The role of the calmodulin-binding and calmodulin-like domains of the epidermal growth factor receptor in tyrosine kinase activation

Depósito de investigación de la Universidad de Sevilla https://idus.us.es/ “This is the peer reviewed version of the following article: Abdelli F, Jellali K, Anguita E, et al. The role of the calmodulin-binding and calmodulin-like domains of the epidermal growth factor receptor in tyrosine kinase activation. J Cell Physiol. 2021; 236: 4997–5011., which has been published in final form at https://doi.org/10.1002/jcp.30205 . This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self- Archived Versions. This article may not be enhanced, enriched or otherwise transformed into a derivative work, without express permission from Wiley or by statutory rights under applicable legislation. Copyright notices must not be removed, obscured or modified. The article must be linked to Wiley’s version of record on Wiley Online Library and any embedding, framing or otherwise making available the article or pages thereof by third parties from platforms, services and websites other than Wiley Online Library must be prohibited." The role of the calmodulin-binding and calmodulin-like domains of the epidermal growth factor receptor in tyrosine kinase activation Faten Abdelli1,2,§, Karim Jellali1,2,§, Estefanía Anguita2,3, María González-Muñoz2, Eduardo Villalobo4, Ivan Madroñal2, Juan Alcalde2,‡, Mamdouh Ben Ali1, Jihene Elloumi-Mseddi1,2, Ikram Jemel1,2, Francesc Tebar5, Carlos Enrich5, Sami Aifa1* and Antonio Villalobo2,6* 1Centre of Biotechnology of Sfax, P.O. Box 1177, 3018 Sfax, Tunisia. 2Instituto de Investigaciones Biomédicas, Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid, c/ Arturo Duperier 4, E-28029 Madrid, Spain. 3Molecular Biology and Biochemistry Research Center, CIBBIM-Nanomedicine, Vall d'Hebron Hospital Research Institute, Passeig Vall d'Hebron 119-129, Barcelona 08035, Spain. 4Facultad de Biología, Departamento de Microbiología, Universidad de Sevilla, Ave. Reina Mercedes 6, E-41012 Sevilla, Spain. 5Departament de Biomedicina, Unitat de Biologia Cel·lular, Facultat de Medicina i Ciències de la Salut, Centre de Recerca Biomèdica CELLEX, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Universitat de Barcelona, Casanova, 143, E-08036 Barcelona, Spain. 6Cancer and Human Molecular Genetics Area–Oto-Neurosurgery Research Group,University Hospital La Paz Research Institute (IdiPAZ), Paseo de la Castellana 261, E-28046, Madrid, Spain §These authors equally contributed to this work ‡Present address: Oncologica UK, Little Chesterford, Cambridge CB10 1XL, United Kingdom *Correspondence to: Antonio Villalobo (E-mail: [email protected]) and Sami Aifa (E-mail: [email protected]) Running Title: Calmodulin-binding and calmodulin-like domains in EGFR Abbreviations CaM, calmodulin; CaM-BD, CaM-binding domain; CaM-LD, CaM-like domain; c-Cbl, cellular Casitas B-lineage lymphoma protein; DMEM, Dulbecco’s modified Eagle’s medium; ECL, enhanced chemiluminescence; EDTA, ethylenediaminetetraacetic acid; EGF, epidermal growth factor; EGFR, EGF receptor; EGTA, ethylene glycol-bis(2- aminoethylether)-N,N,N′,N′-tetraacetic acid; Eps15, epidermal growth factor pathway substrate 15; ErbB1/2/3/4, avian erythroblastic leukemia viral oncogene B homologues 1-4; FBS, fetal bovine serum; GPDH, glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein; GPCR, G protein-coupled receptor; HB-EGF, heparinbinding EGF-like growth factor; HER1/2/3/4, human epidermal growth factor receptors 1-4; HRP, horseradish peroxidase; IgG, immunoglobulin G; InvCaM-LD, invert CaM-like domain; JM, juxtamembrane; MAPK, mitogen-activated protein kinase; MEM, minimum essential medium; NLS, nuclear localization sequence/signal; PAE, porcine aortic endothelial; PBS, phosphate buffer saline; PI3K, phosphatidylinositol 3-kinase; PLA2, phospholipase A2; PLC, phospholipase C; PLD1, phospholipase D1; PTB, phospho-tyrosine binding; PVDF, polyvinylidenedifluoride; RTK, receptor tyrosine kinase; SDS-PAGE, polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate; SH2, Src homology domain 2; STAT, signal transducer and activator of transcription; TMR, tetramethylrhodamine; TGF , transforming/tumor growth factor ; TMR-EGF, tetramethylrhodamine-EGF; Tris, tris(hydroxymethyl)aminomethane. Keywords calmodulin, calmodulin-binding domain, calmodulin-like domain, epidermal growth factor receptor, receptor internalization, tyrosine kinase activity. Acknowledgments We thank Lucía Sánchez and Laura Molero for technical assistance with the confocal microscope and flow cytometer, respectively. Authors contribution FA, KJ, EA, MG-M, EV, IV and JA performed experiments; MBL, JE-M and IJ designed and prepared the structural models; FT and CE designed and constructed the GFP chimera receptor mutant and provided the stable transfected PAE cell lines; SA and AV conceived and supervised the project. All authors analyzed the results, read and approved the paper. AV and SA wrote the paper. Funding This work was supported by grants (to AV and SA) from the i-COOP+ 2014 program of the Consejo Superior de Investigaciones Científicas (COOPA20053) and the Agencia Española de Cooperación Internacional para el Desarrollo (A/5444/06, A/8197/07 and A/019018/08), and grants (to AV) from the Secretaría de Estado de Investigación, Desarrollo e Innovación (SAF2014-52048-R) and the Consejería de Educación, Juventud y Deportes – Comunidad de Madrid (B2017/BMD-36) involving contributions from the European Funds for Regional Development (EFRD) and the Social European Fund (SEF). Conflict of interest The authors declare no conflict of interest. ABSTRACT The epidermal growth factor receptor (EGFR) harbors a calmodulin (CaM)-binding domain (CaM-BD) and a CaM-like domain (CaM-LD) upstream and downstream, respectively, of the tyrosine kinase domain. We demonstrate in this paper that deletion of the positively charged CaM-BD (EGFR/CaM-BD∆) inactivated the tyrosine kinase activity of the receptor. Moreover, deletion of the negatively charged CaM-LD (EGFR/CaM-LD∆), leaving a single negative residue (glutamate), reduced the activity of the receptor. In contrast, substituting the CaM-LD with a histidine/valine-rich peptide (EGFR/InvCaM-LD) caused full inactivation. We also demonstrated using confocal microscopy and flow cytometry that the chimera EGFR-green fluorescent protein (GFP)/CaM-BD∆, the EGFR/CaM-LD∆ and EGFR/InvCaM-LD mutants all bind tetramethylrhodamine-labelled EGF. These EGFR mutants were localized at the plasma membrane as the wild-type receptor does. However, only the EGFR/CaM-LD∆ and EGFR/InvCaM-LD mutants appear to undergo ligand-dependent internalization, while the EGFR-GFP/CaM-BD∆ mutant seems to be deficient in this regard. The obtained results and in silico modelling studies of the asymmetric structure of the EGFR kinase dimer support a role of a CaM-BD/CaM-LD electrostatic interaction in the allosteric activation of the EGFR tyrosine kinase. INTRODUCTION The epidermal growth factor receptor (EGFR/ErbB1/HER1) is overexpressed and/or mutated in many solid tumor cells. This results in hyperactive and/or ligandindependent constitutively active receptors, enhancing in this manner cell proliferation and enhanced cell survival, which give advantage to the dissemination of cancerous cells. The EGFR is composed of an extracellular domain containing the ligand-binding site, a single transmembrane domain, and an intracellular region starting with the cytosolic juxtamembrane (JM) domain, followed by the tyrosine kinase (TK) domain and a C-terminal tail containing tyrosine residues serving as auto(trans)-phosphorylation sites. The EGFR forms homodimers and heterodimers with other ErbB receptor family members (ErbB2/HER2/neu, ErbB3/HER3 or ErbB4/HER4) (Carpenter, 1987; Jorissen et al., 2003; Lemmon & Schlessinger, 2010). The EGFR is activated through ligand binding and dimerization, followed by auto(trans)-phosphorylation and recruitment of proteins harboring SH2 or PTB domains for downstream signaling and crosstalk (Carpenter, 1987; Citri & Yarden, 2006; Jorissen et al., 2003; Lemmon & Schlessinger, 2010). The dimerization of the EGFR occurs after binding of a variety of polypeptide ligands including: EGF, TGF, heregulins, epiregulin, -cellulin or HB-EGF, among others (Alroy & Yarden, 1997; Harris, 2003). EGFR signaling is mediated by activation of the MAPK and the PI3K/Akt pathways, although the receptor also crosstalks with other pathways implicating distinct phospholipases (e.g. PLC, PLA2, and PLD1) (Hack et al., 1991; Han et al., 2002; Haugh et al., 1999; Wahl et al., 1989), STAT (Ruff- Jamison et al., 1995; Zhong et al., 1994) and a variety of GPCRs (Schäfer et al., 2004). EGFR signaling is downregulated by protein tyrosine phosphatases (Forbes et al., 2012; Mattila et al., 2005), or effector proteins that directly interact with the receptor, as for example Mig-6 (Hackel et al., 2001; Reschke et al., 2010; Zhang et al., 2007). Moreover, calmodulin (CaM) binds to the cytosolic JM region of the receptor (Martín- Nieto & Villalobo, 1998) and positively modulates the ligand-dependent activation of the EGFR in living cells (H. Li et al., 2012). Furthermore, phospho-(Tyr)-CaM also plays a relevant activating role in this regard after binding to the same site (Stateva et al., 2015; Villalobo, 2018). We have demonstrated that the EGFR has a CaM-binding domain (CaM-BD) in the cytosolic JM region of the receptor, upstream of its TK domain, containing eight positively charged amino acids (645RRRHIVRKRTLRRLLQ660) (underlined) (Aifa et al., 2002; Martín-Nieto & Villalobo, 1998; McLaughlin et al., 2005). It also contains an acidic region downstream of its TK domain with nine negatively charged residues (979DEEDMDDVVDADEY992) (underlined) (Martín-Nieto et al., 2002). We dubbed this acidic region the CaM-like domain (CaM-LD), in analogy to similar segments in other CaM-binding proteins (Harmon et al., 1994; Harper et al., 1994; Hrabak et al., 1996; Villalobo et al., 2019), because of its resemblance to a CaM segment located at its C-terminal, just adjacent and upstream of the IV Ca2+-binding site. This CaM region contains three acidic amino acid clusters (118DEEVDEMIREADI130) (underlined) (Watterson et al., 1980) which have high homology to the ones in the CaM-LD of the EGFR (979DEEDMDDVVDADEY992) (Martín-Nieto et al., 2002). Ligand-dependent receptor dimerization started to be understood thanks to the crystallization of the extracellular domain of the EGFR (Garrett et al., 2002; Ogiso et al., 2002). The crystal structure of the EGFR tyrosine kinase showed the occurrence of an open activation loop (Stamos et al., 2002). Thereafter, an asymmetric dimerization mechanism was proposed based on the interaction of the N-lobe of the TK domain of one monomer with the corresponding TK domain C-lobe of the partner monomer, resulting in the allosteric activation of the EGFR (Zhang et al., 2006). The JM region was shown to be indispensable for the allosteric activation of the EGFR (Thiel & Carpenter, 2007). This was attributed to a latch that the distal segment of the JM of the monomer denoted the ‘activator’ fixes on the TK domain of the apposed monomer denoted the ‘receiver’ (Jura et al., 2009; Red Brewer et al., 2009). These results show the importance of the JM region as the allosteric activator. This allosteric mechanism leads to EGFR auto(trans)-phosphorylation by which the C-terminal tail of each monomer gains access to the catalytic loop. Nevertheless, what happens during the conformational change responsible for the allosteric activation, bringing the C-terminal tyrosine residues to the catalytic loop, is unknown. Landau et al. (Landau et al., 2004) have suggested a symmetric model based on the interaction of the negatively charged CaM-LD with the positively charged domain in the N-lobe of the tyrosine kinase to allow the auto(trans)-phosphorylation of the EGFR dimer. The possible interaction between the positive residues of the CaM-BD and the negative residues of the CaM-LD to attain an auto-inhibited conformation of the monomeric receptor in the absence of ligand has been suggested (Martín-Nieto et al., 2002). However, Aifa et al. (Aifa et al., 2005, 2006) noticed that the symmetric model could be further supported by the electrostatic interaction between CaM-BD and the CaM-LD to allow the intracellular dimerization of EGFR and the auto(trans)-phosphorylation process (Sánchez-González et al., 2010; Villalobo et al., 2019). The deletion of the CaM-BD abolished CaM interaction with the EGFR and yielded an inactive receptor unable to conduct auto(trans)-phosphorylation (Aifa et al., 2005, 2006; Poppleton et al., 2000; Yamane et al., 1992) and incompetent to get into the nucleus, since this segment was also described as the NLS of the receptor (Hsu & Hung, 2007; Lin et al., 2001). However, no deletion or sequence substitution of the CaM-LD has been conducted so far to study the functionality of this acidic segment on EGFR activation. This work presents results on the functional effect of the deletion of the CaM-BD, and deletion and mutation of the CaM-LD. We propose, based on in silico modelling studies, that the negatively charged CaM-LD participates in the allosteric activation process by interacting with the positively charged CaM-BD. This may stabilize the active dimer, facilitating in this manner the auto(trans)-phosphorylation of the receptor. Material and Methods Reagents MEM alpha, DMEM, FBS, trypsin/EDTA solution, Lipofectamine® 2000, pre-stained molecular mass standards for SDS-PAGE were purchased from Gibco/Invitrogen. TMR-EGF was obtained from Thermo Fisher Scientific. Rabbit monoclonal anti-EGFR (clone E235, isotype IgG) antibody recognizing the C-terminal region of the human EGFR, mouse monoclonal pan anti-phospho-tyrosine (clone 4G10, isotype IgG2b) antibody recognizing all EGFR phospho-Tyr residues, and anti-Eps15 rabbit polyclonal antibody recognizing the human/mouse/rat proteins were purchased from Millipore. Rabbit monoclonal anti-phospho-Akt antibody recognizing phospho-Ser473, mouse monoclonal anti-Akt (pan) antibody (clone 40D4) recognizing the C-terminal of the human/mouse/rat proteins, mouse monoclonal anti-phospho-Erk1/2 antibody recognizing phospho-Thr202/Tyr204, rabbit monoclonal anti-Erk1/2 (p42/p44) antibody recognizing the C-terminal of p44, and rabbit monoclonal anti-GAPDH (clone 14C10, isotype IgG) antibody recognizing the C-terminal of the protein were obtained from Cell Signaling Technology. Mouse monoclonal anti-GFP (clones 7.1 and 13.1, isotype 20 IgG1) antibody was from Roche. Mouse monoclonal anti-phospho-PLCγ1 antibody recognizing phospho-Tyr783 was obtained from Cell Signaling Technology (clone D6M95) or Santa Cruz (clone pY783.27). Mouse monoclonal anti-c-Cbl antibody (clone 17, isotype IgG1) recognizing residues 595-810 was from Biosciences-US. Secondary anti-mouse IgG antibody coupled to HRP was purchased from Santa Cruz Biotechnology, and the anti-rabbit IgG antibody coupled to HRP was from GeneTex, Inc. Fast Green FCF and Crystal Violet were obtained from Sigma-Aldrich. The PVDF membranes were obtained from Pall Corporation, the ECL kit from GE Healthcare- Amersham, and the X-ray films from Konika Minolta or AGFA Healthcare. Site-directed mutagenesis and plasmid constructions Escherichia coli DH5 was used for plasmid construction and maintenance. Mutations were performed using the QuickChange mutagenesis kit (Stratagene). We constructed an EGFR deletion mutant lacking the CaM-LD encoding sequence (EGFR/CaM-LD using as template the human EGFR cDNA. This deletion was performed by a two-steps mutagenesis protocol. First, we created an XhoI site upstream of the CaM-LD sequence of the EGFR cloned in the pcDNA6 vector using the following primers 5’- CCAACTTCTACCGTGCCCTGCTCGAGGAAGAAGACATGGACG-3’ (forward) and 5’- CGTCCATGTCTTCTTCCTCGAGCAGGGCACGGTAGAAGTTGG-3’ (reverse). The XhoI inserted site is underlined. Second, we created within the obtained mutant vector, a second XhoI site downstream of the CaM-LD sequence, using the following primers:5’- CGACGTGGTGGATGCCCTCGAGTACCTCATCCC-3’ (forward) and 5’- GGGATGAGGTACTCGAGGGCATCCACCACGTCG-3’ (reversed). The XhoI inserted site is underlined. The obtained plasmid was cut by XhoI in order to remove the CaM-LD encoding sequence and ligated on itself allowing the production of EGFR/CaM-LD∆. Moreover, we inserted in place of the deleted CaM-LD a histidine/valine-rich peptide (LEGIHHVVHVFFLE) just by re-cloning the deleted XhoI fragment in the inverted direction (EGFR/InvCaM-LD). DNA sequencing using the primer 5’-ATGTCTACATGATCATGGTC-3’ ascertained the correctness of the mutagenesis procedure and related constructions. Dr. Mien-Chie Hung from the University of Texas, MD Anderson Cancer Center (Houston, TX) kindly provided the vector for the EGFR/CaM-BD∆ deletion mutant lacking the 645RRRHIVRKRTLRR657 sequence, as previously described (Hsu & Hung, 2007). The 645RRRHIVRKRTLRRLLQ660 sequence was deleted in the EGFR-GFP construct to obtain the EGFR-GFP/CaM-BD∆ deletion mutant as previously described (Martín- Nieto & Villalobo, 1998). Cell culture and transfection Chinese hamster ovary (CHO) cells and human epidermoid carcinoma A431 cells were grown in MEM alpha or DMEM, and porcine aortic endothelial (PAE) cells in RPMI 1640 medium, at 37 °C in a humidified atmosphere containing 5% CO2. All media were supplemented with 10% (v/v) FBS, 2 mM L-glutamine and 40 μg/ml gentamicin. One day before transfection 4-6 x 106 CHO cells were seeded in p100 dishes with complete medium to give the cells the chance to attach and grow. At ~80% confluence, cells were washed with PBS (pH 7.4) and transfected using the Lipofectamine® 2000 transfection kit in medium without FBS or antibiotic, as described by the manufacturer, using the following plasmids: pcDNA6A (empty vector), pcDNA6A-EGFR wild-type, pcDNA6A-EGFR/CaM-BD, pcDNA6A-EGFR/CaM-LD, pcDNA6A- EGFR/InvCaM-LD, pEGFR-GFP and pEGFR-GFP/CaM-BD∆. After 6 hours, cells were trypsinized and seeded in p6 multi-well dishes in complete medium. After attachment cells were incubated overnight in FBS-free medium to perform the EGF activation experiments. EGFR activation experiments Twenty-four hours post-transfection, serum-deprived CHO cells were incubated with 10 nM EGF at different time periods (2-30 min) as indicated in the figures. Control A431 cells, that overexpress the EGFR, were incubated with 10 nM EGF for 2 min. The reaction was arrested removing the medium and adding 1 ml of ice-cold 10% (w/v) trichloroacetic acid. Cells were collected and centrifuged for 10 min at 16,000 g in a bench-top centrifuge. The pellets were homogenized in 90 µl of Laemmli buffer and 10 µl 1.5 M Tris-HCl (pH 8.8) to neutralize the acid. After heating for 5 min at 100 °C, the samples were processed for SDS-PAGE. Electrophoresis and Western blot Proteins were separated by SDS-PAGE in 5-20% (w/v) polyacrylamide linear gradient gels containing 0.1% (w/v) SDS (pH 8.3) at 6 mA during ~17 h. Proteins were electrotransferred from the gels to PVDF membranes for 2 h at 300 mA in a solution containing 48 mM Tris-base, 36.6 mM L-glycine, 0.04% (w/v) SDS and 20% (v/v) methanol; membranes were fixed with 0.2% (v/v) glutaraldehyde in 25 mM Tris-HCl (pH 8), 150 mM NaCl and 2.7 mM KCl (TNK buffer) for 10 min and transiently stained with Fast Green FCF to ascertain the regularity of the transfer procedure. The PVDF membranes were blocked with 5% (w/v) bovine serum albumin or 5% (w/v) fat-free powdered milk, according to the instructions of the antibodies manufacturers in 10 mM Tris-HCl (pH 8), 150 mM NaCl and 0.1% (w/v) Tween-20 (TBS-T medium) and probed overnight at 4 °C using a 1/2000 dilution of the corresponding primary antibody and thereafter for 1 h at room temperature using a 1/5000 dilution of the appropriate secondary anti-IgG antibody conjugated to HRP. The bands were visualized upon development with the ECL reagents following instructions from the manufacturer and exposure of X-ray films for appropriate periods of time. The intensity of the bands was quantified using the ImageJ (version 1.46r) program (National Institutes of Health, USA). Confocal microscopy CHO cells transiently transfected with EGFR wild-type, EGFR/CaM-LD∆ and EGFR/InvCaM- LD mutants were maintained 24 h post-transfection in p60 plates containing several glass coverslips in DMEM supplemented with 10% (v/v) FBS, 2 mM L-glutamine and 40 μg/ml gentamicin for 24 h as above. The plates were washed three times with PBS (pH 7.4) supplemented with 1 mM MgCl2, 1 mM CaCl2, 20 mM glucose and 1% (w/v) bovine serum albumin. Thereafter, 2 µg/ml TMR-EGF was added and incubated at room temperature for the times indicated in the figure legends. The coverslips containing attached cells were washed twice with the supplemented PBS indicated above, and fixed with 4% (v/v) paraformaldehyde in the same medium for 5 min. Coverslips were washed twice with PBS (pH 7.4) and 1/1000 dilution of DAPI in PBS (pH 7.4) was added, incubated for 3 min at room temperature, and washed again in the same medium. Thereafter, samples were mounted using ProLong antifade reagent, dried for 24 h in the dark at 4 ºC and observed in a Zeiss confocal microscope model LSM710, using a 63x/1.4 oil-immersion objective. The 561 nm and 405 nm lasers were used to observe TMR-EGF and DAPI, respectively. No signal was detected in nontransfected cells. Alternatively, CHO cells transiently transfected with EGFR-GFP wildtype and the EGFR-GFP/CaM-BD∆ mutant were seeded in plates with a glass window, grown in DMEM supplemented with 10% (v/v) FBS, 2 mM L-glutamine and 40 μg/ml gentamicin for 24 h as above. Thereafter, 0.66 µg/ml TMR-EGF was added and incubated at room temperature for the times indicated in the figure legends to follow in living cells the location and ligand-dependent internalization of the TMR-EGF/receptor complex, as observed in a LSM710 Zeiss confocal microscope using a 63x/1.4 oilimmersion objective and the 561 nm and 488 nm lasers to observe TMR-EGF and EGFR-GFP, respectively. No signal was detected in non-transfected cells. Flow cytometry CHO cells transiently transfected with EGFR wild-type or the chimera EGFR-GFP wild-type, and the mutants EGFR/CaM-LD∆, EGFR/InvCaM-LD or EGFR-GFP/CaM- BD∆ were maintained 24 h post-transfection in DMEM supplemented with 10% (v/v) FBS, 2 mM L-glutamine and 40 μg/ml gentamicin as above. The cells were washed twice in PBS (pH 7.4) and gently detached with a plastic scraper in the same medium containing 1 mM EGTA to facilitate cell detachment avoiding trypsin treatment. The cells were collected in PBS (pH 7.4) by centrifugation at 600 g for 5 min and finally suspended in the same medium. The cell suspension was incubated with 0.17 µg/ml TMR-EGF for 15 min, and thereafter, passed through a FACSCANTO II flow cytometer (Becton Dickinson) using the 488 nm laser and the 585 nm 525 nm filters to detect TMR-EGF and EGFR-GFP, respectively. EGFR signaling Stably transfected PAE cells expressing wild-type EGFR-GFP and EGFR-GFP/CaM- BD∆mutant were grown at 37 °C and 5% CO2 in DMEM supplemented with 10% (v/v) FBS until confluence in 6-well plates. Cells were washed with PBS (pH 7.4) and incubated with serum-free medium for 3 h. Thereafter, cells were incubated in the absence or presence of 10 nM EGF for 2, 5, 10, 15 and 30 min. The reaction was arrested upon addition of ice-cold 10% (w/v) trichloroacetic acid. The cells were recollected and incubated on ice for 10 min. The tubes were centrifuged at 16,000 g and 4 °C for 10 minutes and supernatants were discarded. After addition of Laemmli buffer and boiling for 5 minutes, the samples were processed by SDS-PAGE and Western Autoinhibitor in CDPK, a Protein Kinase with a Calmodulin-like Domain. Biochemistry, 33(23), 7267–7277. https://doi.org/10.1021/bi00189a031 Harris, R. (2003). EGF receptor ligands. 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Deletion of the CaM-BD and CaM-LD impair EGFR activation in different extent. CHO cells were transiently transfected with wild-type (wt) EGFR, the EGFR/CaM-BDΔ (BDΔ) or the EGFR/CaM-LDΔ (LDΔ) mutants, and 24 hours posttransfection were incubated in the absence or presence of 10 nM EGF for 2-30 min. Negative control using non-transfected (NT) cells and a positive control using A431 cells stimulated with 10 nM EGF for 2 min are also shown (inset). The samples were subjected to SDS-PAGE and Western blot using an anti-EGFR antibody recognizing the C-terminal region, and a pan anti-phospho-Tyr antibody to detect all phosphorylated Tyr residues in the EGFR (P-EGFR). An anti-GAPDH antibody and a blot segment of proteins stained with Fast Green were used as loading controls. The numbers represents the fold increment phosphorylation induced by EGF measuring the densitometric P- EGFR/EGFR ratio. N/S, not significant phosphorylation. Similar results were obtained in three distinct experiments. Fig. 2. Substitution of the CaM-LD with a histidine/valine-rich peptide inactivates the EGFR. CHO cells were transiently transfected with wild-type (wt) EGFR, the EGFR/CaM-BDΔ (BDΔ), EGFR/CaM-LDΔ (LDΔ) or the EGFR/InvCaM-LD (InvLD) mutants, and were incubated 24 hours post-transfection in the absence (-) or presence (+) of 10 nM EGF for 10 min. A negative control using non-transfected (NT) cells and a control using A431 cells stimulated with 10 nM EGF for 2 min are also shown. The samples were subjected to SDS-PAGE and Western blot using an anti-EGFR antibody the C-terminal region, and a pan anti-phospho-Tyr antibody to detect all Tyr residues in the EGFR (P-EGFR). An anti-GAPDH antibody and a membrane blot segment of proteins stained with Fast Green were used as loading controls. The numbers represents the fold increment phosphorylation induced by EGF measuring the densitometric P- EGFR/EGFR ratio. N/S, not significant phosphorylation. Similar results were obtained in two distinct experiments. Fig. 3. Location and ligand binding capacity of an EGFR-GFP mutant lacking the CaM-BD. (A) CHO cells were transiently transfected with wild-type (wt) EGFR-GFP or the EGFR-GFP/CaM-BDΔ mutant, and incubated 24 hours post-transfection in the presence of TMR-EGF, fixed and observed in a confocal microscope as described in Material and Methods. The images show the EGFR-GFP (green) and TMR-EGF (red) signals, and a merge of both signals to illustrate co-localization. A control with cells non-transfected with EGFR-GFP treated with TMR-EGF and counterstained with DAPI is shown. Similar results were obtained in three distinct experiments. (B) CHO cells were transiently transfected with wild-type (wt) EGFR-GFP or the EGFR-GFP/CaM- BDΔ mutant, and subjected 24 hours post-transfection to flow cytometry after incubation in the absence and presence of TMR-EGF as described in Material and Methods. The dot plots show the occurrence of events corresponding to double-labelled (GFP plus TMR) cells in quadrant Q2. (C) The plot presents the mean ± range of the percentage of double-labelled (GFP plus TMR) cells in two independent experiments as those shown in B. (D) The plot presents the number of double-labelled (GFP plus TMR) cells versus the intensity of the signals from an experiment as the one shown in B corresponding to the wild-type EGFR-GFP (wt, blue) and EGFR-GFP/CaM-BD∆ mutant (BD∆, red). Fig.4. The EGFR-GFP/CaM-BD∆ mutant has impaired ligand-dependent capacity. CHO cells were transiently transfected with wild-type (wt) EGFR-GFP (A) or the EGFR-GFP/CaM-BDΔ mutant (B), and 24 hours post-transfection the living cells were observed in a confocal microscope at 10 min or 60 min after addition of TMR-EGF as described in Material and Methods. The images show the EGFR-GFP (green) and TMR-EGF (red) signals, and a merge of both signals to illustrate co-localization. The arrows point to the internalized TMR-EGF:EGFR-GFP complex in vesicles. Similar results were obtained in two distinct experiments. Fig. 5. Location and ligand binding capacity of the EGFR/CaM-LD∆ and EGFR/InvCaM-LD mutants. (A) CHO cells were transiently transfected with wildtype (wt) EGFR, the EGFR/CaM-LDΔ or EGFR/InvCaM-LD mutants, and 24 hours post-transfection were incubated in the presence TMR-EGF and observed in a confocal microscope as described in Material and Methods. The images show the TMR-EGF (red) signal. Similar results were obtained in two distinct experiments. (B) CHO cells were transiently transfected with EGFR wt, the EGFR/CaM-LDΔ or EGFR/InvCaM-LD mutants, and 24 hours post-transfection were subjected to flow cytometry after incubation in the absence and presence of TMR-EGF as described in Material and Methods. A control with non-transfected (NT) cells was also included. The dot plots show the occurrence of events corresponding to cells labelled with TMR in quadrant Q1. (C) The plot presents mean ± range of the percentage of cells labelled with TMREGF in two independent experiments as those shown in B. (D) The plot presents the number of cells labelled with TMR-EGF versus the intensity of the signal from an experiment as the one shown in B corresponding to wild-type EGFR (wt, blue), the EGFR/CaM-LD∆ (LD∆, green) and the EGFR/InvCaM-BD (Inv, red) mutants. The grey area corresponds to cell debris non-specifically labelled with TMR. Fig. 6. The EGFR/CaM-LD∆ and EGFR/InvCaM-LD mutants preserve liganddependent internalization capacity. CHO cells were transiently transfected with wildtype (wt) EGFR, the EGFR/CaM-LDΔ or EGFR/InvCaM-LD mutants, and 24 hours post-transfection the cells were incubated in the presence TMR-EGF for 2 min or 60 min as indicated, fixed and observed in a confocal microscope as described in Material and Methods. The images show the TMR-EGF (red) signal. The arrows point to internalized TMR-EGF in endosome vesicles. Similar results were obtained in two distinct experiments. Fig. 7. Functional effect of CaM-BD deletion in EGFR signaling. (A) Serum-starved stably transfected PAE cells expressing wild-type EGFR-GFP and the EGFRGFP/CaM-BD∆ mutant were activated for the indicated time with 10 nM EGF. Samples were subjected to SDS-PAGE and Western blot using an anti-EGFR antibody (recognizing the C-terminal region) and an anti-GFP antibody to detect the transfected chimera receptor, a pan anti-phospho-Tyr antibody to detect all phosphorylated Tyr residues in the EGFR (P-EGFR), anti-phospho-PLCγ1 recognizing phospho-Tyr783, anti-phospho-Akt recognizing phospho-Ser473, anti-Akt (total), anti-phospho-ERK1/2 recognizing phospho-Thr202/Tyr204 and anti-ERK1/2 (total) antibodies. (B) Serumstarved stably transfected PAE cells expressing wild-type EGFR-GFP and the EGFRGFP/CaM-BD∆ mutant were activated with 10 nM EGF for 5 min. The receptor was immunoprecipitated (IP) and subjected to Western blot using an anti-EGFR antibody (recognizing the C-terminal region) and a pan anti-phospho-Tyr antibody to detect all phosphorylated Tyr residues in the EGFR (P-EGFR), an anti-c-Cbl (recognizing residues 595-810) and an anti-Eps15 antibody. (C) Total cell extract (TCE) and mock immunoprecipitation without anti-EGFR antibodies were processed as input and negative controls, respectively. The numbers represents the percent phosphorylation induced by EGF measuring the densitometric signal of either the P-EGFR-GFP/EGFR and/or P-EGFR-GFP/GFP, the PLC/Fast Green, the P-Akt/Akt and the P- ERK1/2:ERK1/2 ratios, respectively. Similar results were obtained in four different experiments. Fig. 8. Model of the EGFR transmembrane-juxtamembrane segment in active and inactive (relaxed) conformations. (A) Model of the asymmetric structure of the EGFR kinase dimer showing that the CaM-BD (blue surface) and CaM-LD (red surface) are well positioned to establish an electrostatic interaction. (B) A top view of the model shows the displacement caused by the conformational change starting from JM harboring the CaM-BD and ends at the activation loop. (C) A back view of the model illustrates the conformational state of the EGFR transmembrane-juxtamembrane segment in active and inactive context whereas the rest of the molecule is in perfect superimposition. The active structures 2JIT (blue) and 3GOP (cyan) and the inactive structures 2GS2 (pink) and 2EB3 (yellow) of the EGFR were used to construct the model. The EGFR monomer receiver is colored in green. A dotted grey box delimits the EGFR transmembrane-juxtamembrane segment. The graphical presentation was prepared using the PyMOL software. Fig. 9. Models showing the CaM-BD/CaM-LD electrostatic interaction in wild-type EGFR and the effect of deletion or substitution of the CaM-LD in the mutants. (A) Model of the wild-type EGFR where the positively charged CaM-BD and negatively charged CaM-LD represented as blue and red surfaces, respectively, electrostatically interact leading to an active receptor. (B) Model of the EGFR/CaM-LD∆ mutant where the CaM-LD was deleted leaving a single glutamic acid (red surface), in which this acidic residue may interact with the CaM-BD leading to a reduction of the auto(trans)- phosphorylation of the receptor. (C) Model of the EGFR/InvCaM-LD mutant where the CaM-LD was substituted by a histidine/valine-rich peptide in which a steric hindrance and/or electrostatic repulsion lead to the inactivation of the EGFR tyrosine kinase. Hydrophobic residues (white surface), basic residues (blue surface) and acidic residues (red surface) are shown.