RESEARCH ARTICLE OFFICIAL JOURNAL

RASopathy-Associated CBL Germline Mutations Cause Aberrant Ubiquitylation and Trafficking of EGFR www.hgvs.org

Kristina Brand, Hendrik Kentsch, Christina Glashoff, and Georg Rosenberger∗ Institute of Human Genetics, University Medical Center Hamburg-Eppendorf, Hamburg, Germany

Communicated by Andrew O.M. Wilkie Received 17 December 2013; accepted revised manuscript 21 August 2014. Published online 1 September 2014 in Wiley Online Library (www.wiley.com/humanmutation). DOI: 10.1002/humu.22682

ABSTRACT: Noonan syndrome, a congenital disorder comprising a characteristic face, short stature, heart defects, learning difficulties, and a predisposition to malignancies, is caused by heterozygous germline mutations in genes encoding components of RAS-MAPK signaling pathways. Mutations in the CBL tumor suppressor gene have been reported in patients with a Noonan syndromelike phenotype. CBL encodes a multivalent adaptor protein with ubiquitin ligase activity, which promotes ubiquitylation and vesicle-mediated internalization and degradation of the epidermal growth factor (EGF) receptor (EGFR). We investigated the functional consequences of diseaseassociated CBL amino acid changes p.K382E, p.D390Y, and p.R420Q on ligand-induced EGFR trafficking. Expression of CBLK382E , CBLD390Y , or CBLR420Q in COS-7 cells resulted in increased levels of surface EGFR and reduced amounts of intracellular EGFR; both consequences indicate ineffective EGFR internalization. Accordingly, receptor-mediated uptake of EGF was decreased. Furthermore, the p.K382E, p.D390Y, and p.R420Q lesions impaired CBL-mediated EGFR ubiquitylation and degradation. Together, these data indicate that pathogenic CBL mutations severely affect vesicle-based EGFR trafficking. Since we detected enhanced ERK phosphorylation in cells expressing mutant CBL, we conclude that aberrant EGFR trafficking contributes to augmented RAS-MAPK signaling, the common trait of Noonan syndrome and related RASopathies. Thus, our data suggest that EGFR trafficking is a novel disease-relevant regulatory level in the RASopathy network. C 2014 Wiley Periodicals, Inc. Hum Mutat 35:1372–1381, 2014. 

KEY WORDS: Noonan syndrome; RASopathies; CBL; c-Cbl; EGFR trafficking

Introduction Noonan syndrome is an autosomal dominant congenital disorder that encompasses effects on multiple organ systems, including distinctive facial features, heart defects, a short stature, learning difficulties, a predisposition to malignancies, and other clinical manifestations [Roberts et al., 2013]. It belongs to a group of genetic syndromes, the RASopathies or RAS pathway disorders, which are ∗

Correspondence to: Georg Rosenberger, Institute of Human Genetics, University

Medical Center Hamburg-Eppendorf, Campus Forschung N27, Martinistraße 52, 20246 Hamburg, Germany. E-mail: [email protected]

caused by mutations in genes that encode proteins involved in RAS-MAPK (mitogen-activated protein kinase) signaling pathways [Zenker, 2011]. Germline mutations in PTPN11, SOS1, KRAS, NRAS, RAF1, BRAF, MAP2K1, or RIT1 underlie Noonan syndrome [Aoki et al., 2013; Roberts et al., 2013]. Disease-causing mutations usually enhance signal flow through RAS-MAPK cascades [Schubbert et al., 2007]. Moreover, mutations in two genes, SHOC2 and CBL (MIM# 165360), have been reported in patients with Noonanlike phenotypes and additional untypical features [Cordeddu et al., 2009; Martinelli et al., 2010; Niemeyer et al., 2010; Perez et al., 2010]. Besides, somatic CBL mutations have been found in various myeloproliferative disorders [Caligiuri et al., 2007; Sargin et al., 2007; Loh et al., 2009; Makishima et al., 2009; Muramatsu et al., 2010]. CBL (also known as c-Cbl) is a multifunctional adaptor protein with ubiquitin ligase (E3) activity, thereby catalyzing ubiquitylation of substrate proteins [Schmidt and Dikic, 2005]. CBL function has been implicated in ligand-mediated internalization and postendocytic sorting of various cell-surface proteins such as the epidermal growth factor (EGF) receptor (EGFR) [Piper and Lehner, 2011], which is a major cellular access to the RAS-MAPK signaling highway. In the present model, CBL is recruited to the activated EGFR directly by interaction with the receptor at a phosphotyrosine residue [Levkowitz et al., 1999] and indirectly through the adaptor protein Grb2 [Waterman et al., 2002; Jiang et al., 2003]. E2 ubiquitin-conjugating enzymes are then recruited to the RING domain of CBL to promote receptor ubiquitylation; subsequently, ubiquitin-binding proteins (e.g., Epsin) and other endocytic proteins (e.g., AP-2) attach and mediate EGFR internalization [Sorkin and Goh, 2009]. After trafficking into endosomes, EGFR can either be recycled back to the plasma membrane or transported into lysosomes for degradation, and ubiquitylation of EGFR is a prerequisite for degradative receptor sorting, a process also called receptor downregulation [Sorkin and Goh, 2009]. Cells use EGFR internalization and sorting into a functional degradative pathway in order to attenuate the magnitude and duration of signaling events [Sorkin and Goh, 2009]. Thus, CBL negatively regulates EGFR-RAS-MAPK signal traffic downstream of EGFR by controlling receptor internalization and degradation. For the presented report, our main objectives were to determine the functional consequences of pathogenic CBL mutations affecting evolutionary conserved amino acids on EGFR ubiquitylation and trafficking and to relate these data to RAS-MAPK pathway dysregulation in Noonan syndrome and other RASopathies.

Materials and Methods Plasmids We used a human wild-type CBL construct (RefSeq: NM 005188.3) as a template and CBL-specific PCR primers  C

2014 WILEY PERIODICALS, INC.

to generate a wild-type CBL cDNA amplicon by PCR. Mutant CBL constructs were established by PCR-mediated mutagenesis. Purified PCR amplicons (CBLWT , CBLC381A , CBLK382E , CBLD390Y , and CBLR420Q ) were cloned into pENTR/D-TOPO (Life Technologies, Darmstadt, Germany) according to the protocol provided. Constructs were sequenced for integrity and used for the transfer into GATEWAYTM -compatible destination vector pcDNA3-DEST, following the manufacturer’s instructions. Wild-type CBL construct was a gift from Mirko Schmidt (Goethe University School of Medicine, Frankfurt am Main, Germany). Wild-type pcDNA3EGFR construct (RefSeq: NM 005228.3) was a gift of Dr. Sarah J. Parsons (University of Virginia, Charlottesville, VA). pRK5-HAubiquitin (RefSeq: NM 021009.5) was purchased from Addgene (plasmid 17608).

Cell culture and transfection COS-7 cells were cultured in Dulbecco’s modified Eagle medium (Life Technologies) supplemented with 10% (v/v) fetal bovine serum (FBS; PAA Laboratories, C¨olbe, Germany) and penicillinstreptomycin (100 U/ml and 100 μg/ml, respectively; Life Technologies) and incubated at 37°C in a humidified atmosphere with 5% CO2 . Cells were transfected with pcDNA3-DEST-CBL constructs using LipofectamineTM 2000 Reagent (Life Technologies) or TurboFectTM (Thermo Scientific, Bonn, Germany) according to manufacturer’s protocols.

Immunoblotting After transfection with expression constructs, cells were incubated under serum-starved (0.1% FBS) conditions overnight. Next morning, cells were stimulated with 10 or 20 ng/ml EGF (Sigma–Aldrich, Taufkirchen, Germany) for different times or left untreated. Cells were washed with ice-cold 1× PBS and harvested in modified RIPA (radioimmunoprecipitation assay) buffer (150 mM NaCl, 50 mM Tris–HCl, pH 8.0, 0.5% sodium desoxycholate [w/v], 1% Nonidet P40 [v/v], 0.1% sodium dodecyl sulfate [w/v]) containing protease inhibitor cocktail (Roche, Mannheim, Germany) and, if applicable, phosphatase inhibitor cocktail (Roche) or N-ethylmaleimide (Sigma–Aldrich). Cell debris was removed by centrifugation at 21,000 g for 10 min at 4°C and protein solutions were supplemented with sample buffer. Proteins were separated on SDS-polyacrylamide gels and transferred to PVDF (polyvinylidene difluoride) membranes. Following blocking (20 mM Tris–HCl, pH 7.4; 150 mM NaCl; 0.1% Tween-20; 4% nonfat dry milk) and washing (20 mM Tris–HCl, pH 7.4; 150 mM NaCl; 0.1% Tween-20) membranes were incubated in primary antibody solution (20 mM Tris–HCl, pH 7.4; 150 mM NaCl; 0.1% Tween-20; 5% BSA or 0.5% nonfat dry milk) containing the appropriate antibodies. Next, membranes were washed and incubated with peroxidase-coupled secondary antibody. After final washing, immunoreactive proteins were visualized using the Immobilon Western Chemiluminescent HRP Substrate (Millipore, Schwalbach, Germany).

Antibodies and reagents Following primary antibodies and dilutions were used: rabbit anti-Cbl (C-15, WB 1:1,000; IF 1:200; Santa Cruz Biotechnology, Heidelberg, Germany); rabbit anti-EGFR (clone 1005, WB 1:300; Santa Cruz Biotechnology); rat anti-HA-HRP (WB 1:12,000; Roche); rabbit anti-p44/42 MAP kinase (ERK 1/2, approved pro-

tein symbols MAPK3 and MAPK1) antibody (WB 1:1,000; Cell Signaling Technology, Danvers, MA), rabbit anti-phospho-p44/42 MAP kinase (pERK 1/2) (Thr202/Tyr204) antibody (WB 1:1,000; Cell Signaling Technology), mouse anti-α-tubulin antibody (clone DM 1A; WB 1:7,500; Sigma–Aldrich). As secondary antibodies, horseradish peroxidase-conjugated anti-mouse or anti-rabbit antibodies (1:5,000–1:30,000 dilution; GE Healthcare, Munich, Germany) were used. Further reagents used are as follows: Cycloheximide (10 μg/ml; Sigma–Aldrich), N-ethylmaleimide (10 mM; Sigma–Aldrich), EGF complexed with Alexa Fluor 488 (100 ng/ml; Life Technologies), and unlabelled human EGF (10 ng/ml or 20 ng/ml; Sigma–Aldrich). High EGF concentrations were chosen because only higher concentrations of EGF (>10 ng/ml) induce nonclathrin-mediated endocytosis (NCE) in addition to clathrinmediated endocytosis (CME) [Sigismund et al., 2008; Sigismund et al., 2013]. Notably, EGFR ubiquitylation seems to be indispensable for NCE [Sigismund et al., 2013], but not for CME [Madshus and Stang, 2009; Sorkin and Goh, 2009; Haglund and Dikic, 2012; Sigismund et al., 2012].

Biotinylation-based EGFR cell surface assay COS-7 cells were transiently transfected with CBL expression constructs and incubated under serum-starved (0.1% FBS) culture conditions overnight. Next day, cells were stimulated with starvation medium supplemented with 10 ng/ml EGF for 30 min or left untreated. Subsequently, cells were transferred on ice and washed three times with ice-cold HBSS (Life Technologies). Next, cell surface proteins were biotinylated using 0.5 mg/ml sulfo-NHS-SS biotin (Thermo Scientific Pierce, Bonn, Germany) in HBSS for 15 min at 4°C. Unbound biotin was removed by washing twice with ice-cold HBSS containing 5 mM Tris–HCl (pH 7.4) followed by rinsing with PBS. Subsequent to biotinylation, cells were harvested in ice-cold RIPA buffer containing protease inhibitor cocktail and the cell debris was removed by centrifugation. Aliquots of the total cell lysates were kept for immunoblotting and the remaining supernatants were subjected to precipitation with streptavidin agarose (Sigma–Aldrich) overnight at 4°C. Biotin-labeled surface EGFR fractions coupled to agarose beads were collected by centrifugation for 1 min at 8,000 g at 4°C, and agarose pellets were washed twice with ice-cold RIPA buffer. Precipitates and total cell lysates were separated on SDS-polyacrylamide gels, transferred to PVDF membranes, and subjected to immunodetection.

Biotinylation-based EGFR internalization assay COS-7 cells were transiently transfected with CBL expression constructs and incubated under serum-starved (0.1% FBS) culture conditions overnight. Cells were transferred to ice, rinsed three times with ice-cold HBSS, and cell surface proteins were biotinylated using 0.5 mg/ml biotin (Thermo Scientific Pierce) in HBSS for 15 min at 4°C. Unbound biotin was quenched by washing three times with 5 mM Tris–HCl (pH 7.4) in HBSS. Internalization of biotinylated EGF receptors was induced by stimulation with 10 ng/ml EGF in prewarmed starvation medium (0.1% FBS) for 30 min at 37°C. To stop endocytosis and remove remaining cell surface-bound biotin, cells were transferred on ice and immediately covered and washed with ice-cold glutathionecontaining buffer (50 mM glutathione, 1 mM EDTA, 75 mM NaCl, 10% FBS [v/v], 75 mM NaOH). After washing with ice-cold PBS, cells were lysed in RIPA buffer; as controls and to demonstrate the efficiency of biotin-stripping, unstimulated HUMAN MUTATION, Vol. 35, No. 11, 1372–1381, 2014

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cultures were lysed in RIPA buffer. Biotinylated proteins were precipitated using streptavidin-conjugated agarose beads; intracellular EGFR fractions were collected in the precipitates. After washing precipitates with RIPA buffer, both total cell lysates and precipitates were separated by SDS-PAGE and subjected to immunoblotting.

NaCl, 5 mM EDTA, 50 mM HEPES, pH 7.5) supplemented with 0.05% (w/v) SDS. Precipitates and total cell lysates were separated on SDS-polyacrylamide gels, transferred to PVDF membranes, and subjected to immunodetection.

Scanning densitometry and statistical analysis EGFR degradation assay COS-7 cells were transiently transfected with CBL expression constructs and incubated under serum-starved (0.1% FBS) culture conditions overnight. Next day, cells were incubated with 10 μg/ml cycloheximide in starvation medium for 30 min to block protein synthesis. Then, parallel cultures were stimulated by incubation in starvation medium supplemented with 20 ng/ml EGF for 60, 120, or 240 min at 37°C. As controls, parallel cultures were harvested without EGF stimulation. Cells were rinsed with ice-cold PBS and lysed with ice-cold RIPA buffer (150 mM NaCl, 1% Nonidet P40 [v/v], 0.5% sodium deoxycholate [w/v], 0.1% SDS [w/v], 50 mM Tris, pH 8). Finally, lysates were subjected to SDS-PAGE and Western blot analysis.

Signals on microscopic images and on autoradiographs were quantified by densitometric analysis using the ImageJ software (NIH; http://rsb.info.nih.gov/ij/index.html). Relative EGF488 and EGFR levels were assessed as described in the figure legends. Oneway analyses of variance (ANOVAs) were used to compare means of all conditions (CBL protein variants) for statistical significance. Means were considered significant different at P value