Journal of Molecular Biology
Intrinsically Unstructured Domains of Arf and Hdm2 Form Bimolecular Oligomeric Structures In Vitro and In Vivo
Introduction
The Arf tumor suppressor (p14Arf in humans and p19Arf in mice) is activated by abnormal mitogenic signaling in mammalian cells (e.g., through activation of the Ras,1 Myc,2 E2F1,3 or BCR-ABL4 oncogene). Arf prevents tumorigenesis through p53-dependent and p53-independent mechanisms.4 In the absence of mitogenic signals, Hdm2 (Mdm2 in mice) inhibits p53-dependent cell cycle arrest or apoptosis via a negative feedback mechanism.4 Upon oncogene activation, Arf is expressed and directly binds Hdm2. Several studies have suggested that the Hdm2/Arf complex is sequestered in nucleoli, where Hdm2 can no longer inhibit p53 activity.5, 6 However, while their direct association is required, nucleolar colocalization of Arf and Hdm2 is dispensable for p53 activation.7, 8 Arf loss is associated with tumorigenesis in mice,9 and genetic loss or silencing of Arf is frequently observed in human cancer.10 Moreover, Arf function is overridden in 5–10% of all human cancers through Hdm2 overexpression.11 Thus, maintaining the integrity of the Arf–Hdm2–p53 pathway is critical for tumor suppression. Knowledge of the physical basis of Arf and Hdm2 interactions will provide critical mechanistic insights into the most commonly altered pathway in human cancer.4
p19Arf and p14Arf are comprised of 22% and 18% arginine residues, respectively, and do not exhibit significant sequence similarity to other proteins. This high Arg content is associated with the ability to interact with more than 25 other proteins,4 often through the formation of heterooligomers in vitro and in vivo.12 For example, Arf forms heterooligomers (2–5 MDa) in nucleoli with nucleophosmin13 and forms β-strand-rich heterooligomers when bound to Hdm2.14, 15 Previous studies have shown that a ∼ 60-residue N-terminal fragment of Arf encoded by exon 1β recapitulates the functions of full-length Arf mediated through interactions with Hdm2,5 N-Myc and c-Myc,16 and nucleophosmin.17 Furthermore, a 37-residue intrinsically unstructured N-terminal fragment of p19Arf (mArf-N37)18 bound to Mdm2 and caused p53-dependent cell cycle arrest in mouse fibroblasts.6, 18 Finally, a peptide comprised of residues 1–20 of p14Arf fused to green fluorescent protein interacted with Hdm2 and activated p53 in vivo.19, 20 The first 13 residues are highly conserved among mammalian Arf sequences, highlighting their importance in Arf-mediated tumor suppression (Fig. 1a).
Hdm2 is comprised of p53 binding (residues 26–108),21 C4 zinc finger (residues 297–329),22 and C2H2C4 RING domains (residues 429–491)23 that are connected by intrinsically unstructured linker segments. Hdm2 inhibits p53 through two mechanisms: (1) the N-terminal domain binds the p53 transcriptional activation domain, inhibiting p53-dependent gene transcription,24 and (2) the E3 ubiquitin ligase RING domain polyubiquitinates p53, promoting its degradation by the 26S proteasome.25, 26 Arf inhibits Hdm2-dependent inactivation of p53 by binding to an Hdm2 domain spanning residues 210–304 (termed “Hdm2-ABD”).6, 14 The human and mouse sequences of this segment are 89% identical and enriched in acidic residues (32% Asp/Glu in Hdm2) (Fig. 1b). Mapping studies revealed that Arf and Hdm2 each contain two conserved segments that mediate their interaction;14 these are A1 (residues 3–10) and A2 (residues 21–29) of p19Arf, and H1 (residues 235–259) and H2 (residues 270–289) of Hdm2.14 Segments A1 and A2 exhibit a conserved motif comprised of five mostly hydrophobic residues flanked by Arg residues18 (Fig. 1a). In contrast, segments H1 and H2 of Hdm2 are rich in acidic amino acids, and H1, in particular, exhibits a conserved alternating pattern of acidic (or hydrophilic) residues and hydrophobic residues14 (Fig. 1b).
mArf-N37 and Hdm2-ABD, the domains of Arf and Hdm2 that have been shown to mediate their interactions in vivo, are intrinsically unstructured in isolation.14, 18 When combined in solution, however, these polypeptides coassembled to form β-strand-rich supramolecular structures that, under certain conditions, resemble amyloid fibrils.14 Detailed structural characterization of these coassemblies using either NMR spectroscopy or X-ray crystallography was not feasible.14, 15, 27 To overcome this difficulty, we studied complexes formed by coassembly of a peptide containing a single Arg-rich motif (residues 2–10 of p19Arf) (Fig. 1a) with Hdm2-ABD. The resulting coassemblies, while recapitulating many of the structural hallmarks of those comprised of larger fragments of Arf, remained soluble at high concentration (> 1 mM). We report herein a detailed characterization of A1-mini/Hdm2-ABD coassemblies using CD, NMR spectroscopy, and analytical ultracentrifugation (AUC), providing the first insights into the structural organization of these bimolecular β-strand-rich oligomers. In addition, we show that fluorescently tagged A1-mini exhibited nucleolar localization in mouse fibroblasts and, in some cells, was colocalized with endogenous Mdm2. Overall, these data suggest strongly that Arf inhibits Hdm2 through the formation of bimolecular β-strand-rich oligomeric structures.
Section snippets
A1-mini and Hdm2-ABD cofold into β-strands
A 15-residue peptide containing the A1 segment of p14Arf was previously shown to coassemble with Hdm2-ABD,14 and with a 15-residue peptide containing the H1 segment of Hdm2,15 into bimolecular, β-strand-rich oligomeric structures. These structures, however, could not be studied by solution NMR spectroscopy due to their large size and poor solubility. Consequently, we surveyed the Hdm2-ABD coassembly properties of several truncated A1 peptides (containing 7–15 residues). A peptide containing
Discussion
Oncogene activation and inappropriate mitogenic signaling activate the expression of Arf, which binds to Hdm2 and relieves inhibition of p53, ultimately leading to p53-dependent and p53-independent cell cycle arrest or apoptosis.38, 39 While the importance of Arf and Hdm2 in tumor biology is well established, little information on the molecular basis of Arf and Hdm2 interactions is available. This dearth of knowledge stems from experimental difficulties associated with lack of structure within
Preparation of Hdm2-ABD and A1-mini
Hdm2-ABD was expressed in Escherichia coli using a pET-28a vector (Novagen) and was purified from the soluble fraction after lysis in 20 mM Tris–HCl (pH 8.0) and 500 mM NaCl and centrifugation, as previously described.14 Urea was added to the clarified extract to a concentration of 6 M, followed by purification of His-tagged Hdm2-ABD using Ni2 +-affinity chromatography with buffers containing 6 M urea. Fractions containing Hdm2-ABD were exhaustively dialyzed against 20 mM Tris–HCl (pH 8.0) and
Acknowledgements
The authors acknowledge Dr. Rensheng Luo for assistance with the diffusion NMR experiments and Dr. Charles Galea for helpful discussion. This work was supported by grants from the National Institutes of Health (CA 82491 awarded to R.W.K. and P30CA21765 awarded to St. Jude Children's Research Hospital) and the American Lebanese Syrian Associated Charities of St. Jude Children's Research Hospital.
References (50)
- et al.
Oligomerization of the human ARF tumor suppressor and its response to oxidative stress
J. Biol. Chem.
(2003) - et al.
Defining the molecular basis of Arf and Hdm2 interactions
J. Mol. Biol.
(2001) - et al.
p14ARF interacts with N-Myc and inhibits its transcriptional activity
FEBS Lett.
(2007) - et al.
Tumor suppressor ARF degrades B23, a nucleolar protein involved in ribosome biogenesis and cell proliferation
Mol. Cell
(2003) - et al.
Contribution of two independent MDM2-binding domains in p14(ARF) to p53 stabilization
Curr. Biol.
(2000) - et al.
Solution structure of the Hdm2 C2H2C4 RING, a domain critical for ubiquitination of p53
J. Mol. Biol.
(2006) - et al.
The Mdm-2 oncogene product forms a complex with the p53 protein and inhibits p53-mediated transactivation
Cell
(1992) - et al.
Macromolecular size-and-shape distributions by sedimentation velocity analytical ultracentrifugation
Biophys. J.
(2006) Size-distribution analysis of macromolecules by sedimentation velocity ultracentrifugation and Lamm equation modeling
Biophys. J.
(2000)- et al.
Size-distribution analysis of proteins by analytical ultracentrifugation: strategies and application to model systems
Biophys. J.
(2002)
Tumor suppression by Ink4a-Arf: progress and puzzles
Curr. Opin. Genet. Dev.
Oncoprotein MDM2 is a ubiquitin ligase E3 for tumor suppressor p53
FEBS Lett.
Functional amyloid—from bacteria to humans
Trends Biochem. Sci.
Oncogenic ras activates the ARF–p53 pathway to suppress epithelial cell transformation
Proc. Natl Acad. Sci. USA
Myc signaling via the ARF tumor suppressor regulates p53-dependent apoptosis and immortalization
Genes Dev.
E2F1 suppresses skin carcinogenesis via the ARF–p53 pathway
Oncogene
Divorcing ARF and p53: an unsettled case
Nat. Rev. Cancer
Nucleolar Arf sequesters Mdm2 and activates p53
Nat. Cell Biol.
Cooperative signals governing ARF–mdm2 interaction and nucleolar localization of the complex
Mol. Cell. Biol.
Stabilization of p53 by p14ARF without relocation of MDM2 to the nucleolus
Nat. Cell Biol.
ARF function does not require p53 stabilization or Mdm2 relocalization
Mol. Cell. Biol.
Tumor suppression at the mouse INK4a locus mediated by the alternative reading frame product p19ARF
Cell
Hypermethylation of the p14(ARF) gene in ulcerative colitis-associated colorectal carcinogenesis
Cancer Res.
MDM2 and prognosis
Mol. Cancer Res.
Physical and functional interactions of the Arf tumor suppressor protein with nucleophosmin/B23
Mol. Cell. Biol.
Cited by (28)
Direct detection of carbon and nitrogen nuclei for high-resolution analysis of intrinsically disordered proteins using NMR spectroscopy
2018, MethodsCitation Excerpt :This allowed for very high resolution in the directly detected dimensions and moderate resolution in the indirectly detected dimensions and also balanced considerations regarding sensitivity and total acquisition time. Hdm2-ABD is a relatively small and well-behaved IDR and, despite its extensive disorder [21], yielded well resolved 2D 1H-15N and 13C-15N correlation spectra that did not provide the opportunity to illustrate the full potential of 15N-detection for improved resolution. However, the Surf6-N IDR presented greater resolution challenges due to its larger size (185 versus 99 residues for Surf6-N and Hdm2-ABD, respectively), low sequence complexity (17.7% Ala, 15.5% Lys, 12.6% Glu and 11.0% Arg residues), and tendency for self-association (unpublished results, E. Gibbs and R. Kriwacki).
Cellular Strategies for Regulating Functional and Nonfunctional Protein Aggregation
2012, Cell ReportsCitation Excerpt :These aggregates could be found as extracellular or intracellular deposits in different human tissues and have been linked to human pathologies such as neurodegenerative diseases or systemic amyloidosis (Caughey and Lansbury, 2003; Chiti and Dobson, 2006). While the harmful effects of aggregation are generally accepted, recent findings indicate that a variety of organisms take advantage of the particular properties (e.g., yield strength, protease and detergent resistance) of amyloid-like protein aggregates, i.e., they use them as “functional” aggregates (Badtke et al., 2009; Decker et al., 2007; Fiumara et al., 2010; Fowler et al., 2006; Fowler et al., 2007; Franks and Lykke-Andersen, 2008; Herter et al., 2005; Kaiser et al., 2008; Kentsis et al., 2002; Kopito, 2000; Lelouard et al., 2002; Maji et al., 2009; Olzscha et al., 2011; Ozgur et al., 2010; Reijns et al., 2008; Salazar et al., 2010; Sivakolundu et al., 2008; Spector, 2006). Bacteria such as Escherichia coli or Steptomyces coelicolor use fibrils formed from different proteins to mediate binding to host proteins and enable hyphae growth into the air, respectively.
Inhibition of p53 DNA binding function by the MDM2 protein acidic domain
2011, Journal of Biological ChemistryCitation Excerpt :Here we have shown that ARF binding to the MDM2 acidic domain also prevents p53 misfolding and stimulates p53 DNA binding. ARF binding to MDM2 has been shown to promote stable β sheet formation (58, 59), which may trap MDM2 acidic domain in a conformation that cannot bind p53 core domain or induce p53 misfolding. It is still not clear how p53-MDM2-ARF complex can activate transcription after binding DNA.
Linking folding and binding
2009, Current Opinion in Structural BiologyExpanding the proteome: Disordered and alternatively folded proteins
2011, Quarterly Reviews of Biophysics
- 1
Present addresses: B. Bothner, Department of Chemistry and Biochemistry, Montana State University, Bozeman, MT 59717, USA; C. Ashley, College of Medicine, University of Tennessee Health Science Center, Memphis, TN 38163, USA; J. Satumba, Cargill Incorporated, Global Scientific Resources, 2500 Shadywood Road, Navarre, MN 55331, USA.