Unlike its targeting of c-Jun, JNK requires additional cellular components, yet to be identified, to target the ubiquitination of ATF2. Elk1 is phosphorylated by JNK, but JNK neither associates with nor targets Elk1 for ubiquitination. The implications for the dual role of JNK in the regulation of ubiquitination and stability of c-Jun, ATF2, and JunB in normally growing versus stressed cells are discussed. Regulatory proteins are often ubiquitinated, depending on their phosphorylation status as well as on their association with ancillary proteins that serve as adapters of the ubiquitination machinery. We previously demonstrated that c-Jun is targeted for ubiquitination by its association with inactive c-Jun NH2-terminal kinase (JNK). Phosphorylation by activated JNK protects c-Jun from ubiquitination, thus by prolonging its half-life. In the study reported here, we determined the ability of JNK to target ubiquitination of its other substrates (Elk1 and activating transcription factor 2 (ATF2)) and associated proteins (ATF2 and JunB). We demonstrate that phosphorylation by JNK protects ATF2, but not Elk1, from JNK-targeted ubiquitination. We also show that association of inactive JNK with JunB or ATF2 is necessary to target them for ubiquitination. Unlike its targeting of c-Jun, JNK requires additional cellular components, yet to be identified, to target the ubiquitination of ATF2. Elk1 is phosphorylated by JNK, but JNK neither associates with nor targets Elk1 for ubiquitination. The implications for the dual role of JNK in the regulation of ubiquitination and stability of c-Jun, ATF2, and JunB in normally growing versus stressed cells are discussed. The cellular response to stress activates early response proteins by both transcription and post-translational modifications which dictate the cell's ability to undergo cell cycle arrest for DNA damage repair or to initiate programmed cell death. Among the stress-modulated factors that contribute to the cell's ability to cope with stress are c-Jun and ATF2, 1The abbreviations used are: ATF2, activating transcription factor 2; JNK, c-Jun NH2-terminal kinase; PCR, polymerase chain reaction; Ub-HA, hemagglutinin-tagged ubiquitin; PAGE, polyacrylamide gel electrophoresis; RL, reticulocyte lysate; HA, hemagglutinin; ATPγS, adenosine 5′-O-(thiotriphosphate; NTA, nitrilotriacetic acid. 1The abbreviations used are: ATF2, activating transcription factor 2; JNK, c-Jun NH2-terminal kinase; PCR, polymerase chain reaction; Ub-HA, hemagglutinin-tagged ubiquitin; PAGE, polyacrylamide gel electrophoresis; RL, reticulocyte lysate; HA, hemagglutinin; ATPγS, adenosine 5′-O-(thiotriphosphate; NTA, nitrilotriacetic acid. both of which are activated by their NH2-terminal phosphorylation via stress-activated protein kinases (c-Jun NH2-terminal kinases; JNK) (1Hibi M. Lin A. Smeal T Minden A. Karin M. Genes Dev. 1994; 7: 2135-2148Crossref Scopus (1697) Google Scholar, 2Kyriakis J.M. Banerjee P. Nikolakaki E. Dai T. Rubie E.A. Ahmad M.F. Avruch J. Woodgett J.R. Nature. 1994; 369: 156-160Crossref PubMed Scopus (2403) Google Scholar, 3Han J. Lee J.-D. Bibbs L. Ulevitch R.J. Science. 1994; 265: 808-811Crossref PubMed Scopus (2390) Google Scholar). JNKs are proline-directed serine/threonine kinases, which are activated by a wide variety of stimuli, including physical and chemical DNA-damaging agents and inhibitors of protein synthesis as well as heat and osmotic shock (reviewed in Kyriakis and Avruch (4Kyriakis J.M. Avruch J. J. Biol. Chem. 1996; 271: 24313-24316Abstract Full Text Full Text PDF PubMed Scopus (1024) Google Scholar)). Different forms of stress utilize alternate cellular pathways for JNK activation (5Adler V. Schaffer A. Kim J. Dolan L. Ronai Z. J. Biol. Chem. 1995; 270: 26071-26077Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar). For example, JNK activation by UV irradiation requires their phosphorylation by the upstream kinase, mitogen-activated protein-kinase kinase 4 (6Lin A. Minden A. Martinetto H. Claret F.X. Lange-Carter C. Mercurio F. Johnson G.L. Karin M. Science. 1995; 268: 286-290Crossref PubMed Scopus (706) Google Scholar), the association of JNK with p21 ras (7Adler V. Pincus M. Brandt-Rauf P.W. Ronai Z. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10585-10589Crossref PubMed Scopus (80) Google Scholar), the presence of nuclear DNA lesions (8Adler V. Fuchs S.Y. Kim J. Kraft A. King M.P. Pelling J. Ronai Z. Cell Growth Differ. 1995; 6: 1437-1446PubMed Google Scholar, 9Saleem A. Datta R. Yuan Z.-M. Kharbanda S. Kufe D. Cell Growth Differ. 1995; 6: 1651-1658PubMed Google Scholar), and inactivation of a redox-sensitive inhibitor. 2V. Adler, S. Y. Fuchs, M. R. Pincus, K. D. Tew, R. J. Davis, and Z. Ronai, manuscript submitted for publication. 2V. Adler, S. Y. Fuchs, M. R. Pincus, K. D. Tew, R. J. Davis, and Z. Ronai, manuscript submitted for publication. JNK were first identified and named as a Jun-associated kinases (11Adler V. Unlap T. Kraft A. J. Biol. Chem. 1994; 269: 11186-11191Abstract Full Text PDF PubMed Google Scholar), reflecting their strong hydrophobic interaction with c-Jun. JNK-c-Jun association is ATP-independent and is required for efficient ATP-dependent phosphorylation of c-Jun at flanking phosphoacceptor sites (Ser63 and Ser73). The mechanism by which JNK phosphorylation confers transcriptional activities of c-Jun remains largely unknown. One of the key mechanisms for regulating protein's activity is tight control of its stability. Many regulatory proteins are selectively degraded by the proteasome pathway at specific phases of cell growth. Polyubiquitination, i.e. covalent attachment of multiple ubiquitin residues to ε-lysil amino groups of lysine, serves as a marker for proteasome recognition (reviewed in Hochstrasser (12Hochstrasser M. Curr. Opin. Cell Biol. 1995; 7: 215-223Crossref PubMed Scopus (775) Google Scholar)). The ubiquitination process is regulated by several mechanisms, including degradation of inhibitors, processing of inactive precursors, and stabilization of activated proteins. For example, activation of NFκB requires the ubiquitination and degradation of its inhibitor IκB as well as the processing of its precursor p105 (13Palombela V.J. Rando O.J. Goldberg A.L. Maniatis T. Cell. 1994; 78: 773-785Abstract Full Text PDF PubMed Scopus (1908) Google Scholar). Conversely, it is the DNA damaged-induced stabilization of the tumor suppressor protein p53 that acquires its activities (14Kuerbitz S.J. Plunkett B.S. Walsh W.V. Kastan M.B. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7491-7495Crossref PubMed Scopus (1838) Google Scholar). Central to JNK's association with c-Jun is the δ domain of c-Jun (amino acids 30–57), which is deleted in its oncogenic counterpart v-Jun. The δ domain is also essential for c-Jun ubiquitination, which explains the mechanism underlying the greater stability of v-Jun as compared with its cellular homologue (15Treier M. Staszewski L. Bomann D. Cell. 1994; 78: 787-798Abstract Full Text PDF PubMed Scopus (845) Google Scholar). Using an in vitromodel system, we previously demonstrated that c-Jun is targeted for ubiquitination by association with JNK. However, phosphorylation of c-Jun on Ser73 by JNK is sufficient to protect c-Jun from ubiquitination, resulting in a prolonged half-life (16Fuchs S.Y. Dolan L. Davis R.J. Ronai Z. Oncogene. 1996; 13: 1531-1535PubMed Google Scholar). The dual activity of JNK in targeting c-Jun ubiquitination via physical association and in protecting it from entering this pathway via phosphorylation points to the role of JNK in controlling c-Jun's stability in cells exposed to environmental stress or inflammatory cytokines. In light of finding phosphorylation-dependent targeting of c-Jun ubiquitination, in the present study, we have compared JNK target ubiquitination of its substrates and associated protein (ATF2, c-Jun) with nonassociated substrate (Elk1) and associated non-substrate (JunB). Our results provide the foundation for the model in which (i) JNK-targeted ubiquitination requires tight association and (ii) the degree of targeting is affected by the extent of phosphorylation on JNK-associated protein. Constructs encoding c-Junhis (15Treier M. Staszewski L. Bomann D. Cell. 1994; 78: 787-798Abstract Full Text PDF PubMed Scopus (845) Google Scholar), c-JunhisΔ1–72 (16Fuchs S.Y. Dolan L. Davis R.J. Ronai Z. Oncogene. 1996; 13: 1531-1535PubMed Google Scholar) and Elk1his (17Gupta S. Barrett T. Whitmarsh A.J. Cavanagh J. Sluss H.K. Derijard B. Davis R.J. EMBO J. 1996; 15: 2760-2770Crossref PubMed Scopus (1166) Google Scholar) were previously described. JunB open reading frame was amplified by PCR using the wild type JunB mammalian expression vector (18Deng T. Karin M. Genes Dev. 1993; 7: 479-490Crossref PubMed Scopus (287) Google Scholar) as a template and cloned into the pET15b vector (Novagen) at the NdeI site. A BamHI digest of the same amplification product has been cloned into pET15b at the BamHI site, providing the JunBhis construct, which lacks the first 44 amino acids (JunBhisΔ1–44). Full-length ATF2 as well as ATF2 with mutated JNK phosphoacceptor sites Thr → Ala69 and Ala71 open reading frames were amplified by PCR using pECE-ATF2 plasmids (4Kyriakis J.M. Avruch J. J. Biol. Chem. 1996; 271: 24313-24316Abstract Full Text Full Text PDF PubMed Scopus (1024) Google Scholar, 19Gupta S. Campbell D. Derijard B. Davis R.J. Science. 1995; 267: 389-393Crossref PubMed Scopus (1333) Google Scholar) as templates followed by unidirectionally cloning them into pET15b at NdeI/BamHI sites, resulting in ATF2his and ATF2hisΔ69,71 constructs, respectively. The ATF2 mutant lacking JNK binding site (ATF2hisΔ40–66) has been created using a QuickChange site-directed mutagenesis kit (Stratagene). An HA-tagged ubiquitin encoding construct was generated by PCR-mediated cloning. The sequence encoding ASYPYDVDPYASLSR followed by the second codon of ubiquitin open reading frame was used as a 5′ primer for PCR amplification and cloned unidirectionally into pET15b at NdeI/BamHI sites. Open reading frames of all final constructs were verified by dideoxy sequencing (Sequenase kit, U. S. Biochemical Corp.). Histidine-fusion proteins were expressed in the BL21(D:E3)3pLysS bacterial strain and purified by affinity chromatography using nickel resins under denaturing conditions, as recommended by the manufacturer (Qiagen). Proteins attached to the beads were refolded by excessive (20 volumes) column washings with mixtures of 8 m urea in sodium phosphate buffer (pH 8.0) and renaturation buffer (Tris-HCl, pH 7.8, 150 mm NaCl, 1 mm EDTA, 1 mmdithiothreitol, 10% EtOH, and 0.2% Nonidet P-40) at the subsequent ratios of 3:1, 1:1, 1:3, and 1:7. The columns were then washed three times with 20 volumes of renaturation buffer, followed by two additional washes using the same buffer without alcohol. The beads were then washed with 2 × storage buffer (40 mm HEPES, pH 7.6, 150 mm NaCl, 10 mm EDTA, 1 mmdithiothreitol, 0.5 mm phenylmethylsulfonyl fluoride, and 0.2% Nonidet P-40) to block the remaining nonoccupied nickel binding sites, which are not protected by bound proteins. Bead-bound proteins were then resuspended in 50% glycerol and stored at −20 °C. HA-tagged ubiquitin (Ub-HA)-bound beads were washed three more times (rather than resuspended in glycerol) with thrombin cleavage buffer (40 mm Tris-HCl, pH 8.5, 150 mm NaCl, 2.5 mm CaCl2) and incubated in 10 volumes of the same buffer with thrombin (Sigma; 2 units/mg of recombinant protein) for 16 h at 20 °C. Resins were pelleted, and the supernatant containing Ub-HA was incubated at 90 °C for 15 min, chilled on ice, and cleared by centrifugation at 15,000 × g for 20 min at 4 °C. The resulting supernatant was concentrated and washed in double distilled water using Ultra-free-15 centrifugation units (Sigma) with 5000 cutoff membranes to obtain pure Ub-HA (verified by silver staining of SDS-PAGE gel and immunoblotting with antibodies against ubiquitin and HA). 4A-3T3 and NIH-3T3 mouse fibroblasts were grown in Dulbecco's modified Eagle's medium supplemented with 10% calf serum (Life Technologies, Inc.) and antibiotics at 37 °C and 5% CO2. Calpain inhibitor LLM (Sigma) and proteasome inhibitor MG132 (Peptide International Co.) were added to the cells at 50 and 10 μm (respectively) in Me2SO (less than 0.1% of medium volume) 16 h before harvesting. UV exposure (50 J/m2) or sham irradiation was performed as described previously (16Fuchs S.Y. Dolan L. Davis R.J. Ronai Z. Oncogene. 1996; 13: 1531-1535PubMed Google Scholar). Rabbit blood enriched with reticulocytes was purchased (Pel-Freeze). Cell lysates and reticulocyte lysates (RL) were prepared as described previously (16Fuchs S.Y. Dolan L. Davis R.J. Ronai Z. Oncogene. 1996; 13: 1531-1535PubMed Google Scholar). Since RL were found to contain trace amounts of JNK they were immunodepleted of JNK prior to their use (16Fuchs S.Y. Dolan L. Davis R.J. Ronai Z. Oncogene. 1996; 13: 1531-1535PubMed Google Scholar). For JNK purification, 600 mg of protein extract from 4A-3T3 cells were prepared from UV-irradiated 4A-3T3 cells (60 J/m2) 45 min after the treatment and subjected to the purification procedure as described previously (11Adler V. Unlap T. Kraft A. J. Biol. Chem. 1994; 269: 11186-11191Abstract Full Text PDF PubMed Google Scholar). Briefly, the protein extract was loaded on a sizing column (Sepharose 6B; Pharmacia Biotech Inc.) and active fractions (measured in the solid phase kinase assay) (11Adler V. Unlap T. Kraft A. J. Biol. Chem. 1994; 269: 11186-11191Abstract Full Text PDF PubMed Google Scholar) between 30 and 70 kDa were pooled, preincubated with glutathione S-transferase-bound beads, and loaded on the glutathione S-transferase-c-Jun (amino acids 5–89) affinity columns. After extensive washes with kinase buffer, the proteins were eluted with 3% n-octyl β-d-glucopyranoside (Sigma), dialyzed against kinase buffer, and loaded onto a phenyl Sepharose column (Pharmacia). The bound material was eluted with 0.2m (NH4)2SO4 and separated on a SuperX gel filtration column (Pharmacia). The middle active fraction containing ∼80% of JNK2 and 20% of JNK1 (revealed by immunoblotting) was used in in vitro ubiquitination assays (∼0.1 μg/assay). The substrates were phosphorylated by means of the purified JNK2 (20 ng/reaction) or active bacterially expressed JNK (Biomol; 400 ng/reaction) in the solid phase kinase reaction as described elsewhere (16Fuchs S.Y. Dolan L. Davis R.J. Ronai Z. Oncogene. 1996; 13: 1531-1535PubMed Google Scholar). HA11 monoclonal antibodies were purchased (Babco) and used in immunoblotting at 1:1000 dilution. Anti-c-Jun, anti-JunB, and anti-Elk1 polyclonal and anti-ATF2 monoclonal antibodies were purchased (Santa Cruz Biochemicals) and used in immunoblotting at 1:2000 dilution. Anti-ubiquitin monoclonal antibodies, described elsewhere (20Murti K.G. Smith H.T. Fried V.A. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 3019-3023Crossref PubMed Scopus (73) Google Scholar), were used at 1:50 dilution. Anti-JNK monoclonal (clones 666 and 333) antibodies were provided by Dr. C. Monel (PharMingen). The immmunoblotting procedure was performed as described elsewhere (16Fuchs S.Y. Dolan L. Davis R.J. Ronai Z. Oncogene. 1996; 13: 1531-1535PubMed Google Scholar). For determination of c-Jun, JunB, and ATF2 co-precipitated with JNK, 1.5 mg of NIH-3T3 lysates, precleared with protein G-bound beads (Santa Cruz), were incubated with anti-JNK monoclonal (clone 333) antibody. Material precipitated using protein G-beads was washed four times in phosphate-buffered saline with 0.1% of Nonidet P-40, resolved on 10% SDS-PAGE, and analyzed by means of immunoblotting with respective antibodies (Santa Cruz). Previously we established an in vitro system for studying the ubiquitination of c-Jun (16Fuchs S.Y. Dolan L. Davis R.J. Ronai Z. Oncogene. 1996; 13: 1531-1535PubMed Google Scholar). In this system, after preincubation of recombinant c-Jun with whole cell extract, the unbound proteins are washed off and the targeting effects of c-Jun-bound proteins on its ubiquitination by rabbit RL are monitored. The extent of ubiquitination is reflected by the intensity of the multi-ubiquitin chain that appears as a smear of ubiquitin immunoreactive material produced from the position of the substrate to the top of the gel. Using this system, we demonstrated that binding of cellular proteins from rat fibroblasts to recombinant c-Jun increases its ubiquitination. Immunodepletion of JNK reduced the targeting activity of protein extracts, which could be restored by adding immunopurified JNK (16Fuchs S.Y. Dolan L. Davis R.J. Ronai Z. Oncogene. 1996; 13: 1531-1535PubMed Google Scholar). However, in the absence of any fibroblast proteins, the purified form of JNK was capable of mediating only a marginal increase in c-Jun ubiquitination. The latter has been attributed to low sensitivity of our detection system which relied on antibodies to ubiquitin. To improve the sensitivity of ubiquitination detection, we generated a construct that allows expression and purification of Ub-HA. The NH2-terminal fusion of HA peptide was shown not to interfere with formation of polyubiquitin chains in vivo(15Treier M. Staszewski L. Bomann D. Cell. 1994; 78: 787-798Abstract Full Text PDF PubMed Scopus (845) Google Scholar), and yet it enabled tracking ubiquitination of different substrates with highly specific and sensitive anti-HA antibody. Fifty micrograms of whole cell lysates from NIH-3T3 cells (immunodepleted with normal rabbit serum or antibody against JNK as described previously (16Fuchs S.Y. Dolan L. Davis R.J. Ronai Z. Oncogene. 1996; 13: 1531-1535PubMed Google Scholar)) or 0.1 μg of purified JNK were incubated on ice with bacterially expressed substrates (1–5 μg) bound to nickel beads for 45 min. After extensive washes (four times with 1 ml of kinase buffer) (16Fuchs S.Y. Dolan L. Davis R.J. Ronai Z. Oncogene. 1996; 13: 1531-1535PubMed Google Scholar), the substrate-bound beads were equilibrated with 1 × ubiquitination buffer (50 mm Tris-HCl, pH 8.0, 5 mm MgCl2, 0.5 mmdithiothreitol, 2 mm NaF, and 3 mm okadaic acid) and incubated in the same buffer supplemented with 2 mm ATP, 10 mm creatine phosphate, 0.02 unit of creatine phosphokinase, 2 μg of Ub-HA, 1.5 mm ATPγS (Sigma) and 33% RL (v/v) in a total volume of 30 ml at 30 °C for 5 min. Ubiquitin-aldehyde, synthesized as described previously (21Lam Y.A. Xu W. DeMartino G.N. Cohen R.E. Nature. 1997; 385: 737-740Crossref PubMed Scopus (365) Google Scholar), was added at 1 μm final concentration as indicated under “Results.” The reaction was stopped by adding 0.5 ml of 8m urea in sodium phosphate buffer (pH 6.3) with 0.1% of Nonidet P-40. The beads were washed three times with the stop-buffer and once with phosphate-buffered saline supplemented with 0.5% of Triton X-100, and the protein moiety was eluted with Laemmli sample buffer at 100 °C. Samples were resolved on 8% SDS-PAGE and electrotransferred onto a nitrocellulose filter. When anti-ubiquitin antibody was used, a polyvinylidene difluoride membrane served as a filter. Nitrocellulose filters were boiled in double distilled water for 10 min, blocked with 5% nonfat milk, and probed with HA11 antibody. After their detection via chemiluminescence (ECL, Amersham Corp.) the blots were stripped and reprobed with antibody against the specific substrate, followed by alkaline phosphatase detection to ensure equal loading of the substrate. To analyze the association of JNK with JunB and JunBΔ1–44, 250 μg of lysates obtained from UV-irradiated 4A-3T3 cells were incubated with NTA bead-bound JunB proteins for 45 min on ice. After four washes with kinase buffer, proteins were eluted by boiling in Laemmli sample buffer, separated on 10% SDS-PAGE, and transferred onto a nitrocellulose filter. The filter was probed with antibodies to JNK (clone 666, PharMingen) and reprobed with polyclonal anti-JunB (Santa Cruz) antibody. Preincubation of c-Junhis with JNK purified from 4A-3T3 mouse fibroblasts led to substantial increase in c-Junhis ubiquitination by RL immunodepleted of JNK (Fig.1; compare lanes 1 and 2). A further increase was noted when the whole cellular extract, immunodepleted with normal rabbit serum, was added as a source of targeting proteins (Fig. 1, lane 3). Immunodepletion of whole cellular extract with antibody to JNK substantially decreased its ability to target c-Junhis ubiquitination; adding purified JNK to the JNK-depleted protein extract restored the original level of c-Jun ubiquitination (Fig. 1). The addition of ubiquitin aldehyde to the ubiquitination reaction did not change the pattern of results (data not shown), suggesting that addition and removal of JNK affected the conjugation of ubiquitin rather than isopeptidase activity. As negative control nickel resins were incubated with protein lysates of uninduced bacterial strain BL21(D;E)3pLysS and purified, as c-Junhisdid not exhibit any HA-detectable smear, providing evidence of the substrate specificity of the reaction (Fig. 1, lane NTA). The same results were observed when an immunoblot from a parallel experiment was probed with anti-ubiquitin monoclonal antibody (not shown). Since the physical association between JNK and c-Jun targets the latter for ubiquitination, their in vivo interaction is expected to be unstable because of c-Jun degradation. To modulate the steady state level of the c-Jun-JNK complex, mouse fibroblasts were pretreated with potent proteasome inhibitor MG132 (20Murti K.G. Smith H.T. Fried V.A. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 3019-3023Crossref PubMed Scopus (73) Google Scholar). Fig.2 A demonstrates that pretreatment with MG132 substantially increased the amount of c-Jun which could be co-immunoprecipitated with antibody against JNK. MG132 treatment did not affect the JNK level (Fig. 2 B) but increased the amount of c-Jun measured in the whole cell extracts (Fig.2 A). These findings suggest that the c-Jun-JNK complex in vivo is a target for proteasome activity. UV irradiation of mouse fibroblast cells neither affected the JNK-c-Jun association nor led to retardation of the electrophoretic mobility of c-Jun bound to JNK (Fig. 2 A). These data provide further support for the role of JNK as a targeting molecule in c-Jun ubiquitination and, therefore, in determining its stability. In addition to its ability to bind and phosphorylate c-Jun, JNK is known to associate with JunB and ATF2 and to phosphorylate Elk1 and ATF2. We therefore examined the possible involvement of JNK in the regulation of ATF2, Elk1, and JunB ubiquitination. In all cases, the His-tagged substrates were used in our in vitro ubiquitination assay. To study Elk-1 ubiquitination we used a bacterially expressed histidine-tagged Elk-1 protein, which was previously shown to be a functional sequence specific DNA binding protein (17Gupta S. Barrett T. Whitmarsh A.J. Cavanagh J. Sluss H.K. Derijard B. Davis R.J. EMBO J. 1996; 15: 2760-2770Crossref PubMed Scopus (1166) Google Scholar). As is evident from the data presented in Fig. 3, Elk1 is efficiently ubiquitinated by RL, even in the absence of targeting molecules. Preincubation with either purified JNK or with whole cell lysate did not alter the extent of ubiquitination (Fig. 3). These findings suggest that Elk1, which is not capable of association with JNK (17Gupta S. Barrett T. Whitmarsh A.J. Cavanagh J. Sluss H.K. Derijard B. Davis R.J. EMBO J. 1996; 15: 2760-2770Crossref PubMed Scopus (1166) Google Scholar), cannot be targeted for ubiquitination by JNK (purified or in the content of the cell lysate). The ability of RL to mediate a high degree of Elk1 ubiquitination suggests that RL provides all necessary components for Elk1 ubiquitination, including enzymes of the ubiquitination machinery and targeting molecule(s), which were not depleted by the antibodies to JNK (16Fuchs S.Y. Dolan L. Davis R.J. Ronai Z. Oncogene. 1996; 13: 1531-1535PubMed Google Scholar). As JNK phosphorylation of c-Jun protects it from subsequent targeting for ubiquitination (16Fuchs S.Y. Dolan L. Davis R.J. Ronai Z. Oncogene. 1996; 13: 1531-1535PubMed Google Scholar, 22Musti A.M. Treier M. Bohmann D. Science. 1997; 275: 400-402Crossref PubMed Scopus (406) Google Scholar), we tested whether Elk1 phosphorylation affects its degree of ubiquitination. Unlike c-Jun, extensive phosphorylation of Elk1 by JNK (data not shown) did not alter its ubiquitination (Fig. 3). JunB preserves a δ domain-like sequence within its NH2 terminus, thus enabling JNK binding (17Gupta S. Barrett T. Whitmarsh A.J. Cavanagh J. Sluss H.K. Derijard B. Davis R.J. EMBO J. 1996; 15: 2760-2770Crossref PubMed Scopus (1166) Google Scholar, 23Kallunki T. Deng T. Hibi M. Karin M. Cell. 1997; 87: 929-939Abstract Full Text Full Text PDF Scopus (399) Google Scholar). Basal levels of JunBhisubiquitination by RL were higher than those found with c-Jun (data not shown). As in the instance of c-Jun, preincubation with JNK or with whole cellular extract increased the extent of JunBhisubiquitination (Fig. 4 A, compare lanes 1–3). Whole cell extract immunodepleted of JNK mediated the decreased extent of JunBhisubiquitination, whereas addition of a purified form of JNK to these extracts restored the original degree of ubiquitination (Fig.4 A, lanes 4 and 5). To further confirm JNK's role in targeting JunB ubiquitination, we performed experiments using JunBhisΔ1–44, which lacks the first 44 amino acids. This mutant lacks the ability to associate with JNK as suggested by available data (23Kallunki T. Deng T. Hibi M. Karin M. Cell. 1997; 87: 929-939Abstract Full Text Full Text PDF Scopus (399) Google Scholar) and evidenced by an in vitro binding assay (Fig. 4 B). Ubiquitination of JunBhisΔ1–44 was severely impaired. Neither JNK nor cell extract could increase JunBhisΔ1–44 ubiquitination (Fig.4 C). Although JNK binds JunB, it cannot phosphorylate this transcription factor because of the absence of phosphoacceptor sites (17Gupta S. Barrett T. Whitmarsh A.J. Cavanagh J. Sluss H.K. Derijard B. Davis R.J. EMBO J. 1996; 15: 2760-2770Crossref PubMed Scopus (1166) Google Scholar, 23Kallunki T. Deng T. Hibi M. Karin M. Cell. 1997; 87: 929-939Abstract Full Text Full Text PDF Scopus (399) Google Scholar). Indeed, preincubation of JunB with JNK and ATP in a solid phase kinase reaction followed by JNK removal (with 3%n-octyl β-d-glucopyranoside as described previously (16Fuchs S.Y. Dolan L. Davis R.J. Ronai Z. Oncogene. 1996; 13: 1531-1535PubMed Google Scholar)) did not result in incorporation of [32P]phosphate into JunB nor did it yield any protection from subsequent ubiquitination (data not shown). Transcription factor ATF2 can associate with JNK and is a substrate for JNK-mediated phosphorylation. Surprisingly, preincubation of ATF2hiswith JNK2 purified from 4A-3T3 cells did not target ubiquitination of this recombinant protein (Fig.5 A, compare lanes 2 and 3). Addition of cell extract as a source of targeting molecules led to a clear increase in the extent of ATF2 ubiquitination (Fig. 5, lane 4). Immunodepletion of this protein extract with antibody to JNK substantially decreased ATF2his ubiquitination. Conversely, reconstituting this protein extract with purified JNK restored the extent of ATF2 ubiquitination (Fig. 5, lanes 5 and 6). To further support the role of JNK in ATF2 ubiquitination, we performed experiments using ATF2hisΔ40–60, which cannot bind JNK (19Gupta S. Campbell D. Derijard B. Davis R.J. Science. 1995; 267: 389-393Crossref PubMed Scopus (1333) Google Scholar, 24Livingstone C. Patel G. Jones N. EMBO J. 1995; 14: 1785-1797Crossref PubMed Scopus (471) Google Scholar). Although ATF2hisΔ40–60 exhibits some basal ubiquitination by RL, it cannot be increased by whole cell extract (Fig. A, lanes 7 and 8). Phosphorylation of ATF2his by JNK leads to a modest yet highly reproducible increase in the extent of its basal ubiquitination (Fig. 5 B, lane 1 versus 2). However, such phosphorylation prevents an increase in the degree of ATF2his ubiquitination by mouse fibroblast protein extracts (compare lanes 3 and 4). Additional studies to confirm the protective effect of ATF2 phosphorylation by JNK in ATF2 ubiquitination utilized an ATF2 mutant in which JNK phosphorylation sites (Thr69 and Thr71) were replaced with Ala residues. Phosphorylation of this mutant by JNK was not capable of preventing whole cell extract-targeted ubiquitination (Fig.5 B; compare lanes 3 and 4 versus 7 and 8). Treatment of mouse fibroblasts with proteasome inhibitor MG132 (but not with calpain inhibitor LLM) increased the amount of JunB co-immunoprecipitated with anti-JNK antibody (Fig. 6 A). Blocking the proteasome pathway did not alter the total amount of JunB, suggesting that only a small portion of JunB is bound to JNK and susceptible for JNK-targeted ubiquitination and proteasome-mediated degradation. We cannot rule out the possibility that calcium-dependent proteases play a role in a JunB degradation in the JNK-independent manner. Immunoblot" @default.
• W2092762008 created "2016-06-24" @default.
• W2092762008 creator A5000363681 @default.
• W2092762008 creator A5013600050 @default.
• W2092762008 creator A5026512599 @default.
• W2092762008 creator A5028742170 @default.
• W2092762008 creator A5071155236 @default.
• W2092762008 creator A5089573644 @default.
• W2092762008 date "1997-12-01" @default.
• W2092762008 modified "2023-10-13" @default.
• W2092762008 title "c-Jun NH2-terminal Kinases Target the Ubiquitination of Their Associated Transcription Factors" @default.
• W2092762008 cites W1510515534 @default.
• W2092762008 cites W1555489233 @default.
• W2092762008 cites W1572174728 @default.
• W2092762008 cites W1692197226 @default.
• W2092762008 cites W1749711544 @default.
• W2092762008 cites W1969158591 @default.
• W2092762008 cites W1973588973 @default.
• W2092762008 cites W1977302628 @default.
• W2092762008 cites W1982284863 @default.
• W2092762008 cites W1984665850 @default.
• W2092762008 cites W1993983676 @default.
• W2092762008 cites W2001399809 @default.
• W2092762008 cites W2002223631 @default.
• W2092762008 cites W2009644729 @default.
• W2092762008 cites W2014792240 @default.
• W2092762008 cites W2015997780 @default.
• W2092762008 cites W2019556865 @default.
• W2092762008 cites W2026951979 @default.
• W2092762008 cites W2030283142 @default.
• W2092762008 cites W2033030646 @default.
• W2092762008 cites W2033529039 @default.
• W2092762008 cites W2036046756 @default.
• W2092762008 cites W2036985660 @default.
• W2092762008 cites W2042260090 @default.
• W2092762008 cites W2042479492 @default.
• W2092762008 cites W2047263793 @default.
• W2092762008 cites W2065477683 @default.
• W2092762008 cites W2077880395 @default.
• W2092762008 cites W2081892455 @default.
• W2092762008 cites W2087586960 @default.
• W2092762008 cites W2092716869 @default.
• W2092762008 cites W2101759139 @default.
• W2092762008 doi "https://doi.org/10.1074/jbc.272.51.32163" @default.
• W2092762008 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/9405416" @default.
• W2092762008 hasPublicationYear "1997" @default.
• W2092762008 type Work @default.
• W2092762008 sameAs 2092762008 @default.
• W2092762008 citedByCount "140" @default.
• W2092762008 countsByYear W20927620082012 @default.
• W2092762008 countsByYear W20927620082013 @default.
• W2092762008 countsByYear W20927620082014 @default.
• W2092762008 countsByYear W20927620082015 @default.
• W2092762008 countsByYear W20927620082016 @default.
• W2092762008 countsByYear W20927620082017 @default.
• W2092762008 countsByYear W20927620082018 @default.
• W2092762008 countsByYear W20927620082020 @default.
• W2092762008 countsByYear W20927620082021 @default.
• W2092762008 countsByYear W20927620082022 @default.
• W2092762008 crossrefType "journal-article" @default.
• W2092762008 hasAuthorship W2092762008A5000363681 @default.
• W2092762008 hasAuthorship W2092762008A5013600050 @default.
• W2092762008 hasAuthorship W2092762008A5026512599 @default.
• W2092762008 hasAuthorship W2092762008A5028742170 @default.
• W2092762008 hasAuthorship W2092762008A5071155236 @default.
• W2092762008 hasAuthorship W2092762008A5089573644 @default.
• W2092762008 hasBestOaLocation W20927620081 @default.
• W2092762008 hasConcept C104317684 @default.
• W2092762008 hasConcept C138885662 @default.
• W2092762008 hasConcept C179926584 @default.
• W2092762008 hasConcept C184235292 @default.
• W2092762008 hasConcept C185592680 @default.
• W2092762008 hasConcept C25602115 @default.
• W2092762008 hasConcept C2779087070 @default.
• W2092762008 hasConcept C2779664074 @default.
• W2092762008 hasConcept C31258907 @default.
• W2092762008 hasConcept C41008148 @default.
• W2092762008 hasConcept C41895202 @default.
• W2092762008 hasConcept C55493867 @default.
• W2092762008 hasConcept C86339819 @default.
• W2092762008 hasConcept C86803240 @default.
• W2092762008 hasConcept C95444343 @default.
• W2092762008 hasConceptScore W2092762008C104317684 @default.
• W2092762008 hasConceptScore W2092762008C138885662 @default.
• W2092762008 hasConceptScore W2092762008C179926584 @default.
• W2092762008 hasConceptScore W2092762008C184235292 @default.
• W2092762008 hasConceptScore W2092762008C185592680 @default.
• W2092762008 hasConceptScore W2092762008C25602115 @default.
• W2092762008 hasConceptScore W2092762008C2779087070 @default.
• W2092762008 hasConceptScore W2092762008C2779664074 @default.
• W2092762008 hasConceptScore W2092762008C31258907 @default.
• W2092762008 hasConceptScore W2092762008C41008148 @default.
• W2092762008 hasConceptScore W2092762008C41895202 @default.
• W2092762008 hasConceptScore W2092762008C55493867 @default.
• W2092762008 hasConceptScore W2092762008C86339819 @default.
• W2092762008 hasConceptScore W2092762008C86803240 @default.
• W2092762008 hasConceptScore W2092762008C95444343 @default.
• W2092762008 hasIssue "51" @default.

• next