Chapter 2

Chapter 2 Regulation of Transcription Factor Function by Targeted Protein Degradation: An Overview Focusing on p53, c-Myc, and c-Jun Jukka Westermarck Abstract Regulation of protein degradation is an important mechanism by which concentrations of proteins is controlled in cells. In addition to proteins involved in cell cycle regulation or mitosis, protein levels of many transcription factors are regulated by targeted proteosomal degradation. Regulation of protein degradation and stability is usually linked to post-translational modification of the target protein by phosphorylation. The resulting phosphoaminoacid in the context of the adjacent protein sequence is then recognized by E3 ubiquitin ligase enzymes that covalently attach small ubiquitin protein to the target protein and thereby direct them to be degraded by the proteosomes. Here, we present an overview of mechanisms regulating stability of p53, c-Myc, and c-Jun transcription factors. Especially, the purpose is to highlight the role of protein phosphorylation in the regulation of stability of these transcription factors. We also present examples where phosphorylation can either enhance or inhibit protein degradation. Lastly, we discuss the common theme among p53, c-Myc, and c-Jun proteins that the N-terminal phosphorylation both increases the transactivation capacity of the protein and protects the protein from proteolytic degradation. Key words: c-Jun, c-Myc, p53, Phosphorylation, Proteosomal degradation, Ubiquitin

1. Introduction Protein stability is an important mechanism by which concentrations of biologically active proteins is controlled in cells. Changes in protein stability occurs usually when rapid fluctuations in protein amounts are needed. Some proteins are intrinsically very stable but become degraded in response to specific physiological signals or cellular state. One example of such proteins are those involved in cell cycle regulation. Other proteins are usually very short lived and degraded at all times, but they Paul J. Higgins (ed.), Transcription Factors: Methods and Protocols, Methods in Molecular Biology, vol. 647, DOI 10.1007/978-1-60761-738-9_2, © Springer Science+Business Media, LLC 2010

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become stabilized when a rapid increase in protein amount is required and induction of protein expression by increased gene transcription is not sufficient. This type of mechanism is best exemplified by the very rapid increase in p53 transcription factor stability in response to cellular stress (1). However, often both gene transcription and protein stability are coupled synchronously to either repress or increase protein amounts. c-myc gene transcription and stabilization of MYC protein, for example, are modulated in response to phosphorylation by ERK kinase (2, 3). Control of protein stability is therefore a powerful mechanism to regulate protein amount and therefore this mechanism has been hijacked by cancer cells to promote oncogenic behavior. In general, alterations linked to protein stabilization in cancer tend to accelerate degradation of tumor suppressor proteins and in turn protect oncoproteins from degradation (4, 5). Regulation of protein stability is very often linked to protein phosphorylation. The resulting phosphoaminoacid in the context of the adjacent protein sequence (phospho-degron) is recognized by E3 ubiquitin ligase enzymes that covalently attach small ubiquitin proteins moieties the lysine residue of the target protein (6). This is followed by sequential attachment of new ubiquitin molecules to the ubiquitins already linked to specific target protein to build up a polyubiquitin chain. A protein marked with a polyubiquitin chain is consequently transported to cellular organelles responsible for protein degradation, the 26S proteosomes, and through a multi-step process digested into short peptides (6). Several families of ubiquitin ligases exist, and the same target protein can be subjected to ubquitination by several different ubiquitin ligases potentially resulting in different biological outcomes (6, 7).

2. Regulation of Transcription Factor Expression by Targeted Protein Degradation

Protein levels of many transcription factors are regulated by targeted proteosomal degradation. The contribution of mechanisms regulating protein stability to overall protein levels vary greatly between different proteins and cellular conditions. Although biologically intelligible, the simultaneous increase or decrease in mRNA and protein levels may mask the influence of protein stabilization, and thus mislead the interpretations regarding mechanisms that are truly relevant for regulation of steady-state levels of the studied protein. Therefore, should mRNA and protein expression levels, or the trend in their regulation, does not readily correlate, it is relevant to consider if protein stability is affected. On the other hand, regulation of protein stability would

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appropriately be considered in the cases where protein expression is lost without parallel regulation of corresponding mRNA, or when steady-state levels of both endogenous and exogenously expressed forms of the same protein are regulated in a similar fashion. In all these cases, protein stability can be easily assessed by experiments where protein translation is blocked with cycloheximide treatment, and the decline of protein amounts is followed by standard Western blotting or radioactive protein labeling techniques, as described previously (8–10). Here I present an overview of mechanisms regulating stability of p53, c-Myc, and c-Jun transcription factors. Rather than a complete review, the aim here is to introduce p53, c-Myc, and c-Jun as examples how transcription factor expression is regulated by proteosomal degradation. The purpose is to highlight the role of protein phosphorylation in the regulation of transcription factor protein stability. For more details, and for information about other ubiquitin ligases regulating of p53, c-Myc, and c-Jun activities, reader is encouraged to become acquainted with the following articles (1, 3, 6, 7, 9, 11, 12). 2.1. p53

Transcription factor p53 is a short-lived protein in normal quiescent cells. Its half-life is approximately 20 min and it is continuously degraded by the proteosomal system. p53 degradation is mostly controlled by its association with the ubiquitin E3 ligase Mdm2 which binds to the aminoterminal domain of p53 and targets the newly synthesized protein for degradation by tagging it with ubiquitin (1). Interestingly, the Mdmd2 binding domain of p53 is phosphorylated by several kinases regulating p53 transcriptional activity and stability. The kinases known to phosphorylate p53 include Chk1, Chk2, ATM/ATR, the activity of which are rapidly induced in response to genotoxic stress. Phosphorylation of the aminoterminal amino acids of p53 changes structure of the aminoterminal domain inhibiting Mdm2 binding. Interestingly, phosphorylation of Mdm2 also regulates its association with p53, and thereby p53 stability (1). Survival promoting signals, through Akt kinase-mediated phosphorylation, stimulates Mdm2 binding to p53, and consequently enhances p53 degradation. Conversely, some of the stress-activated kinases described above (Chk2, ATM/ ATR) can, in addition to directly phosphorylating p53, inactivate Mdm2 and thereby promote p53 stabilization. Taken together, phosphorylation-dependent regulation of Mdm2 binding to p53, by any of the mechanisms described above, provides a very elegant way to regulate p53 activity, and by these means allows cells to respond rapidly to cellular stress and survival signals.

2.2. c-Myc

Oncogenic transcription factor c-Myc is expressed at low levels in normal cells, but both c-myc mRNA expression and protein

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stability is increased in response to mitogenic stimuli and cellular transformation. Like, p53, regulation of c-Myc stability is closely linked with protein phosphorylation. c-Myc harbors a phosphodegron motif recognized by Fbw7 ubiquitin ligase in its N-terminal domain (5). Within this motif, serine 62 phosphorylation by ERK kinase is required as “priming phosphorylation” for GSK3mediated phosphorylation of an adjacent threonine 58 (3, 5). In vitro, c-Myc peptide that is double phosphorylated on serine 62 and threonine 58 binds to Fbw7 (9), but experiments in cultured cells demonstrate that if serine 62 is phosphorylated, the protein is protected from proteosomal degradation (10, 13). Additionally, serine 62 has been shown to be dephosphorylated by tumor suppressor protein phosphatase 2A (PP2A) and this leads to c-Myc destabilization (10, 13). Therefore, the stability of c-Myc is regulated by a complex interplay between kinases phosphorylating threonine 58 and serine 62, and PP2A phosphatase activity. Interestingly, recent studies have identified new proteins involved in the regulation of c-Myc stability through Fbw7 phosphodegron. Cancerous inhibitor of PP2A (CIP2A) was shown to inhibit c-Myc serine 62 dephosphorylation by PP2A and to stabilize c-Myc (8). Ubiquitin-specific protease 28 in turn was shown to antagonize Fbw7-mediated c-Myc ubiquitination and by these means to prevent c-Myc degradation (14). Both CIP2A and USP28 are upregulated in human cancers illustrating an additional level of regulation of protein stability to promote tumorigenesis. 2.3. c-Jun

c-Jun is an AP-1 family transcription factor implicated in the regulation of cell death and survival as well as in neurological degeneration and cellular transformation (15). Similarly to c-myc, c-jun is an immediate-early gene whose mRNA expression is rapidly induced by both mitogenic and stress signals, and which protein stability is enhanced by the same stimuli that increases gene expression. Activation of stress-activated JNK kinases results in phosphorylation of c-Jun on serines 63/73 and on threonines 91/93. c-Jun N-terminal phosphorylation has been shown to increase the transactivation potential of c-Jun, but it also inhibits ubiquitination and degradation of the protein (15, 16). Whereas JNK-mediated N-terminal phosphorylation stabilizes the c-Jun protein, GSK3-mediated phosphorylation of c-Jun on threonine 239 induces Fbw7 E3-ligase recruitment and protein degradation. Interestingly, the Fbw-7 phospho-degron in c-Jun and c-Myc is highly similar in sequence (9). In both proteins, GSK3mediated phosphorylation of the threonine (239 in c-Jun and 58 in c-Myc) requires a priming phosphorylation at the +4 position. However, whereas in c-Myc the priming phosphorylation is done by ERK kinase and this phosphorylation has to be removed in order for the protein to become ubiquitinylated (10),

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in c-Jun the kinase responsible for serine 243 phosphorylation is not known, and this phosphorylation stimulates protein ubiquitinylation and degradation (9).

3. Conclusions In addition to examples presented above, expression of many other transcription factors, such as b-catenin, HIF-1a, and Smad proteins, is regulated by targeted proteosomal degradation. One common theme among proteins targeted for ubiquitination is that post-translational modification of the E3 ubiquitin ligasebinding site determines the efficiency of protein ubiquitination and degradation. Whereas in the case of most of the other transcription factors this post-translational modification is phosphorylation, in the case of HIF-1a, it is prolyl hydroxylation (17). Moreover, as exemplified with c-Jun above, one protein can be subjected to phosphorylation-dependent regulation of ubiquitination in more than one protein domain, and depending on the site, phosphorylation can either enhance or inhibit ubiquitination and protein stability. On the other hand, in addition to the specific transcription factor to be modified by phosphorylation and ubiquitination, phosphorylation of the ubiquitin ligase regulates its activity and thereby target protein stability. The best established examples of this are phosphorylation of p53 and c-Jun ubiquitin ligases Mdm2 and Itch, respectively (1, 11). Lastly, an additional common theme shared by p53, c-Myc, and c-Jun is that the N-terminal phosphorylation that increases the transactivation capacity of the protein also prevents the protein from proteolytic degradation (1, 16, 18). Even though this complicates conclusions regarding the biological role of such ­phosphorylations it clearly suggests that natue has evolved such system to enhance activity of these transcription factors in the most economical manner. References 1. Lavin MF, Gueven N (2006) The complexity of p53 stabilization and activation. Cell Death Differ 13:941–950 2. Sears R, Leone G, DeGregori J, Nevins JR (1999) Ras enhances Myc protein stability. Mol Cell 3:169–179 3. Sears RC (2004) The life cycle of C-myc: from synthesis to degradation. Cell Cycle 3:1133–1137 4. Junttila MR, Westermarck J (2008) Mechanisms of MYC stabilization in human malignancies. Cell Cycle 7:592–596

5. Welcker M, Clurman BE (2008) FBW7 ubiquitin ligase: a tumour suppressor at the crossroads of cell division, growth and differentiation. Nat Rev Cancer 8:83–93 6. Welchman RL, Gordon C, Mayer RJ (2005) Ubiquitin and ubiquitin-like proteins as multifunctional signals. Nat Rev Mol Cell Biol 6:599–609 7. Dai MS, Jin Y, Gallegos JR, Lu H (2006) Balance of Yin and Yang: ubiquitylationmediated regulation of p53 and c-Myc. Neoplasia 8:630–644

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8. Junttila MR, Puustinen P, Niemela M, Ahola R, Arnold H, Bottzauw T, Ala-Aho R, Nielsen C, Ivaska J, Taya Y, Lu SL, Lin S, Chan EK, Wang XJ, Grenman R, Kast J, Kallunki T, Sears R, Kähäri VM, Westermarck J (2007) CIP2A inhibits PP2A in human malignancies. Cell 130:51–62 9. Wei W, Jin J, Schlisio S, Harper JW, Kaelin WG Jr (2005) The v-Jun point mutation allows c-Jun to escape GSK3-dependent recognition and destruction by the Fbw7 ubiquitin ligase. Cancer Cell 8:25–33 10. Yeh E, Cunningham M, Arnold H, Chasse D, Monteith T, Ivaldi G, Hahn WC, Stukenberg PT, Shenolikar S, Uchida T, Counter CM, Nevins JR, Means AR, Sears R (2004) A signalling pathway controlling c-Myc degradation that impacts oncogenic transformation of human cells. Nat Cell Biol 6:308–318 11. Gao M, Labuda T, Xia Y, Gallagher E, Fang D, Liu YC, Karin M (2004) Jun turnover is controlled through JNK-dependent phosphorylation of the E3 ligase Itch. Science 306:271–275 12. Wertz IE, O’Rourke KM, Zhang Z, Dornan D, Arnott D, Deshaies RJ, Dixit VM (2004) Human De-etiolated-1 regulates c-Jun by assembling a CUL4A ubiquitin ligase. Science 303:1371–1374

13. Arnold HK, Sears RC (2006) Protein phosphatase 2A regulatory subunit B56alpha associates with c-myc and negatively regulates c-myc accumulation. Mol Cell Biol 26:2832–2844 14. Popov N, Wanzel M, Madiredjo M, Zhang D, Beijersbergen R, Bernards R, Moll R, Elledge SJ, Eilers M (2007) The ubiquitin-specific protease USP28 is required for MYC stability. Nat Cell Biol 9:765–774 15. Shaulian E, Karin M (2002) AP-1 as a regulator of cell life and death. Nat Cell Biol 4:E131–E136 16. Musti AM, Treier M, Bohmann D (1997) Reduced ubiquitin-dependent degradation of c-Jun after phosphorylation by MAP kinases. Science 275:400–402 17. Jaakkola P, Mole DR, Tian YM, Wilson MI, Gielbert J, Gaskell SJ, Kriegsheim A, Hebestreit HF, Mukherji M, Schofield CJ, Maxwell PH, Pugh CW, Ratcliffe PJ (2001) Targeting of HIF-alpha to the von Hippel–Lindau ubiquitylation complex by O2-regulated prolyl hydroxylation. Science 292:468–472 18. Benassi B, Fanciulli M, Fiorentino F, Porrello A, Chiorino G, Loda M, Zupi G, Biroccio A (2006) c-Myc phosphorylation is required for cellular response to oxidative stress. Mol Cell 21:509–519