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W1966227162 abstract "Pinin is a cell adhesion-associated and nuclear protein that has been shown to localize in the vicinity of intermediate filament (IF) convergence upon the cytoplasmic face of the desmosomal plaque as well as in the nucleus. The localization of pinin to the desmosomes has been correlated with the reinforcement of intercellular adhesion and increased IF organization. In this study, keratins 18, 8, and 19 were identified to interact with the amino end domain of pinin in a two-hybrid screening. Further truncation analyses indicated that the 2B domain of keratin contains the sequence responsible for interacting with pinin. The amino end of pinin (residues 1–98) is sufficient to bind to keratin. Point mutation analyses revealed two essential residues within the pinin fragment 1–98, leucine 8 and leucine 19, for the interaction with keratin. Finally, in vitro protein overlay binding assays confirmed the direct interaction of the amino end domain of pinin with keratins, while pinin mutant L8P GST fusion protein failed to bind to keratins in the overlay assay. Coupled with our previous morphological observations and transfection studies, these data suggest that pinin may play a role in epithelial cell adhesion and the IF complex through a direct interaction with the keratin filaments. Pinin is a cell adhesion-associated and nuclear protein that has been shown to localize in the vicinity of intermediate filament (IF) convergence upon the cytoplasmic face of the desmosomal plaque as well as in the nucleus. The localization of pinin to the desmosomes has been correlated with the reinforcement of intercellular adhesion and increased IF organization. In this study, keratins 18, 8, and 19 were identified to interact with the amino end domain of pinin in a two-hybrid screening. Further truncation analyses indicated that the 2B domain of keratin contains the sequence responsible for interacting with pinin. The amino end of pinin (residues 1–98) is sufficient to bind to keratin. Point mutation analyses revealed two essential residues within the pinin fragment 1–98, leucine 8 and leucine 19, for the interaction with keratin. Finally, in vitro protein overlay binding assays confirmed the direct interaction of the amino end domain of pinin with keratins, while pinin mutant L8P GST fusion protein failed to bind to keratins in the overlay assay. Coupled with our previous morphological observations and transfection studies, these data suggest that pinin may play a role in epithelial cell adhesion and the IF complex through a direct interaction with the keratin filaments. intermediate filament K18, and K19, keratin 8, 18, and 19, respectively polymerase chain reaction activation domain binding domain Pinin was first identified to be a protein associated with the desmosome, which was recruited to the preformed desmosomes of the epithelia but absent at nascent desmosomes (1.Ouyang P. Sugrue S.P. J. Cell Biol. 1992; 118: 1477-1488Crossref PubMed Scopus (53) Google Scholar). Immunofluorescence and immuno-EM studies have shown pinin decorating keratin filaments near the cytoplasmic face of the desmosomal plaque in the vicinity of keratin filament convergence upon the desmosome. Our previous studies have revealed a correlation between the placement of pinin at the desmosome and an increase in the organization/stabilization of desmosome-IF1 complex (1.Ouyang P. Sugrue S.P. J. Cell Biol. 1992; 118: 1477-1488Crossref PubMed Scopus (53) Google Scholar, 2.Ouyang P. Sugrue S.P. J. Cell Biol. 1996; 135: 1027-1042Crossref PubMed Scopus (82) Google Scholar). Presumably, one of the functions of pinin is related to the desmosome-IF complex.The expression level of pinin has been correlated with the overall epithelial phenotype. HEK-293 cells, when transfected with pinin full-length cDNA, exhibited a striking phenotype change from a fibroblast-like spindle shape to cells with extensive cell-cell contact growing in culture as islands (2.Ouyang P. Sugrue S.P. J. Cell Biol. 1996; 135: 1027-1042Crossref PubMed Scopus (82) Google Scholar). Intriguingly, EM analysis of these transfected cells revealed that the array of epithelial cell junctions was enhanced. In addition, carcinoma-derived cells, when transfected with pinin cDNA, exhibited inhibition of anchorage-independent growth in soft agar. Furthermore, pinin's gene locus and dysregulation of pinin expression in primary tumor tissues suggest that pinin may function as a tumor suppressor in certain types of cancer (3.Degen W.G. Agterbos M.A. Muyrers J.P. Bloemers H.P. Swart G.W. Biochim. Biophys. Acta. 1999; 1444: 384-394Crossref PubMed Scopus (18) Google Scholar, 4.Shi Y. Sugrue S.P. Oncogene. 2000; 19: 289-297Crossref PubMed Scopus (46) Google Scholar).Pinin has also been localized in the nucleus in various tissues as well as in cultured cell lines (5.Brandner J.M. Reidenbach S. Franke W.W. Differentiation. 1997; 62: 119-127Crossref PubMed Scopus (33) Google Scholar, 6.Brandner J.M. Reidenbach S. Kuhn C. Franke W.W. Eur J. Cell Biol. 1998; 75: 295-308Crossref PubMed Scopus (33) Google Scholar, 7.Ouyang P. Biochem. Biophys. Res. Commun. 1999; 263: 192-200Crossref PubMed Scopus (34) Google Scholar). Brandner et al. (6.Brandner J.M. Reidenbach S. Kuhn C. Franke W.W. Eur J. Cell Biol. 1998; 75: 295-308Crossref PubMed Scopus (33) Google Scholar) has proposed an involvement of pinin in spliceosomal function. The dual location of pinin may be indicative of the involvement of pinin in multiple cellular activities, both at the desmosome and in the nucleus; however, it is not yet clear whether or not the function of pinin in cell-cell adhesion is coordinated with its function in the nucleus. As a step toward understanding the functions of pinin, we sought to identify proteins that interact with pinin. In this study, we focus on the ability of pinin to bind keratin.Keratin filaments are anchored to the lateral plasma membrane at desmosomes. These intercellular junctions reinforce epithelial adhesion as well as integrate the IF network across the entire epithelium. Numerous structure-function studies of desmosomal proteins have revealed details pertaining to the molecular organization of desmosome-IF complex. The relationships among the desmosomal components have been extensively reviewed elsewhere (8.Kowalczyk A.P. Bornslaeger E.A. Norvell S.M. Palka H.L. Green K.J. Int. Rev. Cytol. 1999; 185: 237-302Crossref PubMed Google Scholar, 9.Troyanovsky S.M. Leube R.E. Subcell. Biochem. 1998; 31: 263-289PubMed Google Scholar, 10.Fuchs E. Yang Y. Dowling J. Kouklis P. Smith E. Guo L. Yu Q.C. Soc. Gen. Physiol. Ser. 1997; 52: 141-148PubMed Google Scholar, 11.Smith E.A. Fuchs E. J. Cell Biol. 1998; 141: 1229-1241Crossref PubMed Scopus (203) Google Scholar). The constitutive components of the desmosome include desmosomal cadherins (desmogleins and desmocollins) and plaque proteins, plakoglobin, desmoplakin, and plakophilin. Among these proteins, desmoplakin (12.Kouklis P.D. Hutton E. Fuchs E. J. Cell Biol. 1994; 127: 1049-1060Crossref PubMed Scopus (243) Google Scholar, 13.Meng J.J. Bornslaeger E.A. Green K.J. Steinert P.M. Ip W. J. Biol. Chem. 1997; 272: 21495-21503Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar) and plakophilin (11.Smith E.A. Fuchs E. J. Cell Biol. 1998; 141: 1229-1241Crossref PubMed Scopus (203) Google Scholar, 14.Hatzfeld M. Kristjansson G.I. Plessmann U. Weber K. J. Cell Sci. 1994; 107: 2259-2270Crossref PubMed Google Scholar) have been shown to bind directly to keratins. In addition, other peripherally associated desmosome proteins such as plectin (15.Foisner R. Leichtfried F.E. Herrmann H. Small J.V. Lawson D. Wiche G. J. Cell Biol. 1988; 106: 723-733Crossref PubMed Scopus (143) Google Scholar,16.Wiche G. Gromov D. Donovan A. Castanon M.J. Fuchs E. J. Cell Biol. 1993; 121: 607-619Crossref PubMed Scopus (114) Google Scholar), envoplakin/periplakin (17.Ruhrberg C. Hajibagheri M.A. Simon M. Dooley T.P. Watt F.M. J. Cell Biol. 1996; 134: 715-729Crossref PubMed Scopus (152) Google Scholar, 18.Ruhrberg C. Hajibagheri M.A. Parry D.A. Watt F.M. J. Cell Biol. 1997; 139: 1835-1849Crossref PubMed Scopus (176) Google Scholar), and pinin (1.Ouyang P. Sugrue S.P. J. Cell Biol. 1992; 118: 1477-1488Crossref PubMed Scopus (53) Google Scholar) are also thought to interact, directly or indirectly, with keratin. Significant questions pertaining to the molecular associations and specific roles of these accessory proteins of the desmosome remain.To identify potential protein-protein interactions of pinin, a two-hybrid screening was performed with either the amino portion or the carboxyl portion of pinin as bait. In this study, we presented a detailed analysis on the binding of the amino end domain of pinin to one group of the identified proteins, the keratins. Keratin 18 (K18), keratin 8 (K8), and keratin 19 (K19) were shown to interact with the amino portion of pinin in the two-hybrid screen. Further truncation analyses defined the specific domain of keratin that mediates the interaction. In addition, the specific domain of pinin molecule sufficient for the interaction was characterized, and through site-directed mutagenesis, the essential residues within this particular domain were investigated. In vitro blot overlay assays were performed to confirm the interaction between the amino end domain of pinin and the keratins. Overall, our data strongly suggest that pinin is capable of binding directly to the intermediate filament proteins, specifically the keratins. These data provide important information on eventual understanding of mechanism by which pinin may affect the assembly/stabilization of epithelial cell adhesion.DISCUSSIONIn this study, we present data demonstrating the direct interaction of the amino end domain of pinin with the 2B domain of keratin from simple epithelial cells. These data are not only consistent with our previous morphological observations but provide biochemical support of pinin-IF association.There are four distinct coiled-coil stretches, 1A, 1B, 2A, and 2B in the central rod domain of a keratin molecule. Our data indicate that pinin binds to the sequence within the 2B domain of keratin. Coil 1 of keratin exhibited no binding to pinin, strongly supporting the conclusion that the interaction between the 2B domain of keratin and pinin amino-terminal domain is indeed specific and not due to nonspecific interaction with coiled-coil-containing proteins. Direct binding to the rod 2B domain of keratin 18 has been reported for BPAG 2, a hemidesmosome-associated protein (33.Aho S. Uitto J. J. Cell. Biochem. 1999; 72: 356-367Crossref PubMed Scopus (40) Google Scholar). While desmoplakin has been shown to bind to the head domain of epidermal keratins, such as keratin 1/keratin 10 and keratin 5/keratin 14 (11.Smith E.A. Fuchs E. J. Cell Biol. 1998; 141: 1229-1241Crossref PubMed Scopus (203) Google Scholar), it has also been shown to be capable of binding to the rod domain of simple epithelial keratin K8/K18 heterodimer (13.Meng J.J. Bornslaeger E.A. Green K.J. Steinert P.M. Ip W. J. Biol. Chem. 1997; 272: 21495-21503Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar).The truncation analyses suggested the amino end of pinin (residues 1–98) contained the sequence responsible for binding to keratin. Although short coiled-coils composed of four to five heptad repeats have been reported (31.Lupas A. Curr. Opin. Struct. Biol. 1997; 7: 388-393Crossref PubMed Scopus (203) Google Scholar), it is not clear whether the four and a half heptad repeats at the amino end of pinin are actually sufficient to form a coiled-coil structure in vivo. The amino end of pinin does not contain a “trigger sequence” (34.Kammerer R.A. Schulthess T. Landwehr R. Lustig A. Engel J. Aebi U. Steinmetz M.O. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13419-13424Crossref PubMed Scopus (155) Google Scholar), so it may not participate in the formation of a coiled-coil. However, data derived from point mutation analyses of the amino-terminal domain of pinin suggest the sequence within the heptad repeats is indeed essential for the interaction with keratin. N′ L8P and N′ L19P completely abolished the binding of pinin to K18, whereas N′ L29P did not, suggesting that the heptad repeats located nearer the amino end of pinin may play a more significant role in pinin-keratin interaction.We have suggested that pinin may function as a tumor suppressor based on chromosomal location of pinin and tumor biological analyses (4.Shi Y. Sugrue S.P. Oncogene. 2000; 19: 289-297Crossref PubMed Scopus (46) Google Scholar). It has been shown that the expression of pinin was absent or greatly reduced in certain carcinomas, including renal cell carcinoma and transitional cell carcinoma. On the other hand, pinin expression was up-regulated in a subset of melanoma tissues (3.Degen W.G. Agterbos M.A. Muyrers J.P. Bloemers H.P. Swart G.W. Biochim. Biophys. Acta. 1999; 1444: 384-394Crossref PubMed Scopus (18) Google Scholar) and a subset of renal cell carcinoma (4.Shi Y. Sugrue S.P. Oncogene. 2000; 19: 289-297Crossref PubMed Scopus (46) Google Scholar). Decreased expression of pinin was correlated with loss of epithelial cell-cell adhesion, while increasing pinin expression by transfection of pinin cDNA was shown to enhance cell-cell adhesion (4.Shi Y. Sugrue S.P. Oncogene. 2000; 19: 289-297Crossref PubMed Scopus (46) Google Scholar, 35.Shi Y. Tabesh M. Sugrue S.P. Ivest. Ophthalmol. Vis. Sci. 2000; 41: 1337-1345PubMed Google Scholar). Interestingly, K18 and K8 have long been considered as cytological markers for carcinomas due to their persistent expression in tumor cells derived from simple epithelia and their aberrant expression in malignant progression of nonepithelial cells (36.Moll R. Franke W.W. Schiller D.L. Geiger B. Krepler R. Cell. 1982; 31: 11-24Abstract Full Text PDF PubMed Scopus (4495) Google Scholar, 37.Hendrix M.J. Seftor E.A. Chu Y.W. Seftor R.E. Nagle R.B. McDaniel K.M. Leong S.P. Yohem K.H. Leibovitz A.M. Meyskens Jr., F.L. Conaway D.H. Welch D.R. Liotta L.A. Stetler-Steveson W. J. Natl. Cancer. Inst. 1992; 84: 165-174Crossref PubMed Scopus (161) Google Scholar, 38.Schussler M.H. Skoudy A. Ramaekers F. Real F.X. Am. J. Pathol. 1992; 140: 559-568PubMed Google Scholar, 39.Trask D.K. Band V. Zajchowski D.A. Yaswen P. Suh T. Sager R. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2319-2323Crossref PubMed Scopus (169) Google Scholar). In addition, several studies suggested that K18/K8 filaments have a role in the tumorigenicity. For example, in K8-deficient mice, adult animals developed pronounced colorectal hyperplasia (40.Baribault H. Penner J. Iozzo R.V. Wilson-Heiner M. Genes Dev. 1994; 8: 2964-2973Crossref PubMed Scopus (270) Google Scholar), and the expression of K8 and K18 in human melanoma cell lines resulted in increased invasive and metastatic properties of the cells (37.Hendrix M.J. Seftor E.A. Chu Y.W. Seftor R.E. Nagle R.B. McDaniel K.M. Leong S.P. Yohem K.H. Leibovitz A.M. Meyskens Jr., F.L. Conaway D.H. Welch D.R. Liotta L.A. Stetler-Steveson W. J. Natl. Cancer. Inst. 1992; 84: 165-174Crossref PubMed Scopus (161) Google Scholar, 41.Zarbo R.J. Gown A.M. Nagle R.B. Visscher D.W. Crissman J.D. Mod. Pathol. 1990; 3: 494-501PubMed Google Scholar). It is tempting to speculate that the tumor suppressor function of pinin is related to the interaction of pinin with keratin.This study did not address the important issue regarding the relationship between the desmosome and pinin. Our initial two-hybrid screens identified other, as of yet uncharacterized, proteins interacting with pinin N′ bait 1–480. 2J. Shi and S. P. Sugrue, unpublished data. One of these clones coded for a protein containing motifs highly homologous to periplakin (18.Ruhrberg C. Hajibagheri M.A. Parry D.A. Watt F.M. J. Cell Biol. 1997; 139: 1835-1849Crossref PubMed Scopus (176) Google Scholar), a desmosome-IF-associated protein forming cornified envelope in the stratified epithelial cells. The possibility of pinin connecting to desmosome through this periplakin-like protein is currently being addressed.In summary, we have demonstrated that pinin can bind to keratin 18, keratin 8, and keratin 19. The 2B domain of keratin contains the sequence mediating the interaction with pinin, and the amino end (residues 1–98) of pinin was sufficient to bind keratin. Identification of specific binding sites within pinin for keratin and for other proteins will be an integral step for future studies. We believe that investigation of the function(s) of pinin in cell adhesion and IF organization will greatly contribute to our current knowledge of epithelial cell-cell adhesion. Pinin was first identified to be a protein associated with the desmosome, which was recruited to the preformed desmosomes of the epithelia but absent at nascent desmosomes (1.Ouyang P. Sugrue S.P. J. Cell Biol. 1992; 118: 1477-1488Crossref PubMed Scopus (53) Google Scholar). Immunofluorescence and immuno-EM studies have shown pinin decorating keratin filaments near the cytoplasmic face of the desmosomal plaque in the vicinity of keratin filament convergence upon the desmosome. Our previous studies have revealed a correlation between the placement of pinin at the desmosome and an increase in the organization/stabilization of desmosome-IF1 complex (1.Ouyang P. Sugrue S.P. J. Cell Biol. 1992; 118: 1477-1488Crossref PubMed Scopus (53) Google Scholar, 2.Ouyang P. Sugrue S.P. J. Cell Biol. 1996; 135: 1027-1042Crossref PubMed Scopus (82) Google Scholar). Presumably, one of the functions of pinin is related to the desmosome-IF complex. The expression level of pinin has been correlated with the overall epithelial phenotype. HEK-293 cells, when transfected with pinin full-length cDNA, exhibited a striking phenotype change from a fibroblast-like spindle shape to cells with extensive cell-cell contact growing in culture as islands (2.Ouyang P. Sugrue S.P. J. Cell Biol. 1996; 135: 1027-1042Crossref PubMed Scopus (82) Google Scholar). Intriguingly, EM analysis of these transfected cells revealed that the array of epithelial cell junctions was enhanced. In addition, carcinoma-derived cells, when transfected with pinin cDNA, exhibited inhibition of anchorage-independent growth in soft agar. Furthermore, pinin's gene locus and dysregulation of pinin expression in primary tumor tissues suggest that pinin may function as a tumor suppressor in certain types of cancer (3.Degen W.G. Agterbos M.A. Muyrers J.P. Bloemers H.P. Swart G.W. Biochim. Biophys. Acta. 1999; 1444: 384-394Crossref PubMed Scopus (18) Google Scholar, 4.Shi Y. Sugrue S.P. Oncogene. 2000; 19: 289-297Crossref PubMed Scopus (46) Google Scholar). Pinin has also been localized in the nucleus in various tissues as well as in cultured cell lines (5.Brandner J.M. Reidenbach S. Franke W.W. Differentiation. 1997; 62: 119-127Crossref PubMed Scopus (33) Google Scholar, 6.Brandner J.M. Reidenbach S. Kuhn C. Franke W.W. Eur J. Cell Biol. 1998; 75: 295-308Crossref PubMed Scopus (33) Google Scholar, 7.Ouyang P. Biochem. Biophys. Res. Commun. 1999; 263: 192-200Crossref PubMed Scopus (34) Google Scholar). Brandner et al. (6.Brandner J.M. Reidenbach S. Kuhn C. Franke W.W. Eur J. Cell Biol. 1998; 75: 295-308Crossref PubMed Scopus (33) Google Scholar) has proposed an involvement of pinin in spliceosomal function. The dual location of pinin may be indicative of the involvement of pinin in multiple cellular activities, both at the desmosome and in the nucleus; however, it is not yet clear whether or not the function of pinin in cell-cell adhesion is coordinated with its function in the nucleus. As a step toward understanding the functions of pinin, we sought to identify proteins that interact with pinin. In this study, we focus on the ability of pinin to bind keratin. Keratin filaments are anchored to the lateral plasma membrane at desmosomes. These intercellular junctions reinforce epithelial adhesion as well as integrate the IF network across the entire epithelium. Numerous structure-function studies of desmosomal proteins have revealed details pertaining to the molecular organization of desmosome-IF complex. The relationships among the desmosomal components have been extensively reviewed elsewhere (8.Kowalczyk A.P. Bornslaeger E.A. Norvell S.M. Palka H.L. Green K.J. Int. Rev. Cytol. 1999; 185: 237-302Crossref PubMed Google Scholar, 9.Troyanovsky S.M. Leube R.E. Subcell. Biochem. 1998; 31: 263-289PubMed Google Scholar, 10.Fuchs E. Yang Y. Dowling J. Kouklis P. Smith E. Guo L. Yu Q.C. Soc. Gen. Physiol. Ser. 1997; 52: 141-148PubMed Google Scholar, 11.Smith E.A. Fuchs E. J. Cell Biol. 1998; 141: 1229-1241Crossref PubMed Scopus (203) Google Scholar). The constitutive components of the desmosome include desmosomal cadherins (desmogleins and desmocollins) and plaque proteins, plakoglobin, desmoplakin, and plakophilin. Among these proteins, desmoplakin (12.Kouklis P.D. Hutton E. Fuchs E. J. Cell Biol. 1994; 127: 1049-1060Crossref PubMed Scopus (243) Google Scholar, 13.Meng J.J. Bornslaeger E.A. Green K.J. Steinert P.M. Ip W. J. Biol. Chem. 1997; 272: 21495-21503Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar) and plakophilin (11.Smith E.A. Fuchs E. J. Cell Biol. 1998; 141: 1229-1241Crossref PubMed Scopus (203) Google Scholar, 14.Hatzfeld M. Kristjansson G.I. Plessmann U. Weber K. J. Cell Sci. 1994; 107: 2259-2270Crossref PubMed Google Scholar) have been shown to bind directly to keratins. In addition, other peripherally associated desmosome proteins such as plectin (15.Foisner R. Leichtfried F.E. Herrmann H. Small J.V. Lawson D. Wiche G. J. Cell Biol. 1988; 106: 723-733Crossref PubMed Scopus (143) Google Scholar,16.Wiche G. Gromov D. Donovan A. Castanon M.J. Fuchs E. J. Cell Biol. 1993; 121: 607-619Crossref PubMed Scopus (114) Google Scholar), envoplakin/periplakin (17.Ruhrberg C. Hajibagheri M.A. Simon M. Dooley T.P. Watt F.M. J. Cell Biol. 1996; 134: 715-729Crossref PubMed Scopus (152) Google Scholar, 18.Ruhrberg C. Hajibagheri M.A. Parry D.A. Watt F.M. J. Cell Biol. 1997; 139: 1835-1849Crossref PubMed Scopus (176) Google Scholar), and pinin (1.Ouyang P. Sugrue S.P. J. Cell Biol. 1992; 118: 1477-1488Crossref PubMed Scopus (53) Google Scholar) are also thought to interact, directly or indirectly, with keratin. Significant questions pertaining to the molecular associations and specific roles of these accessory proteins of the desmosome remain. To identify potential protein-protein interactions of pinin, a two-hybrid screening was performed with either the amino portion or the carboxyl portion of pinin as bait. In this study, we presented a detailed analysis on the binding of the amino end domain of pinin to one group of the identified proteins, the keratins. Keratin 18 (K18), keratin 8 (K8), and keratin 19 (K19) were shown to interact with the amino portion of pinin in the two-hybrid screen. Further truncation analyses defined the specific domain of keratin that mediates the interaction. In addition, the specific domain of pinin molecule sufficient for the interaction was characterized, and through site-directed mutagenesis, the essential residues within this particular domain were investigated. In vitro blot overlay assays were performed to confirm the interaction between the amino end domain of pinin and the keratins. Overall, our data strongly suggest that pinin is capable of binding directly to the intermediate filament proteins, specifically the keratins. These data provide important information on eventual understanding of mechanism by which pinin may affect the assembly/stabilization of epithelial cell adhesion. DISCUSSIONIn this study, we present data demonstrating the direct interaction of the amino end domain of pinin with the 2B domain of keratin from simple epithelial cells. These data are not only consistent with our previous morphological observations but provide biochemical support of pinin-IF association.There are four distinct coiled-coil stretches, 1A, 1B, 2A, and 2B in the central rod domain of a keratin molecule. Our data indicate that pinin binds to the sequence within the 2B domain of keratin. Coil 1 of keratin exhibited no binding to pinin, strongly supporting the conclusion that the interaction between the 2B domain of keratin and pinin amino-terminal domain is indeed specific and not due to nonspecific interaction with coiled-coil-containing proteins. Direct binding to the rod 2B domain of keratin 18 has been reported for BPAG 2, a hemidesmosome-associated protein (33.Aho S. Uitto J. J. Cell. Biochem. 1999; 72: 356-367Crossref PubMed Scopus (40) Google Scholar). While desmoplakin has been shown to bind to the head domain of epidermal keratins, such as keratin 1/keratin 10 and keratin 5/keratin 14 (11.Smith E.A. Fuchs E. J. Cell Biol. 1998; 141: 1229-1241Crossref PubMed Scopus (203) Google Scholar), it has also been shown to be capable of binding to the rod domain of simple epithelial keratin K8/K18 heterodimer (13.Meng J.J. Bornslaeger E.A. Green K.J. Steinert P.M. Ip W. J. Biol. Chem. 1997; 272: 21495-21503Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar).The truncation analyses suggested the amino end of pinin (residues 1–98) contained the sequence responsible for binding to keratin. Although short coiled-coils composed of four to five heptad repeats have been reported (31.Lupas A. Curr. Opin. Struct. Biol. 1997; 7: 388-393Crossref PubMed Scopus (203) Google Scholar), it is not clear whether the four and a half heptad repeats at the amino end of pinin are actually sufficient to form a coiled-coil structure in vivo. The amino end of pinin does not contain a “trigger sequence” (34.Kammerer R.A. Schulthess T. Landwehr R. Lustig A. Engel J. Aebi U. Steinmetz M.O. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13419-13424Crossref PubMed Scopus (155) Google Scholar), so it may not participate in the formation of a coiled-coil. However, data derived from point mutation analyses of the amino-terminal domain of pinin suggest the sequence within the heptad repeats is indeed essential for the interaction with keratin. N′ L8P and N′ L19P completely abolished the binding of pinin to K18, whereas N′ L29P did not, suggesting that the heptad repeats located nearer the amino end of pinin may play a more significant role in pinin-keratin interaction.We have suggested that pinin may function as a tumor suppressor based on chromosomal location of pinin and tumor biological analyses (4.Shi Y. Sugrue S.P. Oncogene. 2000; 19: 289-297Crossref PubMed Scopus (46) Google Scholar). It has been shown that the expression of pinin was absent or greatly reduced in certain carcinomas, including renal cell carcinoma and transitional cell carcinoma. On the other hand, pinin expression was up-regulated in a subset of melanoma tissues (3.Degen W.G. Agterbos M.A. Muyrers J.P. Bloemers H.P. Swart G.W. Biochim. Biophys. Acta. 1999; 1444: 384-394Crossref PubMed Scopus (18) Google Scholar) and a subset of renal cell carcinoma (4.Shi Y. Sugrue S.P. Oncogene. 2000; 19: 289-297Crossref PubMed Scopus (46) Google Scholar). Decreased expression of pinin was correlated with loss of epithelial cell-cell adhesion, while increasing pinin expression by transfection of pinin cDNA was shown to enhance cell-cell adhesion (4.Shi Y. Sugrue S.P. Oncogene. 2000; 19: 289-297Crossref PubMed Scopus (46) Google Scholar, 35.Shi Y. Tabesh M. Sugrue S.P. Ivest. Ophthalmol. Vis. Sci. 2000; 41: 1337-1345PubMed Google Scholar). Interestingly, K18 and K8 have long been considered as cytological markers for carcinomas due to their persistent expression in tumor cells derived from simple epithelia and their aberrant expression in malignant progression of nonepithelial cells (36.Moll R. Franke W.W. Schiller D.L. Geiger B. Krepler R. Cell. 1982; 31: 11-24Abstract Full Text PDF PubMed Scopus (4495) Google Scholar, 37.Hendrix M.J. Seftor E.A. Chu Y.W. Seftor R.E. Nagle R.B. McDaniel K.M. Leong S.P. Yohem K.H. Leibovitz A.M. Meyskens Jr., F.L. Conaway D.H. Welch D.R. Liotta L.A. Stetler-Steveson W. J. Natl. Cancer. Inst. 1992; 84: 165-174Crossref PubMed Scopus (161) Google Scholar, 38.Schussler M.H. Skoudy A. Ramaekers F. Real F.X. Am. J. Pathol. 1992; 140: 559-568PubMed Google Scholar, 39.Trask D.K. Band V. Zajchowski D.A. Yaswen P. Suh T. Sager R. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2319-2323Crossref PubMed Scopus (169) Google Scholar). In addition, several studies suggested that K18/K8 filaments have a role in the tumorigenicity. For example, in K8-deficient mice, adult animals developed pronounced colorectal hyperplasia (40.Baribault H. Penner J. Iozzo R.V. Wilson-Heiner M. Genes Dev. 1994; 8: 2964-2973Crossref PubMed Scopus (270) Google Scholar), and the expression of K8 and K18 in human melanoma cell lines resulted in increased invasive and metastatic properties of the cells (37.Hendrix M.J. Seftor E.A. Chu Y.W. Seftor R.E. Nagle R.B. McDaniel K.M. Leong S.P. Yohem K.H. Leibovitz A.M. Meyskens Jr., F.L. Conaway D.H. Welch D.R. Liotta L.A. Stetler-Steveson W. J. Natl. Cancer. Inst. 1992; 84: 165-174Crossref PubMed Scopus (161) Google Scholar, 41.Zarbo R.J. Gown A.M. Nagle R.B. Visscher D.W. Crissman J.D. Mod. Pathol. 1990; 3: 494-501PubMed Google Scholar). It is tempting to speculate that the tumor suppressor function of pinin is related to the interaction of pinin with keratin.This study did not address the important issue regarding the relationship between the desmosome and pinin. Our initial two-hybrid screens identified other, as of yet uncharacterized, proteins interacting with pinin N′ bait 1–480. 2J. Shi and S. P. Sugrue, unpublished data. One of these clones coded for a protein containing motifs highly homologous to periplakin (18.Ruhrberg C. Hajibagheri M.A. Parry D.A. Watt F.M. J. Cell Biol. 1997; 139: 1835-1849Crossref PubMed Scopus (176) Google Scholar), a desmosome-IF-associated protein forming cornified envelope in the stratified epithelial cells. The possibility of pinin connecting to desmosome through this periplakin-like protein is currently being addressed.In summary, we have demonstrated that pinin can bind to keratin 18, keratin 8, and keratin 19. The 2B domain of keratin contains the sequence mediating the interaction with pinin, and the amino end (residues 1–98) of pinin was sufficient to bind keratin. Identification of specific binding sites within pinin for keratin and for other proteins will be an integral step for future studies. We believe that investigation of the function(s) of pinin in cell adhesion and IF organization will greatly contribute to our current knowledge of epithelial cell-cell adhesion. In this study, we present data demonstrating the direct interaction of the amino end domain of pinin with the 2B domain of keratin from simple epithelial cells. These data are not only consistent with our previous morphological observations but provide biochemical support of pinin-IF association. There are four distinct coiled-coil stretches, 1A, 1B, 2A, and 2B in the central rod domain of a keratin molecule. Our data indicate that pinin binds to the sequence within the 2B domain of keratin. Coil 1 of keratin exhibited no binding to pinin, strongly supporting the conclusion that the interaction between the 2B domain of keratin and pinin amino-terminal domain is indeed specific and not due to nonspecific interaction with coiled-coil-containing proteins. Direct binding to the rod 2B domain of keratin 18 has been reported for BPAG 2, a hemidesmosome-associated protein (33.Aho S. Uitto J. J. Cell. Biochem. 1999; 72: 356-367Crossref PubMed Scopus (40) Google Scholar). While desmoplakin has been shown to bind to the head domain of epidermal keratins, such as keratin 1/keratin 10 and keratin 5/keratin 14 (11.Smith E.A. Fuchs E. J. Cell Biol. 1998; 141: 1229-1241Crossref PubMed Scopus (203) Google Scholar), it has also been shown to be capable of binding to the rod domain of simple epithelial keratin K8/K18 heterodimer (13.Meng J.J. Bornslaeger E.A. Green K.J. Steinert P.M. Ip W. J. Biol. Chem. 1997; 272: 21495-21503Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). The truncation analyses suggested the amino end of pinin (residues 1–98) contained the sequence responsible for binding to keratin. Although short coiled-coils composed of four to five heptad repeats have been reported (31.Lupas A. Curr. Opin. Struct. Biol. 1997; 7: 388-393Crossref PubMed Scopus (203) Google Scholar), it is not clear whether the four and a half heptad repeats at the amino end of pinin are actually sufficient to form a coiled-coil structure in vivo. The amino end of pinin does not contain a “trigger sequence” (34.Kammerer R.A. Schulthess T. Landwehr R. Lustig A. Engel J. Aebi U. Steinmetz M.O. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13419-13424Crossref PubMed Scopus (155) Google Scholar), so it may not participate in the formation of a coiled-coil. However, data derived from point mutation analyses of the amino-terminal domain of pinin suggest the sequence within the heptad repeats is indeed essential for the interaction with keratin. N′ L8P and N′ L19P completely abolished the binding of pinin to K18, whereas N′ L29P did not, suggesting that the heptad repeats located nearer the amino end of pinin may play a more significant role in pinin-keratin interaction. We have suggested that pinin may function as a tumor suppressor based on chromosomal location of pinin and tumor biological analyses (4.Shi Y. Sugrue S.P. Oncogene. 2000; 19: 289-297Crossref PubMed Scopus (46) Google Scholar). It has been shown that the expression of pinin was absent or greatly reduced in certain carcinomas, including renal cell carcinoma and transitional cell carcinoma. On the other hand, pinin expression was up-regulated in a subset of melanoma tissues (3.Degen W.G. Agterbos M.A. Muyrers J.P. Bloemers H.P. Swart G.W. Biochim. Biophys. Acta. 1999; 1444: 384-394Crossref PubMed Scopus (18) Google Scholar) and a subset of renal cell carcinoma (4.Shi Y. Sugrue S.P. Oncogene. 2000; 19: 289-297Crossref PubMed Scopus (46) Google Scholar). Decreased expression of pinin was correlated with loss of epithelial cell-cell adhesion, while increasing pinin expression by transfection of pinin cDNA was shown to enhance cell-cell adhesion (4.Shi Y. Sugrue S.P. Oncogene. 2000; 19: 289-297Crossref PubMed Scopus (46) Google Scholar, 35.Shi Y. Tabesh M. Sugrue S.P. Ivest. Ophthalmol. Vis. Sci. 2000; 41: 1337-1345PubMed Google Scholar). Interestingly, K18 and K8 have long been considered as cytological markers for carcinomas due to their persistent expression in tumor cells derived from simple epithelia and their aberrant expression in malignant progression of nonepithelial cells (36.Moll R. Franke W.W. Schiller D.L. Geiger B. Krepler R. Cell. 1982; 31: 11-24Abstract Full Text PDF PubMed Scopus (4495) Google Scholar, 37.Hendrix M.J. Seftor E.A. Chu Y.W. Seftor R.E. Nagle R.B. McDaniel K.M. Leong S.P. Yohem K.H. Leibovitz A.M. Meyskens Jr., F.L. Conaway D.H. Welch D.R. Liotta L.A. Stetler-Steveson W. J. Natl. Cancer. Inst. 1992; 84: 165-174Crossref PubMed Scopus (161) Google Scholar, 38.Schussler M.H. Skoudy A. Ramaekers F. Real F.X. Am. J. Pathol. 1992; 140: 559-568PubMed Google Scholar, 39.Trask D.K. Band V. Zajchowski D.A. Yaswen P. Suh T. Sager R. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2319-2323Crossref PubMed Scopus (169) Google Scholar). In addition, several studies suggested that K18/K8 filaments have a role in the tumorigenicity. For example, in K8-deficient mice, adult animals developed pronounced colorectal hyperplasia (40.Baribault H. Penner J. Iozzo R.V. Wilson-Heiner M. Genes Dev. 1994; 8: 2964-2973Crossref PubMed Scopus (270) Google Scholar), and the expression of K8 and K18 in human melanoma cell lines resulted in increased invasive and metastatic properties of the cells (37.Hendrix M.J. Seftor E.A. Chu Y.W. Seftor R.E. Nagle R.B. McDaniel K.M. Leong S.P. Yohem K.H. Leibovitz A.M. Meyskens Jr., F.L. Conaway D.H. Welch D.R. Liotta L.A. Stetler-Steveson W. J. Natl. Cancer. Inst. 1992; 84: 165-174Crossref PubMed Scopus (161) Google Scholar, 41.Zarbo R.J. Gown A.M. Nagle R.B. Visscher D.W. Crissman J.D. Mod. Pathol. 1990; 3: 494-501PubMed Google Scholar). It is tempting to speculate that the tumor suppressor function of pinin is related to the interaction of pinin with keratin. This study did not address the important issue regarding the relationship between the desmosome and pinin. Our initial two-hybrid screens identified other, as of yet uncharacterized, proteins interacting with pinin N′ bait 1–480. 2J. Shi and S. P. Sugrue, unpublished data. One of these clones coded for a protein containing motifs highly homologous to periplakin (18.Ruhrberg C. Hajibagheri M.A. Parry D.A. Watt F.M. J. Cell Biol. 1997; 139: 1835-1849Crossref PubMed Scopus (176) Google Scholar), a desmosome-IF-associated protein forming cornified envelope in the stratified epithelial cells. The possibility of pinin connecting to desmosome through this periplakin-like protein is currently being addressed. In summary, we have demonstrated that pinin can bind to keratin 18, keratin 8, and keratin 19. The 2B domain of keratin contains the sequence mediating the interaction with pinin, and the amino end (residues 1–98) of pinin was sufficient to bind keratin. Identification of specific binding sites within pinin for keratin and for other proteins will be an integral step for future studies. We believe that investigation of the function(s) of pinin in cell adhesion and IF organization will greatly contribute to our current knowledge of epithelial cell-cell adhesion." @default.
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W1966227162 title "Dissection of Protein Linkage between Keratins and Pinin, a Protein with Dual Location at Desmosome-Intermediate Filament Complex and in the Nucleus" @default.
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