- ADJUNCT PROFESSOR Oncological Sciences
Karolinska Nobel Conference on Ubiquitin
Hiroshima Cancer Seminar
Golda Meir Fellow
JNK targeted ubiquitination of its associated proteins - the search for JNK-dependent ligase
Former studies in our lab have established the role of JNK in targeting the ubiquitination of degradation of its associated transcription factors substrates. This important phenomena was demonstrated for c-Jun, ATF2 and p53, and occurs under normal growth conditions, when JNK is not active as a kinase. In all cases, activation of JNK by stress, followed by its phosphorylation of associated substrates results in their stabilization. We therefore seek to identify the mechanism underlying JNK ability to target its associated proteins for ubiquitination and degradation. We hypothesize that such protein may be part of E3 ligase complex, or alternatively be a RING finger protein with potent E3 ligase. Recent studies via mass spec analysis of JNK bound proteins enabled the identification of novel partners which could mediate changes in stability of JNK-associated substrates, aspects which are currently under invesitgation.
Fuchs SY, Adler V, Buschmann T, Yin Z, Wu X, Jones SN, Ronai Z. JNK targets p53 ubiquitination and degradation in nonstressed cells. Genes Dev 1998 Sep 1; 12(17): 2658-63.In this study we elucidated the role of nonactive JNK in regulating p53 stability. The amount of p53-JNK complex was inversely correlated with p53 level. A peptide corresponding to the JNK binding site on p53 efficiently blocked ubiquitination of p53. Similarly, p53 lacking the JNK binding site exhibits a longer half-life than p53(wt). Outcompeting JNK association with p53 increased the level of p53, whereas overexpression of a phosphorylation mutant form of JNK inhibited p53 accumulation. JNK-p53 and Mdm2-p53 complexes were preferentially found in G0/G1 and S/G2M phases of the cell cycle, respectively. Altogether, these data indicate that JNK is an Mdm2-independent regulator of p53 stability in nonstressed cells.
Fuchs SY, Ronai Z. Ubiquitination and degradation of ATF2 are dimerization dependent. J Biol Chem 1999; 19: 3289-3298.
Fuchs SY, Chen A, Xiong Y, Ronai Z. HOS, a human homologue of Slimb, forms an SCF complex with Skp1 and Cullin1 and targets the phosphorylation-dependent degradation of IkB and b-catenin. Oncogene 1999 Mar 25; 18(12): 2039-46.SCF E3 ubiquitin ligases mediate ubiquitination and proteasome-dependent degradation of phosphorylated substrates. We identified a human F-box/WD40 repeats protein (HOS), which is homologous to Slimb/h betaTrCP. Being a part of SCF complex with Skp1 and Cullin1, HOS specifically interacted with the phosphorylated IkappaB and beta-catenin, targeting these proteins for proteasome-dependent degradation in vivo. This targeting required Cullin1 as expression of a mutant Cullin1 abrogated the degradation of IkappaB and of beta-catenin. Mutant HOS which lacks the F-box blocked TNF alpha-induced degradation of IkappaB as well as GSK3beta-mediated degradation of beta-catenin. This mutant also inhibited NF-kappaB transactivation and increased the beta-catenin-dependent transcription activity of Tcf. These results demonstrate that SCF(HOS) E3 ubiquitin ligase regulate both NF-kappaB and beta-catenin signaling pathways.
Adler V, Yin Z, Fuchs SY, Benezra M, Rosario L, Tew KD, Pincus MR, Sardana M, Henderson CJ, Wolf CR, Davis RJ, Ronai Z. Regulation of JNK signaling by GSTp. EMBO J 1999 Mar; 18(5): 1321-34.Studies of low basal Jun N-terminal kinase (JNK) activity in non-stressed cells led us to identify a JNK inhibitor that was purified and identified as glutathione S-transferase Pi (GSTp) and was characterized as a JNK-associated protein. UV irradiation or H2O2 treatment caused GSTp oligomerization and dissociation of the GSTp-JNK complex, indicating that it is the monomeric form of GSTp that elicits JNK inhibition. Addition of purified GSTp to the Jun-JNK complex caused a dose-dependent inhibition of JNK activity. Conversely, immunodepleting GSTp from protein extracts attenuated JNK inhibition. Furthermore, JNK activity was increased in the presence of specific GSTp inhibitors and a GSTp-derived peptide. Forced expression of GSTp decreased MKK4 and JNK phosphorylation which coincided with decreased JNK activity, increased c-Jun ubiquitination and decreased c-Jun-mediated transcription. Co-transfection of MEKK1 and GSTp restored MKK4 phosphorylation but did not affect GSTp inhibition of JNK activity, suggesting that the effect of GSTp on JNK is independent of the MEKK1-MKK4 module. Mouse embryo fibroblasts from GSTp-null mice exhibited a high basal level of JNK activity that could be reduced by forced expression of GSTp cDNA. In demonstrating the relationships between GSTp expression and its association with JNK, our findings provide new insight into the regulation of stress kinases.
Buschmann T, Adler V, Matusevich E, Fuchs SY, Ronai Z. p53 phosphorylation and association with murine double minute 2, c-Jun NH2-terminal kinase, p14ARF, and p300/CBP during the cell cycle and after expos. Cancer Res 2000 Feb 15; 60(4): 896-900.p53 phosphorylation and association with proteins is implicated in its stability and activity. We have compared the association of DNA-bound and overall pools of p53 with murine double minute 2 (Mdm2), c-Jun NH2-terminal kinase (JNK), p300/CBP, and p14ARF during cell cycle progression. Whereas DNA-bound p53 associates with JNK at G0-G1 and with Mdm2 and p300 during S and G2-M phases, the general pool of p53 was found in complex with JNK and Mdm2 almost throughout the cell cycle. Phosphorylation of p53 at serines 9, 15, and 20 is at the highest levels at G1 and at serines 37 and 392 during G2-M phase. Whereas a high dose of UV irradiation was required for phosphorylation of serines 15 and 392 between 8 and 24 h after treatment, a low dose caused immediate phosphorylation on serines 9, 20, and 372. These dynamic changes in the phosphorylation of p53 are expected to play a pivotal role in p53 association, stability, and function.
Ivanov VN, Bhoumik A, Krasilnikov M, Raz R, Owen-Schaub LB, Levy D, Ronai Z. Cooperation between STAT3 and c-Jun suppresses Fas transcription. Mol Cell 2001; 7: 517-528.
Cho S, Bhoumik A, Broday L, Ivanov V, Rosenstein B, Warburton P, Ronai Z. TIP49bD a regulator of activating transcription factor 2 (ATF2) response to stress and DNA damage. Mol Cell Biol 2001 December; 21(24): 8398-8413.
Bhoumik A, Ivanov V, Ronai Z. ATF2-derived peptides alter resistance of human tumor cell lines to UV-irradiation and chemical treatment. Clin Cancer Res 2001; 7: 331-342.
Buschmann T, Fuchs SY, Fried VA, Ivanov VN, Alacron-Vargas D, Pincus M, Ronai Z. Jun NH2-Terminal Kinase Phosphorylation of p 53 on Thr-81 Is Important for p53 Stabilization and Transcriptional Activities in Response to Stress. Mol Cel Biol 2001; 21: 2743-2754.
Ivanov I, Bhoumik A, Krasilnikov M, Raz R, Owen-Sachaub LB, Levy D, Ronai Z. Cooperation between STAT3 and c-Jun suppresses Fas transcription. Mol Cell 2001; 7: 517-528.
Habelhah H, Shah K, Huang L, Ostareck-Lederer A, Burlingame AL, Shokat KM, Hentze MW, Ronai Z. ERK phosphorylation drives cytoplasmic accumulation of hnRNP-K and subsequent inhibition of mRNA translation. Nat Cell Biol 2001; 3: 3:325-330.
Habelhah H, Hunag L, Burlingame AL, Shokat MW, Ronai Z. Identification of New JNK Substrate Using ATP Pocket Mutant JNK and a Corresponding ATP Analogue. J Biol Chem 2001; 276: 18090-18095.
Ivanov VN, Fodstad , Ronai Z. Expression of RING finger-deleted TRAF2 sensitizes metastatic melanoma cells to apoptosis via up-regulation of p38, TNFa and suppression of NF-kB activities. Oncogene 2001; 20: 2243-2253.
Ronai Z. An ATF2-derived peptide sensitizes melanomas to apoptosis and inhibits their growth and metastasis.. J. Clinical Invest. 2002;.
Ronai Z. FAP-1 association with Fas (Apo-1) inhibits Fas expression on the cell surface.. Mol Cell Biol 2003;.
Ronai Z. RNF5, a RING Finger Protein That Regulates Cell Motility by Targeting Paxillin Ubiquitination and Altered Localization . Mol Cell Biol 2003;.
Ronai Z. Mdm2: A regulator of cell growth and death.. Adv Cancer Res 2003; 89: 1-34.
Ronai Z. Ubiquitination and translocation of TRAF2 is required for activation of JNK but not of p38 or NF-kappaB.. EMBO J 2004 January;.
Ronai Z. ATF2-derived peptide induces transcriptional switch which sensitizes melanoma to apoptosis and inhibit their tumorigenicity. Proc. Natl. Acd. Sci USA 2004 March;.
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