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• W2144092904 abstract "Migration of keratinocytes requires a regulated and dynamic turnover of hemidesmosomes (HDs). We and others have previously identified three serine residues on the integrin β4 cytoplasmic domain that play a critical role in the regulation of HD disassembly. In this study we show that only two of these residues (Ser-1356 and Ser-1364) are phosphorylated in keratinocytes after stimulation with either PMA or EGF. Furthermore, in direct contrast to previous studies performed in vitro, we found that the PMA- and EGF-stimulated phosphorylation of β4 is not mediated by PKC, but by ERK1/2 and its downstream effector kinase p90RSK1/2. EGF-stimulated phosphorylation of β4 increased keratinocyte migration, and reduced the number of stable HDs. Furthermore, mutation of the two serines in β4 to phospho-mimicking aspartic acid decreased its interaction with the cytoskeletal linker protein plectin, as well as the strength of α6β4-mediated adhesion to laminin-332. During mitotic cell rounding, when the overall cell-substrate area is decreased and the number of HDs is reduced, β4 was only phosphorylated on Ser-1356 by a distinct, yet unidentified, kinase. Collectively, these data demonstrate an important role of β4 phosphorylation on residues Ser-1356 and Ser-1364 in the formation and/or stability of HDs. Migration of keratinocytes requires a regulated and dynamic turnover of hemidesmosomes (HDs). We and others have previously identified three serine residues on the integrin β4 cytoplasmic domain that play a critical role in the regulation of HD disassembly. In this study we show that only two of these residues (Ser-1356 and Ser-1364) are phosphorylated in keratinocytes after stimulation with either PMA or EGF. Furthermore, in direct contrast to previous studies performed in vitro, we found that the PMA- and EGF-stimulated phosphorylation of β4 is not mediated by PKC, but by ERK1/2 and its downstream effector kinase p90RSK1/2. EGF-stimulated phosphorylation of β4 increased keratinocyte migration, and reduced the number of stable HDs. Furthermore, mutation of the two serines in β4 to phospho-mimicking aspartic acid decreased its interaction with the cytoskeletal linker protein plectin, as well as the strength of α6β4-mediated adhesion to laminin-332. During mitotic cell rounding, when the overall cell-substrate area is decreased and the number of HDs is reduced, β4 was only phosphorylated on Ser-1356 by a distinct, yet unidentified, kinase. Collectively, these data demonstrate an important role of β4 phosphorylation on residues Ser-1356 and Ser-1364 in the formation and/or stability of HDs. IntroductionHemidesmosomes (HDs) 3The abbreviations used are: HDhemidesmosomeABDactin binding domainCSconnecting segmentFnIIIfibronectin type IIIMSPmacrophage-stimulating proteinp90RSKp90 ribosomal S6 kinase. are specialized junctional complexes that mediate firm adhesion of epithelial cells to the underlying basement membrane. Two types of HDs have been characterized: type I and II (1Uematsu J. Nishizawa Y. Sonnenberg A. Owaribe K. J. Biochem. 1994; 115: 469-476Crossref PubMed Scopus (73) Google Scholar). Type I (classical) HDs are present in squamous and complex epithelia, such as the skin and the bladder. They contain integrin α6β4, plectin, the bullous pemphigoid antigens 180 (BP180) and 230 (BP230), and the tetraspanin CD151 (2Borradori L. Sonnenberg A. J. Invest Dermatol. 1999; 112: 411-418Abstract Full Text Full Text PDF PubMed Scopus (471) Google Scholar). Type II HDs lack BP180 or BP230 and are present in simple epithelia, such as the intestine (3Orian-Rousseau V. Aberdam D. Fontao L. Chevalier L. Meneguzzi G. Kedinger M. Simon-Assmann P. Dev. Dyn. 1996; 206: 12-23Crossref PubMed Scopus (57) Google Scholar). As the integrin α6β4 binds to Ln-332 in the extracellular matrix (ECM) and associates intracellularly with plectin, which in turn interacts with the keratin filament system, a protein complex is formed that protects the cell against mechanical stress. The importance of this linkage for epidermal-dermal cohesion is substantiated by the finding that in both humans and genetically modified mice, mutations in the genes for these proteins that either prevent their expression or function, result in a skin blistering disorder known as epidermolysis bullosa (2Borradori L. Sonnenberg A. J. Invest Dermatol. 1999; 112: 411-418Abstract Full Text Full Text PDF PubMed Scopus (471) Google Scholar, 4Chung H.J. Uitto J. Dermatol. Clin. 2010; 28: 43-54Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar).The primary interaction between plectin and β4 occurs through the first pair of fibronectin type III (FnIII) domains and a small part of the connecting segment (CS) of β4 and the actin binding domain of plectin (plectin-ABD) (5Niessen C.M. Hulsman E.H. Oomen L.C. Kuikman I. Sonnenberg A. J. Cell Sci. 1997; 110: 1705-1716Crossref PubMed Google Scholar, 6Geerts D. Fontao L. Nievers M.G. Schaapveld R.Q. Purkis P.E. Wheeler G.N. Lane E.B. Leigh I.M. Sonnenberg A. J. Cell Biol. 1999; 147: 417-434Crossref PubMed Scopus (152) Google Scholar, 7de Pereda J.M. Lillo M.P. Sonnenberg A. EMBO J. 2009; 28: 1180-1190Crossref PubMed Scopus (72) Google Scholar). Indeed, mice carrying a specific deletion of the C-terminal portion of the β4 cytoplasmic domain, which still contains the plectin-ABD binding site, can still form normal HDs (8Nikolopoulos S.N. Blaikie P. Yoshioka T. Guo W. Puri C. Tacchetti C. Giancotti F.G. Mol. Cell. Biol. 2005; 25: 6090-6102Crossref PubMed Scopus (111) Google Scholar). However, binding of β4 to the plectin-ABD is stabilized by adjacent binding sites in the CS and the C-tail of the β4 subunit that interact with the plakin domain of plectin (9Rezniczek G.A. de Pereda J.M. Reipert S. Wiche G. J. Cell Biol. 1998; 141: 209-225Crossref PubMed Scopus (209) Google Scholar, 10Koster J. van Wilpe S. Kuikman I. Litjens S.H. Sonnenberg A. Mol. Biol. Cell. 2004; 15: 1211-1223Crossref PubMed Scopus (102) Google Scholar). In type I HDs, the interaction of β4 with plectin is further reinforced through additional interactions with BP180 and BP230 (11Litjens S.H. de Pereda J.M. Sonnenberg A. Trends Cell Biol. 2006; 16: 376-383Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar). As a result, type I HDs are believed to be less dynamic and more stable than type II HDs. Whereas type I HDs mediate firm adhesion of the epidermis to the underlying basement membrane, the presence of type II HDs in migrating intestinal epithelial cells suggests that these structures are dynamically regulated. One factor implicated in the regulation of type II HD stability is the epidermal growth factor (EGF) (12Rabinovitz I. Tsomo L. Mercurio A.M. Mol. Cell. Biol. 2004; 24: 4351-4360Crossref PubMed Scopus (83) Google Scholar). EGF is one of many cytokines produced during wound healing, stimulating both keratinocyte proliferation and migration (13Barrientos S. Stojadinovic O. Golinko M.S. Brem H. Tomic-Canic M. Wound Rep. Reg. 2008; 16: 585-601Crossref PubMed Scopus (2323) Google Scholar). Whether EGF also regulates type I HDs has not been investigated.Previous studies have shown that activation of pathways downstream of the EGF receptor (EGFR) or protein kinase C (PKC) result in phosphorylation of three serines (Ser-1356, Ser-1360, and Ser-1364) located within the CS of β4 (12Rabinovitz I. Tsomo L. Mercurio A.M. Mol. Cell. Biol. 2004; 24: 4351-4360Crossref PubMed Scopus (83) Google Scholar, 14Wilhelmsen K. Litjens S.H. Kuikman I. Margadant C. van Rheenen J. Sonnenberg A. Mol. Biol. Cell. 2007; 18: 3512-3522Crossref PubMed Scopus (66) Google Scholar). Substitution of the serines by phospho-mimicking aspartic acid residues destabilized the interaction between β4 and plectin and partially prevented the assembly of HDs (14Wilhelmsen K. Litjens S.H. Kuikman I. Margadant C. van Rheenen J. Sonnenberg A. Mol. Biol. Cell. 2007; 18: 3512-3522Crossref PubMed Scopus (66) Google Scholar). On the contrary, substitution of the serines by phosphorylation-resistant alanines resulted in a more stable association between β4 and plectin. PKC-dependent phosphorylation of the β4 cytoplasmic tail was also observed in keratinocytes stimulated with macrophage stimulating protein (MSP), and was suggested to create a binding site for 14-3-3 proteins (15Santoro M.M. Gaudino G. Marchisio P.C. Dev. Cell. 2003; 5: 257-271Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar).Although it has been suggested that at least two of the aforementioned serines are substrates for PKCα phosphorylation downstream of EGFR, bioinformatic analysis showed that only Ser-1360 is part of a consensus sequence for PKC (pSXK/R). Furthermore, this consensus sequence is not evolutionarily conserved, unlike the three serine residues (16Margadant C. Frijns E. Wilhelmsen K. Sonnenberg A. Curr. Opin. Cell Biol. 2008; 20: 589-596Crossref PubMed Scopus (81) Google Scholar). This raised the question of whether phosphorylation of these residues downstream of EGFR is directly dependent on phosphorylation by PKCα. Therefore, we decided to reinvestigate the phosphorylation of residues downstream of the EGFR and PKC and determine their role in HD regulation in more detail.Our results show that EGFR and PKC activation leads to phosphorylation of the β4 subunit on Ser-1356 and Ser-1364 in keratinocytes. Furthermore, we present evidence that ERK1/2 and p90RSK1/2 phosphorylate β4 at these sites, resulting in a destabilization of the binding of β4 to plectin, a reduction in the number of type I and type II HDs formed and in α6β4-mediated strength of adhesion, while it leads to an increased migration speed. Finally, we demonstrate that β4 is phosphorylated on Ser-1356 during mitosis by an as yet unidentified kinase.DISCUSSIONIn this study, we identified ERK1/2 and p90RSK1/2 as the kinases that phosphorylate the integrin β4 subunit at Ser-1356 and Ser-1364 in response to stimulation of keratinocytes with EGF or PMA. We show that the phosphorylation of Ser-1356 and Ser-1364 leads to a reduced number of type I and II HDs in keratinocytes by hindering the association between β4 and the plectin-ABD. Furthermore, we present data that suggest that phosphorylation of these two serine residues downstream of the EGFR decreased α6β4-Ln332-mediated adhesion strength, whereas it increased cell migration. Finally, β4 phosphorylation at Ser-1356 was shown to take place during mitosis.Rabinovitz et al. (12Rabinovitz I. Tsomo L. Mercurio A.M. Mol. Cell. Biol. 2004; 24: 4351-4360Crossref PubMed Scopus (83) Google Scholar) have previously reported that β4 is phosphorylated on three serine residues, Ser-1356, Ser-1360, and Ser-1364 after EGFR activation and suggest that at least two of these residues are directly phosphorylated by PKCα by in vitro analysis. As we previously mentioned, the only serine of these three residues present in a PKC phosphorylation consensus site is Ser-1360; however, in this study we were unable to obtain evidence that β4 is phosphorylated on Ser-1360 downstream of the EGFR. Furthermore, we provide evidence that the phosphorylation of Ser-1356 and Ser-1364 downstream of the EGFR is PKC-independent. Additionally, we were also unable to detect phosphorylation of Ser-1360 after PMA addition in these studies, contrary to our previously published results obtained from in vivo phosphopeptide mapping experiments (14Wilhelmsen K. Litjens S.H. Kuikman I. Margadant C. van Rheenen J. Sonnenberg A. Mol. Biol. Cell. 2007; 18: 3512-3522Crossref PubMed Scopus (66) Google Scholar). We are unable to definitively explain this discrepancy except that we now believe that the reduced phosphorylation of β4 in the phosphopeptide experiment was due to a low expression level of S1360A mutant subunit. Consistent with this revised conclusion, it is of note that in the original mapping experiments mutation of Ser-1360 resulted in the loss of two phosphopeptides, and not just of one as would have been expected. These considerations, combined with the consistent non-reactivity of our phosphospecific antibodies against Ser-1360 with β4 from EGF- and PMA-stimulated lysates, led us to modify our initial conclusion that Ser-1360 is phosphorylated by PKC. We now conclude that this residue is not phosphorylated in keratinocytes stimulated with either PMA or EGF. Our data, however, do not rule out the possibility that Ser-1360 becomes phosphorylated after treatment with other stimuli (e.g. growth factors and cytokines that are released during wound healing).Several reports document the activation of PKC downstream of EGFR activation. Rabinovitz et al. (12Rabinovitz I. Tsomo L. Mercurio A.M. Mol. Cell. Biol. 2004; 24: 4351-4360Crossref PubMed Scopus (83) Google Scholar) argued that the activation of a PKCα-dependent pathway downstream of the EGFR is responsible for the phosphorylation of the β4 subunit. However, this conclusion was based on in vitro phosphopeptide mapping experiments. We have been unable to verify that any of the three aforementioned serine residues are phosphorylated by PKC in vitro, and in fact, we have even witnessed the nonspecific phosphorylation of extracellular β4 peptides in these assays (data not shown). Moreover, a role of PKC in the phosphorylation of either Ser-1356 or Ser-1364 downstream of the EGFR could be excluded by the fact that in the presence of the PKC inhibitor Gö6983, as well as after depletion of PKC by overnight incubation with PMA, EGF was still able to induce the phosphorylation of β4 on these residues. It is interesting to note that the pan-pPKC (βII Ser-660) antibody we used in our previous studies to monitor PKC activation downstream of the EGFR is directed against the phosphorylated serine residue in the hydrophobic loop (bulky ring motif, FXXFSF) of PKCβII (14Wilhelmsen K. Litjens S.H. Kuikman I. Margadant C. van Rheenen J. Sonnenberg A. Mol. Biol. Cell. 2007; 18: 3512-3522Crossref PubMed Scopus (66) Google Scholar). This epitope also happens to be present in the C-terminal kinase of p90RSK1/2 and therefore, it most likely recognizes activated p90RSK1/2 kinase as well.In line with previous work, we confirmed that PKA may phosphorylate β4 at Ser-1364 in vivo (14Wilhelmsen K. Litjens S.H. Kuikman I. Margadant C. van Rheenen J. Sonnenberg A. Mol. Biol. Cell. 2007; 18: 3512-3522Crossref PubMed Scopus (66) Google Scholar). However, we found no evidence that this kinase is activated downstream of PKC or the EGFR. Therefore, it is not likely to be responsible for the phosphorylation of Ser-1364 in cells that have been stimulated with either PMA or EGF. In fact, our data clearly show that two different kinase classes, ERK1/2 and p90RSK1/2, phosphorylate β4 on Ser-1356 and Ser-1364, respectively. There is precedent in the literature for the phosphorylation of proteins by both ERK1/2 and p90RSK1/2, including that of c-Fos and LKB1 (33Chen R.H. Abate C. Blenis J. Proc. Natl. Acad. Sci. U.S.A. 1993; 90: 10952-10956Crossref PubMed Scopus (257) Google Scholar, 34Zheng B. Jeong J.H. Asara J.M. Yuan Y.Y. Granter S.R. Chin L. Cantley L.C. Mol. Cell. 2009; 33: 237-247Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar). It has been suggested that the cooperation between ERK1/2 and p90RSK1/2 in protein regulation serves to ensure that they are selectively activated by the ERK/RSK signaling pathway and not by another kinase that has a substrate specificity overlapping with that of either ERK1/2 or p90RSK1/2 (35Hauge C. Frödin M. J. Cell Sci. 2006; 119: 30210-33023Crossref Scopus (153) Google Scholar).CDK1 recognizes the same phosphorylation motif as ERK1/2 (36Lindqvist A. Rodríguez-Bravo V. Medema R.H. J. Cell Biol. 2009; 185: 193-202Crossref PubMed Scopus (408) Google Scholar). This kinase is active at the transition of G2 to M during mitosis and therefore might well be the kinase that phosphorylates β4 at Ser-1356 at this stage of the cell cycle. We previously have shown that the phosphorylation of only one serine residue in the CS is not sufficient to prevent the interaction between β4 and plectin, and therefore, for the disassembly of HDs in mitotic cells other phosphorylation events might be necessary (14Wilhelmsen K. Litjens S.H. Kuikman I. Margadant C. van Rheenen J. Sonnenberg A. Mol. Biol. Cell. 2007; 18: 3512-3522Crossref PubMed Scopus (66) Google Scholar). In this respect, it is interesting to mention that Geramin et al. (37Germain E.C. Santos T.M. Rabinovitz I. Mol. Biol. Cell. 2009; 20: 56-67Crossref PubMed Scopus (44) Google Scholar) have recently identified a novel phosphorylation site, Ser-1424, on the β4 subunit that plays a role in the disassembly of HDs. Although this site is constitutively phosphorylated and is modestly intensified after EGF stimulation, we do not believe that it is involved in the EGF- or PMA-mediated regulation of HDs since our previous phosphopeptide mapping experiments show that only the two phosphopeptides containing phospho-S1356 and/or Ser-1364 increase in intensity after PMA- or EGF-stimulation of keratinocytes. However, a role of this site in the disassembly of HDs during mitosis cannot be excluded.In a previous article, we reported that there was no difference in the migration rates of keratinocytes expressing the wild-type β4 subunit and those expressing the S1356A/S1360A/S1364A or S1356D/S1360D/S1364D triple mutants (14Wilhelmsen K. Litjens S.H. Kuikman I. Margadant C. van Rheenen J. Sonnenberg A. Mol. Biol. Cell. 2007; 18: 3512-3522Crossref PubMed Scopus (66) Google Scholar). Although double mutants were used in this report, we believe that the discrepancy in the results can be explained as follows. The surface expression levels of the β4 mutants were not assessed in the first report and we now have evidence suggesting they were not equivalent. Additionally, the new data were obtained using tissue culture plates pre-coated with collagen-1 and the cells were stimulated to migrate with ten times more EGF (i.e. 5 ng/ml versus 50 ng/ml). Therefore, the added effects of these differences may account for the observed differences in migration rates.Despite the fact that PMA induces the phosphorylation of β4 at Ser-1356 and Ser-1364, it did not stimulate migration of PA-JEB/β4 keratinocytes. This suggests that phosphorylation of β4 by itself is not sufficient to induce migration and that other signaling events, e.g. those that are activated downstream of the EGFR but not of PKC, are essential. Alternatively, PKC activation may negatively regulate a signaling pathway that is critical for supporting cell migration. Indeed, we have observed that PMA stimulation of PA-JEB/β4 keratinocytes leads to the inactivation of basal PI-3 kinase signaling in these cells, as evidenced by a diminution of Akt phosphorylation. PI3-kinase signaling supports cell migration by Rac-dependent remodeling of the actin cytoskeleton and focal contacts (38Ridley A.J. Schwartz M.A. Burridge K. Firtel R.A. Ginsberg M.H. Borisy G. Parsons J.T. Horwitz A.R. Science. 2003; 302: 1704-1709Crossref PubMed Scopus (3773) Google Scholar).Before epithelial cells can migrate during wound healing, type I HDs have to be completely disassembled. However, the disassembly of type II HDs is likely to be part of a more dynamic process. These structures are found in rapidly migrating intestinal epithelial cells and their dynamic regulation may allow for rapid adhesion and de-adhesion necessary for an efficient migration. The disassembly of HDs in wounds occurs under the influence of many cytokines and growth factors, including EGF, which are released by different cell types present in the wound milieu (13Barrientos S. Stojadinovic O. Golinko M.S. Brem H. Tomic-Canic M. Wound Rep. Reg. 2008; 16: 585-601Crossref PubMed Scopus (2323) Google Scholar). However, because EGF only partially prevents the formation of HDs, it is anticipated that other cytokines also play a role in the prevention of their formation, and/or the disruption of these structures. These cytokines may induce the phosphorylation of β4 on other residues than those whose phosphorylation is induced by EGF, thereby augmenting the disruptive effects of EGF on the interaction between β4 and the plectin-ABD. For example, they may phosphorylate residues important for the binding of β4 to the plakin domain of plectin. Binding of β4 to this domain of plectin is not sufficient to mediate the recruitment of plectin by β4 into HDs, but it may nevertheless facilitate and stabilize the binding of the plectin-ABD to β4 (9Rezniczek G.A. de Pereda J.M. Reipert S. Wiche G. J. Cell Biol. 1998; 141: 209-225Crossref PubMed Scopus (209) Google Scholar, 10Koster J. van Wilpe S. Kuikman I. Litjens S.H. Sonnenberg A. Mol. Biol. Cell. 2004; 15: 1211-1223Crossref PubMed Scopus (102) Google Scholar). Other phosphorylation events may interfere with the binding of β4 to either BP180 or BP230, and/or the binding between BP180, B230, and plectin, and the intermediate filament system (39Kitajima Y. Aoyama Y. Seishima M. J. Investig. Dermatol. Symp. Proc. 1999; 4: 137-144Abstract Full Text PDF PubMed Scopus (97) Google Scholar, 40Malecz N. Foisner R. Stadler C. Wiche G. J. Biol. Chem. 1996; 271: 8203-8208Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar).Several reports have suggested that β4 is also phosphorylated on tyrosine residues. Although most of these studies involved the use of carcinoma cells overexpressing the EGF receptor, data have also been presented that tyrosine phosphorylation may occur in normal keratinocytes (41Mainiero F. Pepe A. Yeon M. Ren Y. Giancotti F.G. J. Cell Biol. 1996; 134: 241-253Crossref PubMed Scopus (166) Google Scholar). We have not observed tyrosine phosphorylation of β4 in PA-JEB/β4 keratinocytes after EGF stimulation and therefore have excluded a role of such kinases in the regulation of HD disassembly. However, the tyrosine phosphorylation of β4 in carcinoma cells has been well documented and is likely to contribute to a more efficient disruption of HDs in these cells (16Margadant C. Frijns E. Wilhelmsen K. Sonnenberg A. Curr. Opin. Cell Biol. 2008; 20: 589-596Crossref PubMed Scopus (81) Google Scholar, 42Wilhelmsen K. Litjens S.H. Sonnenberg A. Mol. Cell. Biol. 2006; 26: 2877-2886Crossref PubMed Scopus (104) Google Scholar). IntroductionHemidesmosomes (HDs) 3The abbreviations used are: HDhemidesmosomeABDactin binding domainCSconnecting segmentFnIIIfibronectin type IIIMSPmacrophage-stimulating proteinp90RSKp90 ribosomal S6 kinase. are specialized junctional complexes that mediate firm adhesion of epithelial cells to the underlying basement membrane. Two types of HDs have been characterized: type I and II (1Uematsu J. Nishizawa Y. Sonnenberg A. Owaribe K. J. Biochem. 1994; 115: 469-476Crossref PubMed Scopus (73) Google Scholar). Type I (classical) HDs are present in squamous and complex epithelia, such as the skin and the bladder. They contain integrin α6β4, plectin, the bullous pemphigoid antigens 180 (BP180) and 230 (BP230), and the tetraspanin CD151 (2Borradori L. Sonnenberg A. J. Invest Dermatol. 1999; 112: 411-418Abstract Full Text Full Text PDF PubMed Scopus (471) Google Scholar). Type II HDs lack BP180 or BP230 and are present in simple epithelia, such as the intestine (3Orian-Rousseau V. Aberdam D. Fontao L. Chevalier L. Meneguzzi G. Kedinger M. Simon-Assmann P. Dev. Dyn. 1996; 206: 12-23Crossref PubMed Scopus (57) Google Scholar). As the integrin α6β4 binds to Ln-332 in the extracellular matrix (ECM) and associates intracellularly with plectin, which in turn interacts with the keratin filament system, a protein complex is formed that protects the cell against mechanical stress. The importance of this linkage for epidermal-dermal cohesion is substantiated by the finding that in both humans and genetically modified mice, mutations in the genes for these proteins that either prevent their expression or function, result in a skin blistering disorder known as epidermolysis bullosa (2Borradori L. Sonnenberg A. J. Invest Dermatol. 1999; 112: 411-418Abstract Full Text Full Text PDF PubMed Scopus (471) Google Scholar, 4Chung H.J. Uitto J. Dermatol. Clin. 2010; 28: 43-54Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar).The primary interaction between plectin and β4 occurs through the first pair of fibronectin type III (FnIII) domains and a small part of the connecting segment (CS) of β4 and the actin binding domain of plectin (plectin-ABD) (5Niessen C.M. Hulsman E.H. Oomen L.C. Kuikman I. Sonnenberg A. J. Cell Sci. 1997; 110: 1705-1716Crossref PubMed Google Scholar, 6Geerts D. Fontao L. Nievers M.G. Schaapveld R.Q. Purkis P.E. Wheeler G.N. Lane E.B. Leigh I.M. Sonnenberg A. J. Cell Biol. 1999; 147: 417-434Crossref PubMed Scopus (152) Google Scholar, 7de Pereda J.M. Lillo M.P. Sonnenberg A. EMBO J. 2009; 28: 1180-1190Crossref PubMed Scopus (72) Google Scholar). Indeed, mice carrying a specific deletion of the C-terminal portion of the β4 cytoplasmic domain, which still contains the plectin-ABD binding site, can still form normal HDs (8Nikolopoulos S.N. Blaikie P. Yoshioka T. Guo W. Puri C. Tacchetti C. Giancotti F.G. Mol. Cell. Biol. 2005; 25: 6090-6102Crossref PubMed Scopus (111) Google Scholar). However, binding of β4 to the plectin-ABD is stabilized by adjacent binding sites in the CS and the C-tail of the β4 subunit that interact with the plakin domain of plectin (9Rezniczek G.A. de Pereda J.M. Reipert S. Wiche G. J. Cell Biol. 1998; 141: 209-225Crossref PubMed Scopus (209) Google Scholar, 10Koster J. van Wilpe S. Kuikman I. Litjens S.H. Sonnenberg A. Mol. Biol. Cell. 2004; 15: 1211-1223Crossref PubMed Scopus (102) Google Scholar). In type I HDs, the interaction of β4 with plectin is further reinforced through additional interactions with BP180 and BP230 (11Litjens S.H. de Pereda J.M. Sonnenberg A. Trends Cell Biol. 2006; 16: 376-383Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar). As a result, type I HDs are believed to be less dynamic and more stable than type II HDs. Whereas type I HDs mediate firm adhesion of the epidermis to the underlying basement membrane, the presence of type II HDs in migrating intestinal epithelial cells suggests that these structures are dynamically regulated. One factor implicated in the regulation of type II HD stability is the epidermal growth factor (EGF) (12Rabinovitz I. Tsomo L. Mercurio A.M. Mol. Cell. Biol. 2004; 24: 4351-4360Crossref PubMed Scopus (83) Google Scholar). EGF is one of many cytokines produced during wound healing, stimulating both keratinocyte proliferation and migration (13Barrientos S. Stojadinovic O. Golinko M.S. Brem H. Tomic-Canic M. Wound Rep. Reg. 2008; 16: 585-601Crossref PubMed Scopus (2323) Google Scholar). Whether EGF also regulates type I HDs has not been investigated.Previous studies have shown that activation of pathways downstream of the EGF receptor (EGFR) or protein kinase C (PKC) result in phosphorylation of three serines (Ser-1356, Ser-1360, and Ser-1364) located within the CS of β4 (12Rabinovitz I. Tsomo L. Mercurio A.M. Mol. Cell. Biol. 2004; 24: 4351-4360Crossref PubMed Scopus (83) Google Scholar, 14Wilhelmsen K. Litjens S.H. Kuikman I. Margadant C. van Rheenen J. Sonnenberg A. Mol. Biol. Cell. 2007; 18: 3512-3522Crossref PubMed Scopus (66) Google Scholar). Substitution of the serines by phospho-mimicking aspartic acid residues destabilized the interaction between β4 and plectin and partially prevented the assembly of HDs (14Wilhelmsen K. Litjens S.H. Kuikman I. Margadant C. van Rheenen J. Sonnenberg A. Mol. Biol. Cell. 2007; 18: 3512-3522Crossref PubMed Scopus (66) Google Scholar). On the contrary, substitution of the serines by phosphorylation-resistant alanines resulted in a more stable association between β4 and plectin. PKC-dependent phosphorylation of the β4 cytoplasmic tail was also observed in keratinocytes stimulated with macrophage stimulating protein (MSP), and was suggested to create a binding site for 14-3-3 proteins (15Santoro M.M. Gaudino G. Marchisio P.C. Dev. Cell. 2003; 5: 257-271Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar).Although it has been suggested that at least two of the aforementioned serines are substrates for PKCα phosphorylation downstream of EGFR, bioinformatic analysis showed that only Ser-1360 is part of a consensus sequence for PKC (pSXK/R). Furthermore, this consensus sequence is not evolutionarily conserved, unlike the three serine residues (16Margadant C. Frijns E. Wilhelmsen K. Sonnenberg A. Curr. Opin. Cell Biol. 2008; 20: 589-596Crossref PubMed Scopus (81) Google Scholar). This raised the question of whether phosphorylation of these residues downstream of EGFR is directly dependent on phosphorylation by PKCα. Therefore, we decided to reinvestigate the phosphorylation of residues downstream of the EGFR and PKC and determine their role in HD regulation in more detail.Our results show that EGFR and PKC activation leads to phosphorylation of the β4 subunit on Ser-1356 and Ser-1364 in keratinocytes. Furthermore, we present evidence that ERK1/2 and p90RSK1/2 phosphorylate β4 at these sites, resulting in a destabilization of the binding of β4 to plectin, a reduction in the number of type I and type II HDs formed and in α6β4-mediated strength of adhesion, while it leads to an increased migration speed. Finally, we demonstrate that β4 is phosphorylated on Ser-1356 during mitosis by an as yet unidentified kinase." @default.
• W2144092904 created "2016-06-24" @default.
• W2144092904 creator A5011731019 @default.
• W2144092904 creator A5016503881 @default.
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• W2144092904 creator A5057166290 @default.
• W2144092904 date "2010-11-01" @default.
• W2144092904 modified "2023-10-15" @default.
• W2144092904 title "EGF-induced MAPK Signaling Inhibits Hemidesmosome Formation through Phosphorylation of the Integrin β4" @default.
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