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• W2891542466 abstract "Kindler syndrome is an autosomal recessive genodermatosis that results from mutations in the FERMT1 gene encoding t kindlin-1. Kindlin-1 localizes to focal adhesion and is known to contribute to the activation of integrin receptors. Most cases of Kindler syndrome show a reduction or complete absence of kindlin-1 in keratinocytes, resulting in defective integrin activation, cell adhesion, and migration. However, roles for kindlin-1 beyond integrin activation remain poorly defined. In this study we show that skin and keratinocytes from Kindler syndrome patients have significantly reduced expression levels of the EGFR, resulting in defective EGF-dependent signaling and cell migration. Mechanistically, we show that kindlin-1 can associate directly with EGFR in vitro and in keratinocytes in an EGF-dependent, integrin-independent manner and that formation of this complex is required for EGF-dependent migration. We further show that kindlin-1 acts to protect EGFR from lysosomal-mediated degradation. This shows a new role for kindlin-1 that has implications for understanding Kindler syndrome disease pathology. Kindler syndrome is an autosomal recessive genodermatosis that results from mutations in the FERMT1 gene encoding t kindlin-1. Kindlin-1 localizes to focal adhesion and is known to contribute to the activation of integrin receptors. Most cases of Kindler syndrome show a reduction or complete absence of kindlin-1 in keratinocytes, resulting in defective integrin activation, cell adhesion, and migration. However, roles for kindlin-1 beyond integrin activation remain poorly defined. In this study we show that skin and keratinocytes from Kindler syndrome patients have significantly reduced expression levels of the EGFR, resulting in defective EGF-dependent signaling and cell migration. Mechanistically, we show that kindlin-1 can associate directly with EGFR in vitro and in keratinocytes in an EGF-dependent, integrin-independent manner and that formation of this complex is required for EGF-dependent migration. We further show that kindlin-1 acts to protect EGFR from lysosomal-mediated degradation. This shows a new role for kindlin-1 that has implications for understanding Kindler syndrome disease pathology. Kindler syndrome (KS) (OMIM 173650) is a rare autosomal recessive skin disorder for which there is currently no cure. Genome-wide linkage analysis showed that KS is caused by an abnormality in the actin cytoskeleton and its association with the extracellular matrix due to a deficiency or defect in the focal adhesion protein kindlin-1 (also known as fermitin family homologue 1) (Jobard et al., 2003Jobard F. Bouadjar B. Caux F. Hadj-Rabia S. Has C. Matsuda F. et al.Identification of mutations in a new gene encoding a FERM family protein with a pleckstrin homology domain in Kindler syndrome.Hum Mol Genet. 2003; 12: 925-935Crossref PubMed Scopus (197) Google Scholar, Siegel et al., 2003Siegel D.H. Ashton G.H. Penagos H.G. Lee J.V. Feiler H.S. Wilhelmsen K.C. et al.Loss of kindlin-1, a human homolog of the Caenorhabditis elegans actin-extracellular-matrix linker protein UNC-112, causes Kindler syndrome.Am J Hum Genet. 2003; 73: 174-187Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar). Clinical features of KS range from trauma-induced blistering, progressive poikiloderma and skin atrophy, photosensitivity, destructive periodontal disease, severe colitis, and squamous cell carcinoma (Ashton, 2004Ashton G.H. Kindler syndrome.Clin Exp Dermatol. 2004; 29: 116-121Crossref PubMed Scopus (82) Google Scholar, Lai-Cheong et al., 2007Lai-Cheong J.E. Liu L. Sethuraman G. Kumar R. Sharma V.K. Reddy S.R. et al.Five new homozygous mutations in the KIND1 gene in Kindler syndrome.J Invest Dermatol. 2007; 127: 2268-2270Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Since identifying the FERMT1 gene, at least 170 patients and 60 mutations have been reported. These mutations include nonsense, frameshift splice site, and internal deletion changes all resulting in loss of expression (Has et al., 2011Has C. Castiglia D. del Rio M. Diez M.G. Piccinni E. Kiritsi D. et al.Kindler syndrome: extension of FERMT1 mutational spectrum and natural history.Hum Mutat. 2011; 32: 1204-1212Crossref PubMed Scopus (90) Google Scholar, Techanukul et al., 2011Techanukul T. Sethuraman G. Zlotogorski A. Horev L. Macarov M. Trainer A. et al.Novel and recurrent FERMT1 gene mutations in Kindler syndrome.Acta Derm Venereol. 2011; 91: 267-270Crossref PubMed Scopus (24) Google Scholar). The human FERMT1 gene encodes the protein kindlin-1, and other members of this protein family include kindlin-2 and kindlin-3 (Siegel et al., 2003Siegel D.H. Ashton G.H. Penagos H.G. Lee J.V. Feiler H.S. Wilhelmsen K.C. et al.Loss of kindlin-1, a human homolog of the Caenorhabditis elegans actin-extracellular-matrix linker protein UNC-112, causes Kindler syndrome.Am J Hum Genet. 2003; 73: 174-187Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar). Although related, these proteins exhibit differential expression patterns: kindlin-1 expression is predominantly restricted to epithelial cells, kindlin-2 is widely expressed, and kindlin-3 is present in hematopoietic and endothelial cells (Bialkowska et al., 2010Bialkowska K. Ma Y.Q. Bledzka K. Sossey-Alaoui K. Izem L. Zhang X. et al.The integrin co-activator kindlin-3 is expressed and functional in a non-hematopoietic cell, the endothelial cell.J Biol Chem. 2010; 285: 18640-18649Crossref PubMed Scopus (82) Google Scholar, Lai-Cheong et al., 2009Lai-Cheong J.E. Tanaka A. Hawche G. Emanuel P. Maari C. Taskesen M. et al.Kindler syndrome: a focal adhesion genodermatosis.Br J Dermatol. 2009; 160: 233-242Crossref PubMed Scopus (84) Google Scholar, Siegel et al., 2003Siegel D.H. Ashton G.H. Penagos H.G. Lee J.V. Feiler H.S. Wilhelmsen K.C. et al.Loss of kindlin-1, a human homolog of the Caenorhabditis elegans actin-extracellular-matrix linker protein UNC-112, causes Kindler syndrome.Am J Hum Genet. 2003; 73: 174-187Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar, Wiebe et al., 2008Wiebe C.B. Petricca G. Hakkinen L. Jiang G. Wu C. Larjava H.S. Kindler syndrome and periodontal disease: review of the literature and a 12-year follow-up case.J Periodontol. 2008; 79: 961-966Crossref PubMed Scopus (36) Google Scholar). Both kindlin-1 and kindlin-2 localize to focal adhesions, and kindlin-2 is also recruited to cell-cell junctions (Brahme et al., 2013Brahme N.N. Harburger D.S. Kemp-O’Brien K. Stewart R. Raghavan S. Parsons M. et al.Kindlin binds migfilin tandem LIM domains and regulates migfilin focal adhesion localization and recruitment dynamics.J Biol Chem. 2013; 288: 35604-35616Crossref PubMed Scopus (19) Google Scholar, Lai-Cheong et al., 2008Lai-Cheong J.E. Ussar S. Arita K. Hart I.R. McGrath J.A. Colocalization of kindlin-1, kindlin-2, and migfilin at keratinocyte focal adhesion and relevance to the pathophysiology of Kindler syndrome.J Invest Dermatol. 2008; 128: 2156-2165Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar), whereas kindlin-3 localizes to podosomes (Meves et al., 2009Meves A. Stremmel C. Gottschalk K. Fassler R. The kindlin protein family: new members to the club of focal adhesion proteins.Trends Cell Biol. 2009; 19: 504-513Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). All kindlins have a bipartite FERM (i.e., 4.1 protein, ezrin, radixin, moesin) domain consisting of four subdomains (F0, F1, F2, and F3) that are present in many proteins involved in cytoskeletal organization (Baines et al., 2014Baines A.J. Lu H.C. Bennett P.M. The protein 4.1 family: hub proteins in animals for organizing membrane proteins.Biochim Biophys Acta. 2014; 1838: 605-619Crossref PubMed Scopus (92) Google Scholar, Goult et al., 2009Goult B.T. Bouaouina M. Harburger D.S. Bate N. Patel B. Anthis N.J. et al.The structure of the N-terminus of kindlin-1: a domain important for αIIbβ3 integrin activation.J Mol Biol. 2009; 394: 944-956Crossref PubMed Scopus (69) Google Scholar). The kindlin F2 subdomain differs from other FERM domain proteins by an insertion of a pleckstrin homology (i.e., PH) domain that binds phosphoinositide phosphates (Meves et al., 2009Meves A. Stremmel C. Gottschalk K. Fassler R. The kindlin protein family: new members to the club of focal adhesion proteins.Trends Cell Biol. 2009; 19: 504-513Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). Kindlins have all been shown to bind directly to the cytoplasmic domain of β-integrin subunits and contribute to integrin activation (Rognoni et al., 2016Rognoni E. Ruppert R. Fassler R. The kindlin family: functions, signaling properties and implications for human disease.J Cell Sci. 2016; 129: 17-27Crossref PubMed Scopus (144) Google Scholar). In normal skin, kindlin-1 localizes in basal keratinocytes at the dermal-epidermal junction and accumulates at cell-matrix adhesion sites. In isolated keratinocytes, kindlin-1 localizes to the cell leading edge and focal adhesions (Larjava et al., 2008Larjava H. Plow E.F. Wu C. Kindlins: essential regulators of integrin signalling and cell-matrix adhesion.EMBO Rep. 2008; 9: 1203-1208Crossref PubMed Scopus (202) Google Scholar). Depletion of kindlin-1 leads to reduced proliferation, adhesion, and spreading and to reduced directed migration, with the cells displaying multiple leading edges and multipolar shapes (Has et al., 2008Has C. Ludwig R.J. Herz C. Kern J.S. Ussar S. Ochsendorf F.R. et al.C-terminally truncated kindlin-1 leads to abnormal adhesion and migration of keratinocytes.Br J Dermatol. 2008; 159: 1192-1196PubMed Google Scholar, Herz et al., 2006Herz C. Aumailley M. Schulte C. Schlotzer-Schrehardt U. Bruckner-Tuderman L. Has C. Kindlin-1 is a phosphoprotein involved in regulation of polarity, proliferation, and motility of epidermal keratinocytes.J Biol Chem. 2006; 281: 36082-36090Crossref PubMed Scopus (119) Google Scholar, Zhang et al., 2016Zhang G. Gu Y. Begum R. Chen H. Gao X. McGrath J.A. et al.Kindlin-1 regulates keratinocyte electrotaxis.J Invest Dermatol. 2016; 136: 2229-2239Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). The role of kindlin-1 in integrin-mediated processes provides explanation for some of the clinical features observed in patients with KS. Potential non–integrin-related roles for kindlin-1 in controlling cell behavior remain unclear. In this study we performed mass spectrometry analysis of keratinocytes from KS patients and identified significantly reduced levels of the epidermal growth factor receptor (EGFR) in KS samples. Further analysis showed defective downstream signaling of EGFR and attenuated cell responses to EGF stimulation. The expression of kindlin-1 in KS cells was able to restore EGFR expression levels and responses to EGF. Our investigations showed a direct interaction between kindlin-1 and EGFR at the plasma membrane that acts to protect EGFR from lysosomal degradation, independent of kindlin-1 binding to integrins. These data provide new insight into kindlin-1 function in keratinocytes and may provide new avenues for pursuit of therapeutic strategies to treat KS patients. To identify new pathways downstream of kindlin-1, we profiled lysates of keratinocytes from healthy donors (wild type [WT]) and two different KS patients using mass spectrometry. This analysis showed a reduction in protein levels of EGFR in KS keratinocytes, which was verified using Western blotting (Figure 1a). However, no change in mRNA levels of EGFR was detected in KS cells by semiquantitative reverse transcriptase–PCR (Figure 1b). Analysis of normal human lung (16HBE) and breast (MCF10A) epithelial cell lines also showed a reduction of EGFR levels upon small interfering RNA depletion of kindlin-1 (see Supplementary Figure S1a and b online), suggesting a common role for kindlin-1 in regulating EGFR levels in human epithelial cells. Exogenous expression of kindlin-1 in keratinocytes restored EGFR levels (Figure 1c), thereby specifically attributing this phenotype to kindlin-1 expression. Taken together, these findings show a global reduction in EGFR levels when kindlin-1 is absent or depleted. Further analysis by FACS analysis confirmed a reduction in EGFR surface levels in KS keratinocytes (Figure 1d). Moreover, immunostaining of healthy donor and KS patient skin sections showed a striking reduction of EGFR in the basal keratinocytes in KS skin compared with WT skin (Figure 1e). EGFR regulates a number of signaling pathways, which act to regulate keratinocyte survival, growth, adhesion, and migration (Bakker et al., 2017Bakker J. Spits M. Neefjes J. Berlin I. The EGFR odyssey—from activation to destruction in space and time.J Cell Sci. 2017; 130: 4087-4096Crossref PubMed Scopus (86) Google Scholar). To examine the effect of loss of kindlin-1 on EGFR signaling, cells were starved overnight and stimulated with EGF for 10 minutes, and the phosphorylations of EGFR (Figure 1f) and its downstream effector ERK1/2 (Figure 1g) were assessed. As expected, EGFR phosphorylation in response to EGF was significantly reduced in KS keratinocytes, in line with the constitutively lower levels of EGFR in these cells (Figure 1f), with a resulting loss of EGF-dependent ERK1/2 phosphorylation (Figure 1g). To determine whether this loss of EGF responsiveness had an impact on functional cell behavior, we assessed migratory responses to EGF by time lapse microscopy. Data showed that KS cells exhibited higher migration speeds compared with WT cells under starved conditions, as we have shown previously (see Supplementary Figure 1c and d) (Maiuri et al., 2012Maiuri P. Terriac E. Paul-Gilloteaux P. Vignaud T. McNally K. Onuffer J. et al.The first World Cell Race.Curr Biol. 2012; 22: R673-R675Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Addition of EGF led to increased WT keratinocyte migration rates but had no effect on KS cell speed, confirming a failure to respond to EGF in the absence of kindlin-1. Migration speeds were rescued in KS cells re-expressing mCherry-kindlin-1, confirming that the observed phenotypes were due to loss of kindlin-1 expression (see Supplementary Figure 1d and e). Together, these findings show that kindlin-1–deficient human keratinocytes have reduced EGFR levels, resulting in impaired responses to EGF. To determine whether the reduced levels of EGFR in KS cells coincided with altered subcellular distribution, we analyzed the localization of EGFR in sparsely plated WT and KS keratinocytes after EGF stimulation. Total and surface levels of EGFR were quantified by measuring the mean fluorescence intensities of either the whole cell area or plasma membrane. Consistent with the Western blot analyses (Figure 1a and f), EGF stimulation did not alter the relative intensity of EGFR in either cell type, but there was a marked reduction in total EGFR levels in KS cells (Figure 2a–c). In starved WT cells, EGFR was localized at the plasma membrane and cytoplasmic compartments, whereas KS cells showed very weak EGFR staining at the plasma membrane with increased accumulation in perinuclear compartments (Figure 2a–c). After EGF treatment, EGFR redistributed from the plasma membrane into perinuclear compartments in WT cells, coincident with reduced EGFR at the plasma membrane (Figure 2c). In contrast, EGFR remained in the perinuclear compartments of KS cells after EGF stimulation (Figure 2c). Kindlin-2 has been shown previously to be expressed at normal levels in KS patients (Lai-Cheong et al., 2008Lai-Cheong J.E. Ussar S. Arita K. Hart I.R. McGrath J.A. Colocalization of kindlin-1, kindlin-2, and migfilin at keratinocyte focal adhesion and relevance to the pathophysiology of Kindler syndrome.J Invest Dermatol. 2008; 128: 2156-2165Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar), suggesting that it is not disrupted upon loss of kindlin-1 but also cannot functionally replace kindlin-1 in these cells. However, to determine whether loss of kindlin-1 and resulting EGFR mislocalization could be compensated for overexpression of kindlin-2, WT and KS cells were transfected with GFP–kindlin-2, and total and surface EGFR levels were analyzed by confocal microscopy. Data showed that kindlin-2 overexpression had no effect on EGFR levels or localization in either WT or KS keratinocytes (see Supplementary Figure 1e and f), suggesting that kindlin-2 cannot compensate for loss of kindlin-1 in these cells. Indeed, functional, nonredundant roles for kindlin-1 and -2 have also been suggested in the context of integrin binding in keratinocytes (Bandyopadhyay et al., 2012Bandyopadhyay A. Rothschild G. Kim S. Calderwood D.A. Raghavan S. Functional differences between kindlin-1 and kindlin-2 in keratinocytes.J Cell Sci. 2012; 125: 2172-2184Crossref PubMed Scopus (42) Google Scholar), further supporting the notion that these proteins have different roles in epithelial cell function. EGFR is known to undergo endocytosis and, depending on the cell type and EGF concentration, can be recycled back to the plasma membrane or routed for degradation (Bakker et al., 2017Bakker J. Spits M. Neefjes J. Berlin I. The EGFR odyssey—from activation to destruction in space and time.J Cell Sci. 2017; 130: 4087-4096Crossref PubMed Scopus (86) Google Scholar). To determine whether kindlin-1 may play a role in regulating EGFR dynamics at the plasma membrane, we analyzed WT and KS cells stably expressing EGFR-GFP after fluorescence recovery after photobleaching at the plasma membrane under growth conditions. Despite expressing lower levels of EGFR, KS cells showed enhanced early recovery profiles compared with WT and reduced T1/2 speed without changing the mobile fraction (see Supplementary Figure S2a online). We confirmed that this effect of kindlin-1 was not due to global changes in clathrin-mediated endocytosis, because transferrin-Texas Red uptake assays showed no differences between WT and KS cells (see Supplementary Figure S2b), indicating that global receptor internalization was unperturbed by the loss of kindlin-1. Inhibition of dynamin activity, but not recycling (through dynasore and primaquine treatment, respectively), resulted in a slower fluorescence recovery T1/2 and reduced EGFR mobile fraction (see Supplementary Figure 2c and d). These data show that loss of kindlin-1 destabilizes EGFR under steady state conditions and that inhibition of EGFR internalization, but not receptor recycling, reduces EGFR dynamics at the plasma membrane. To determine potential kindlin-1–dependent changes in EGFR subcellular compartmentalization, we used colocalization analysis to study EGFR localization with key endocytic markers at time points after EGFR stimulation: early endosomes (EEA1, 10 minutes), lysosomes (LAMP1, 30 minutes), and recycling endosomes (Rab11a, 1 hour). Pearson correlation analysis showed significantly reduced co-localization between EGFR/EEA1 and EGFR/Rab11 in KS compared with WT cells (Figure 2d and f). In contrast, a significant increase in colocalization between EGFR and LAMP1 was observed in KS cells compared with WT (Figure 2e). To further explore the real-time dynamics of the EGFR-positive compartments after EGF stimulation, we performed live cell imaging on WT and KS cells expressing EGFR-GFP and cherry-Rab11a and labeled with LysoTracker Far Red (Molecular Probes, Eugene, OR), for 30 minutes after EGF stimulation. Upon addition of EGF, EGFR-positive vesicles moved in a retrograde fashion from the plasma membrane into the cell interior, increasing in number and size over time (Figure 2g and h and see Supplementary Movie S1 online). In contrast, EGFR-labeled vesicles in KS cells displayed random movement in the perinuclear region throughout the 30 minutes of observance, with the size and vesicle number remaining largely unaltered (Figure 2g and h, and see Supplementary Movie S2 online). Analysis of overlapping pixels in the EGFR-GFP– and lysotracker-labeled vesicles confirmed the LAMP1 data in fixed cells (Figure 2e), showing a constitutively higher co-localization between EGFR-positive vesicles and lysosomal compartments in KS cells compared with WT cells throughout the period of EGF stimulation (Figure 2i). EGFR is subject to ligand-induced degradation via the lysosomal or proteasomal pathways (Singh and Coffey, 2014Singh B. Coffey R.J. Trafficking of epidermal growth factor receptor ligands in polarized epithelial cells.Annu Rev Physiol. 2014; 76: 275-300Crossref PubMed Scopus (63) Google Scholar). Given the increased EGFR within lysosomal compartments in KS cells, we next analyzed whether EGFR was reduced in KS cells because of enhanced protein degradation. Treatment of WT and KS cells with the proteasome inhibitor MG132 did not change EGFR levels in KS cells (Figure 3a and d). However, treatment with lysosomal inhibitors leupeptin or concanamycin A restored EGFR expression in KS cells up to WT levels (Figure 3b–d), suggesting that loss of kindlin-1 leads to increased lysosomal-dependent EGFR degradation. EGFR binds to the E3 ubiquitin ligase c-Cbl in response to EGF, either at the plasma membrane or on early endosomes, which in turn promotes polyubiquitination of EGFR, resulting in degradation (Duan et al., 2003Duan L. Miura Y. Dimri M. Majumder B. Dodge I.L. Reddi A.L. et al.Cbl-mediated ubiquitinylation is required for lysosomal sorting of epidermal growth factor receptor but is dispensable for endocytosis.J Biol Chem. 2003; 278: 28950-28960Crossref PubMed Scopus (170) Google Scholar). To determine whether kindlin-1–dependent changes to EGFR altered c-Cbl association with the receptor, we analyzed c-Cbl-EGFR binding by co-immunoprecipitation (IP) in WT and KS cells treated with DMSO or concanamycin A under growth conditions. A dramatic increase in c-Cbl binding to EGFR in KS cell lysates was observed, with or without treatment with concanamycin A (Figure 3e), suggesting that increased constitutive c-Cbl binding in the absence of kindlin-1 may result in increased targeting of EGFR for lysosomal degradation. Kindlin-2 has previously been suggested to directly interact with EGFR through an association with the EGFR kinase domain (Guo et al., 2015Guo B. Gao J. Zhan J. Zhang H. Kindlin-2 interacts with and stabilizes EGFR and is required for EGF-induced breast cancer cell migration.Cancer Lett. 2015; 361: 271-281Crossref PubMed Scopus (47) Google Scholar). To determine whether kindlin-1 could interact with EGFR, individual domains of kindlin-1 were generated as GST fusion proteins and used to pull out endogenous EGFR from cell lysates (Figure 4a). Full-length GST–kindlin-1 (GST1) bound to EGFR and a consistently strong binding with the F1 domain of kindlin-1 was also observed (GST3) (Figure 4b). The F1 domain contains an unstructured loop that we postulated could be a potential binding region for EGFR (Bouaouina et al., 2012Bouaouina M. Goult B.T. Huet-Calderwood C. Bate N. Brahme N.N. Barsukov I.L. et al.A conserved lipid-binding loop in the kindlin FERM F1 domain is required for kindlin-mediated αIIbβ3 integrin coactivation.J Biol Chem. 2012; 287: 6979-6990Crossref PubMed Scopus (46) Google Scholar). We tested this hypothesis by expressing a His-tagged F1 loop to capture EGFR from cell lysates. As predicted, the F1 loop bound strongly to EGFR in cell lysates in contrast to the His-kinesin light chain domain that served as a negative control (Figure 4c). To test whether association between kindlin-1 and EGFR was direct, a GST fusion of the EGFR cytoplasmic domain was incubated with His-F1 loop of kindlin-1 in solution. Pulldown of the GST-EGFR cytoplasmic tail showed a strong interaction with the His–kindlin-1 F1 loop (Figure 4d), indicating a direct interaction between the two proteins. Moreover, assessment of binding kinetics between these proteins by microscale thermophoresis (MST) showed a robust interaction between the EGFR cytoplasmic tail membrane proximal region and both full-length and F0F1 domains of kindlin-1 (Figure 4e). Taken together, these data show that kindlin-1 binds directly to the EGFR cytoplasmic domain via the kindlin-1 F1 loop. Moreover, the fact that c-Cbl binding is significantly and constitutively enhanced in cells lacking kindlin-1 (Figure 3e) suggests that binding of kindlin-1 to the EGFR cytoplasmic tail restricts binding of c-Cbl, leading to retention of EGFR at the plasma membrane, enhanced signaling, and reduced degradation. To further define when and where kindlin-1 may associate with EGFR in cells, we analyzed their relative subcellular distributions using live-cell structure illumination microscopy superresolution imaging of KS cells expressing mCherry–kindlin-1 and EGFR-GFP. Images and subsequent analysis showed that co-localization between the two proteins occurred within the first 15 minutes of EGF stimulation at the plasma membrane (Figure 5a, and see Supplementary Figure S3a online). We were also unable to detect any kindlin-1 co-localizing with EGFR within endosomes. IP of endogenous EGFR from KS cells re-expressing mCherry–kindlin-1 also showed that kindlin-1 forms a complex with EGFR in a time-dependent manner, with strongest interactions occurring 5 minutes after EGF stimulation and resuming to basal levels by 60 minutes (Figure 5b). However, we were unable to detect kindlin-2 in these immunoprecipitated complexes (see Supplementary Figure S3b), suggesting that the binding of kindlin-1 may be specific in keratinocytes. Analysis of direct binding between the two proteins using fluorescence lifetime imaging microscopy to analyze fluorescence resonance energy transfer (FRET) also showed a direct interaction between EGFR-GFP and mCherry–kindlin-1 in cells that was increased after 10 minutes of EGF stimulation (Figure 5c). Moreover, kindlin-1–to–EGFR binding was independent of kindlin-1–to–integrin binding, because FRET-fluorescence lifetime imaging microscopy analysis showed strong, constitutive interaction between EGFR-GFP and mCherry–W612Akindlin-1, which is defective in integrin binding (see Supplementary Figure S3c) (Bouaouina et al., 2012Bouaouina M. Goult B.T. Huet-Calderwood C. Bate N. Brahme N.N. Barsukov I.L. et al.A conserved lipid-binding loop in the kindlin FERM F1 domain is required for kindlin-mediated αIIbβ3 integrin coactivation.J Biol Chem. 2012; 287: 6979-6990Crossref PubMed Scopus (46) Google Scholar, Huet-Calderwood et al., 2014Huet-Calderwood C. Brahme N.N. Kumar N. Stiegler A.L. Raghavan S. Boggon T.J. et al.Differences in binding to the ILK complex determines kindlin isoform adhesion localization and integrin activation.J Cell Sci. 2014; 127: 4308-4321Crossref PubMed Scopus (54) Google Scholar). Further analysis of these cells showed that expression of mCherry–W612Akindlin-1 in KS cells was also able to partially restore the migration response to EGF (see Supplementary Figure S3d), further indicating that kindlin-1–to–EGFR binding plays an important role in control of EGF responses and that this can act at least in part independently of kindlin-1–to–integrin complex formation. To explore whether EGFR kinase activity regulates kindlin-1–EGFR binding, we assessed the co-localization between endogenous EGFR and GFP–kindlin-1 expressed in KS cells in the presence of either DMSO or AG1478, an EGFR-specific tyrosine kinase inhibitor. Inhibition of EGFR activity resulted in an increase in co-localization between EGFR and GFP–kindlin-1 (Figure 5d), potentially through enrichment of EGFR at the plasma membrane. Finally, because kindlin-2 has previously been suggested to be tyrosine phosphorylated (Liu et al., 2015Liu Z. Lu D. Wang X. Wan J. Liu C. Zhang H. Kindlin-2 phosphorylation by Src at Y193 enhances Src activity and is involved in migfilin recruitment to the focal adhesions.FEBS Lett. 2015; 589: 2001-2010Crossref PubMed Scopus (21) Google Scholar, Qu et al., 2014Qu H. Tu Y. Guan J.L. Xiao G. Wu C. Kindlin-2 tyrosine phosphorylation and interaction with Src serve as a regulatable switch in the integrin outside-in signaling circuit.J Biol Chem. 2014; 289: 31001-31013Crossref PubMed Scopus (30) Google Scholar), we sought to determine whether the same modification on kindlin-1 could occur through EGFR-mediated signaling. IP analysis showed that GFP–kindlin-1 was tyrosine phosphorylated under basal conditions (Figure 5e). However, treatment with AG1478 had no effect on kindlin-1 tyrosine phosphorylation levels, suggesting that kindlin-1 is constitutively tyrosine phosphorylated in growth conditions and that this does not depend on signals downstream of active EGFR. In summary, our data show a direct interaction between kindlin-1 and EGFR that acts to restrict c-Cbl–EGFR association and thus protect EGFR from lysosomal degradation. Although our data do not allow us to conclusively state that EGFR-Cbl binding in KS cells is constitutive, our data support the notion that the presence of kindlin-1 is required to ensure correct regulation of the EGFR-Cbl complex. Our proposed model is that binding of kindlin-1 to the EGFR cytoplasmic tail can displace Cbl binding and potentially act to stabilize EGFR at the membrane and subsequently control modulation of EGFR routing to the endo-lysosomal system. Loss of kindlin-1 expression in patients with KS results in lower EGFR levels in the skin and isolated keratinocytes, resulting in loss of EGF-induced signaling and migratory behavior. This newly described function for kindlin-1 is very likely to contribute to the clinical features observed in KS patients in agreement with our recent discovery of an EGFR loss-of-function mutation in patients with skin fragility (Campbell et al., 2014Campbell P. Morton P.E. Takeichi T. Salam A. Roberts N. Proudfoot L.E. et al.Epithelial inflammation resulting from an inherited loss-of-function mutation in EGFR.J Invest Dermatol. 2014; 134: 2570-2578Abstract Full" @default.
• W2891542466 created "2018-09-27" @default.
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• W2891542466 date "2019-02-01" @default.
• W2891542466 modified "2023-09-26" @default.
• W2891542466 title "Kindlin-1 Regulates Epidermal Growth Factor Receptor Signaling" @default.
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