Identification of ∆Np63r Protein Interactions by Mass Spectrometry Angela Amoresano,† Antonella Di Costanzo,‡ Gabriella Leo,† Ferdinando Di Cunto,§ Girolama La Mantia,‡ Luisa Guerrini,| and Viola Calabro ` *,‡ Dipartimento di Chimica Organica e Biologica, Universita` Federico II, Napoli, Italy, Dipartimento di Biologia Strutturale e Funzionale, Universita` di Napoli, Federico II, Italy, Centro di Biotecnologie Molecolari, Universita` di Torino, Torino, Italy, and Dipartimento di Scienze Biomolecolari e Biotecnologie, Universita` di Milano, Italy Received December 4, 2009
Abstract: p63, a transcription factor related to the p53 tumor suppressor, plays a key role in epidermal differentiation and limb development. The gene has two distinct promoters that allow the formation of proteins that either contain (TA) or lack (∆N) a transactivation domain. ∆Np63R is the most widely expressed isoform, at all stages of development and in adult tissues. It supports the regenerative capacity of basal keratinocytes and its upregulation is a hallmark of human squamous carcinomas. To get insight into the complex biology of ∆Np63R, we set out to identify ∆Np63R interacting proteins by co-immunoprecipitation in mammalian cells and mass spectrometry analysis. A total of 49 potential ∆Np63R binding proteins, including several heterogeneous ribonucleoproteins (hnRNPs), were identified. Integration of the proteomic data with a Human Coexpression Network highlighted 5 putative p63 protein interactors whose expression is significantly comodulated with p63: hnRNPA/B, hnRNPK, hnRNPQ, FUS/TLS and Keratin 5. hnRNPA/B was already described as a p63 partner, but the others were novel. Interaction of ∆Np63R with hnRNPQ, hnRNPK and FUS/TLS was confirmed by reciprocal co-immunoprecipitations in human keratinocytes. The finding that ∆Np63R exists in complexes with several RNA-binding proteins lays the premises for the analysis of the role of ∆Np63R in mRNA metabolism and transport. Keywords: Epithelial Differentiation • Mass Spectrometry • p53 Gene Family • mRNA Metabolism • Protein Interaction
Introduction The tumor suppressor p53 controls a powerful stress response by integrating upstream signals from different types of DNA damage and oncogenic stimuli. Activated p53 elicits cell cycle arrest and apoptosis, thereby preventing the formation of tumors.1 p53 is the founding member of a family of proteins * To whom correspondence should be addressed. Prof. Viola Calabro`, Dipartimento di Biologia Strutturale e Funzionale, Universita` Federico II, Napoli. Via Cinzia, Monte S Angelo, 80126 Napoli, Italy. Phone: +39 081 679069. Fax: +39 081 679033. E-mail:
[email protected]. † Dipartimento di Chimica Organica e Biologica, Universita` Federico II. ‡ Dipartimento di Biologia Strutturale e Funzionale, Universita` Federico II. § Universita` di Torino. | Universita` di Milano.
2042 Journal of Proteome Research 2010, 9, 2042–2048 Published on Web 01/19/2010
including p63 and p73.2 All three genes can regulate cell cycle and apoptosis after DNA damage. However, mouse knockout models revealed an unexpected functional diversity among them. Indeed, p63 and p73 null mice exhibit severe developmental abnormalities but no increased cancer susceptibility, whereas this picture is reversed for p53 knockouts.3 The structure of the corresponding genes, named TP53, TP63 and TP73, is evolutionarily conserved. In particular, the most conserved regions are those encoding the aminoterminal transactivation domain (TA), the central DNA binding domain (DBD), and the carboxyterminal oligomerization domain (OD).2,3 The TP63 gene has two distinct promoters allowing the synthesis of proteins that either contain (TA) or lack (∆N) a transactivation domain. In addition, alternative splicing at the 3′ end generates proteins with different C-termini, denoted R, β and γ. Only the R-isoforms (TA and ∆N) contain a Sterile Alpha Motif (SAM) domain,4 which is absent in p53. Distinct p63 isoforms are expressed and differentially modulated during epithelial differentiation.5 In particular, ∆Np63R is strongly expressed in basal keratinocytes of stratified epithelia and disappears in differentiating cells. Accordingly, human ∆Np63R is known to support the regenerative capacity of basal keratinocytes.4 Heterozygous germ line mutations of p63 cause rare autosomal dominant developmental disorders associated with ectodermal dysplasia and limb deformities.3 Accordingly, mice lacking p63 are severely compromised in their ability to generate limbs, and stratified epithelia such as skin.2 In stark contrast with the high mutation rate of p53 in a large compendium of cancer types, p63 is not mutated in tumors. Therefore, although p63 is able to activate p53 responsive genes and induce apoptosis, it is debated whether it might act as a tumor suppressor or an oncogene. Remarkably, ∆Np63R gene is upregulated or amplified in a broad range of squamous cell carcinomas, thus, suggesting that p63 might be required to provide cancer cell population with a selective advantage.6 Studies from different groups showed that ∆Np63R overexpression contributes to cell dedifferentiation and induction of metastasis.7,8 Conversely, it has recently been shown that complete loss of p63 is associated with cancer metastasis and poor prognosis, thus, implying that p63 can be a potential marker for cancer progression and invasion.9 Despite its pleiotropic involvement in multiple facets of epithelial cell biology, mechanistic explanation of how p63 accomplishes its functions remains to be defined. The study of protein-protein interaction by mass spectrometry is an increasingly important strategy to understand the role of 10.1021/pr9011156
2010 American Chemical Society
Identification of p63 Interacting Partners multifunctional proteins. To shed light on the role of ∆Np63R in epithelial biology, we set out to identify ∆Np63R binding proteins by co-immunoprecipitation in H1299 cells followed by mass spectrometry analysis. We identified 49 putative ∆Np63R molecular partners, including several hnRNPs. The finding that ∆Np63R associates with multiple RNA-binding proteins suggests that it may play a relevant role in RNA metabolism processes.
Materials and Methods Cell Culture and Transfections. H1299 cells derived from human lung carcinoma were provided by ATCC (CRL-5803). Cells were grown at 37 °C and 5% (v/v) CO2 in Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum (Euroclone). HaCaT cells were maintained in RPMI medium and 10% fetal calf serum. H1299 cells were seeded at a density of about 70% confluence and transfected with 0.2 µg of plasmid encoding myc∆Np63R using LipofectAMINE 2000 (Life Technologies, Inc.). Immunoaffinity Purification of p63-Associated Proteins. p63 complexes were affinity purified from 10 mg of total extracts prepared from Myc∆Np63R transfected H1299 cells. At 24 h after transfection, cells were washed with PBS and lysed in 50 mM Tris/HCl, pH 7.5, 150 mM NaCl, 0.5% NP40, 5 mM EDTA, 10% glycerol, and protease inhibitors (Sigma). Total protein in cell extracts was quantified using the BioRad protein assay and incubated with mouse anti-IgG agarose conjugated beads (Sigma) overnight (o.n.) at 4 °C. Beads were pretreated with 5% nonfat milk. After clarification, extracts were centrifuged at 3000 rpm for 5 min at 4 °C. Cell extracts were then incubated o.n. with immobilized anti-Myc 9E10 antibody (Santa Cruz). The beads were washed extensively with lysis buffer (150 mM NaCl, 10% glycerol, and 50 mM Tris/HCl, pH 7.5). Control samples were prepared in parallel using untransfected cell extracts. Protein samples were eluted with Myc competitor peptide (200 µg/mL in BC100), TCA precipitated, resuspended in 2× loading buffer (Sigma) and loaded on a 12.5% SDS polyacrylamide gel. The gel was run for 1 h in Tris-Glycine buffer at 25 mA and stained with colloidal Coomassie blue (Pierce). Western Blotting Analysis. Each step of the experimental procedure was paralleled by Western blotting step that served as control. Samples aliquots were resuspended in 2× loading buffer (Sigma) and loaded in a SDS-10% polyacrylamide gel. The gel was run in 10× Tris-Glycine buffer at 200 V, and transferred to a PVDF membrane (Hybond-P, Amersham Biosciences). The membrane was blocked with 4% nonfat milk in TBS (25 mM Tris, pH 7.4, and 125 mM NaCl) for 1 h at room temperature, washed 3 times with TBS and then incubated with mouse anti-Myc horseradish peroxidase-conjugated secondary antibodies (Alexis Biochemicals) Protein were revealed by enhanced chemiluminescence (RPN2132; Amersham, Buckinghamshire, U.K.). In Situ Digestion and MALDI Analysis. Trypsin, dithiothreitol (DTT), iodoacetamide and R-cyano-4-hydroxycinnamic acid were purchased from Sigma. NH4HCO3 was from Fluka. Trifluoroacetic acid (TFA)-HPLC grade was from Carlo Erba. All other reagents and solvents were of the highest purity available from Baker. Slices containing protein bands were excised from the gel and destained by repetitive washes with 0.1 M NH4HCO3, pH 7.5, and acetonitrile. Samples were reduced by incubation with 50 µL of 10 mM DTT in 0.1 M NH4HCO3 buffer, pH 7.5, and
technical notes carboxyamidomethylated with 50 µL of 55 mM iodoacetamide in the same buffer. Enzymatic digestion was carried out with trypsin (12.5 ng/µL) in 10 mM ammonium bicarbonate buffer, pH 7.8. Gel pieces were incubated at 4 °C for 2 h. Trypsin solution was then removed and a new aliquot of the same solution was added; samples were incubated for 18 h at 37 °C. A minimum reaction volume was used as to obtain the complete rehydratation of the gel. Peptides were then extracted by washing the gel particles with 10 mM ammonium bicarbonate and 1% formic acid in 50% acetonitrile at room temperature. The resulting peptide mixtures were desalted using ZipTip pipettes from Millipore, following the recommended purification procedure. MALDI-TOF mass spectra were recorded using an Applied Biosystem Voyager STR instrument equipped with a nitrogen laser (337 nm). One microliter of the analyte mixture was mixed (1/1, v/v) with a 10 mg/mL solution of R-cyano-hydroxycinnamic acid in acetonitrile/50 mM citrate buffer (2/3, v/v) and was applied to the metallic sample plate and dried down at room temperature. Acceleration and reflector voltage were set up as follows: target voltage at 20 kV, grid at 66% of target voltage, delayed extraction at 150 ns to obtain the best signal to-noise ratios and the best possible isotopic resolution. Mass calibration was performed using external peptide standards purchased from Applied Biosystems. Raw data were analyzed using the computer software provided by the manufacturer as monoisotopic masses. NanoHPLC-chip MS/MS Analysis. LC/MS/MS analyses were performed on a LC/MSD Trap XCT Ultra (Agilent Technologies, Palo Alto, CA) equipped with a 1100 HPLC system and a chip cube (Agilent Technologies). After loading, the peptide mixture was first concentrated and washed in 40 nL enrichment column (Agilent Technologies chip), with 0.2% formic acid in 2% acetonitrile as the eluent. The sample was then fractionated on a C18 reverse-phase capillary column (Agilent Technologies chip) at flow rate of 300 nL/min, with a linear gradient of eluent B (0.2% formic acid in 95% acetonitrile) in A (0.2% formic acid in 2% acetonitrile) from 7 to 60% in 50 min. Peptide analysis was performed using data-dependent acquisition of one MS scan (mass range from 300 to 1800 m/z) followed by MS/MS scans of the three most abundant ions in each MS scan. Dynamic exclusion was used to acquire a more complete survey of the peptides by automatic recognition and temporary exclusion (2 min) of ions from which definitive mass spectral data was previously acquired. Nitrogen at a flow rate of 3 L/min and heated to 325 °C was used as the dry gas for spray desolvation. MS/MS spectra were measured automatically when the MS signal surpassed the threshold of 50 000 counts. Double charged ions were preferably isolated and fragmented over single charged ions. The acquired MS/MS spectra were transformed in Mascot generic file format and used for peptides identification with a licensed version of MASCOT, in a local database. Protein Identification. Spectral data were analyzed using Analyst software (version 1.4.1) and MSMS centroid peak lists were generated using the MASCOT.dll script (version 1.6b9). MSMS centroid peaks were threshold at 0.1% of the base peak. MSMS spectra having less than 10 peaks were rejected. MSMS spectra were searched against NCBInr database (2009/07/10 version, 211 309 entries) and then converted in Swiss-protein code, using the licensed version of Mascot 2.1 (Matrix Science), after converting the acquired MSMS spectra in mascot generic file format. The Mascot search parameters were taxonomy Journal of Proteome Research • Vol. 9, No. 4, 2010 2043
technical notes human; “trypsin” as enzyme allowing up to 3 missed cleavages, carbamidomethyl on as fixed modification, oxidation of M, pyroGlu N-term Q, as variable modifications, 600 ppm MSMS tolerance and 0.6 Da peptide tolerance and top 20 protein entries. Spectra with a MASCOT score
