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. 1998 Apr 1;17(7):1892–1906. doi: 10.1093/emboj/17.7.1892

Phosphorylation of human keratin 18 serine 33 regulates binding to 14-3-3 proteins.

N O Ku 1, J Liao 1, M B Omary 1
PMCID: PMC1170536  PMID: 9524113

Abstract

Members of the 14-3-3 protein family bind the human intermediate filament protein keratin 18 (K18) in vivo, in a cell-cycle- and phosphorylation-dependent manner. We identified K18 Ser33 as an interphase phosphorylation site, which increases its phosphorylation during mitosis in cultured cells and regenerating liver, and as an in vitro cdc2 kinase phosphorylation site. Comparison of wild-type versus K18 Ser33-->Ala/Asp transfected cells showed that K18 Ser33 phosphorylation is essential for the association of K18 with 14-3-3 proteins, and plays a role in keratin organization and distribution. Mutation of another K18 major phosphorylation site (Ser52) or K18 glycosylation sites had no effect on the binding of K18 to 14-3-3 proteins. The K18 phospho-Ser33 motif is different from several 14-3-3-binding phosphomotifs already described. Antibodies that are specific to K18 phospho-Ser33 or phospho-Ser52 show that although Ser52 and Ser33 phosphorylated K18 molecules manifest partial colocalization, these phosphorylation events reside predominantly on distinct K18 molecules. Our results demonstrate a unique K18 phosphorylation site that is necessary but not sufficient for K18 binding to 14-3-3 proteins. This binding is likely to involve one or more mitotic events coupled to K18 Ser33 phosphorylation, and plays a role in keratin subcellular distribution. Physiological Ser52 or Ser33 phosphorylation on distinct K18 molecules suggests functional compartmentalization of these modifications.

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Selected References

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  1. Aitken A. 14-3-3 and its possible role in co-ordinating multiple signalling pathways. Trends Cell Biol. 1996 Sep;6(9):341–347. doi: 10.1016/0962-8924(96)10029-5. [DOI] [PubMed] [Google Scholar]
  2. Aitken A. 14-3-3 proteins on the MAP. Trends Biochem Sci. 1995 Mar;20(3):95–97. doi: 10.1016/s0968-0004(00)88971-9. [DOI] [PubMed] [Google Scholar]
  3. Aitken A., Howell S., Jones D., Madrazo J., Patel Y. 14-3-3 alpha and delta are the phosphorylated forms of raf-activating 14-3-3 beta and zeta. In vivo stoichiometric phosphorylation in brain at a Ser-Pro-Glu-Lys MOTIF. J Biol Chem. 1995 Mar 17;270(11):5706–5709. doi: 10.1074/jbc.270.11.5706. [DOI] [PubMed] [Google Scholar]
  4. Banik U., Wang G. A., Wagner P. D., Kaufman S. Interaction of phosphorylated tryptophan hydroxylase with 14-3-3 proteins. J Biol Chem. 1997 Oct 17;272(42):26219–26225. doi: 10.1074/jbc.272.42.26219. [DOI] [PubMed] [Google Scholar]
  5. Bonnefoy-Bérard N., Liu Y. C., von Willebrand M., Sung A., Elly C., Mustelin T., Yoshida H., Ishizaka K., Altman A. Inhibition of phosphatidylinositol 3-kinase activity by association with 14-3-3 proteins in T cells. Proc Natl Acad Sci U S A. 1995 Oct 24;92(22):10142–10146. doi: 10.1073/pnas.92.22.10142. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Boyle W. J., van der Geer P., Hunter T. Phosphopeptide mapping and phosphoamino acid analysis by two-dimensional separation on thin-layer cellulose plates. Methods Enzymol. 1991;201:110–149. doi: 10.1016/0076-6879(91)01013-r. [DOI] [PubMed] [Google Scholar]
  7. Braselmann S., McCormick F. Bcr and Raf form a complex in vivo via 14-3-3 proteins. EMBO J. 1995 Oct 2;14(19):4839–4848. doi: 10.1002/j.1460-2075.1995.tb00165.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Celis J. E., Larsen P. M., Fey S. J., Celis A. Phosphorylation of keratin and vimentin polypeptides in normal and transformed mitotic human epithelial amnion cells: behavior of keratin and vimentin filaments during mitosis. J Cell Biol. 1983 Nov;97(5 Pt 1):1429–1434. doi: 10.1083/jcb.97.5.1429. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chou C. F., Omary M. B. Mitotic arrest with anti-microtubule agents or okadaic acid is associated with increased glycoprotein terminal GlcNAc's. J Cell Sci. 1994 Jul;107(Pt 7):1833–1843. doi: 10.1242/jcs.107.7.1833. [DOI] [PubMed] [Google Scholar]
  10. Chou C. F., Omary M. B. Mitotic arrest-associated enhancement of O-linked glycosylation and phosphorylation of human keratins 8 and 18. J Biol Chem. 1993 Feb 25;268(6):4465–4472. [PubMed] [Google Scholar]
  11. Chou C. F., Riopel C. L., Rott L. S., Omary M. B. A significant soluble keratin fraction in 'simple' epithelial cells. Lack of an apparent phosphorylation and glycosylation role in keratin solubility. J Cell Sci. 1993 Jun;105(Pt 2):433–444. doi: 10.1242/jcs.105.2.433. [DOI] [PubMed] [Google Scholar]
  12. Chou C. F., Smith A. J., Omary M. B. Characterization and dynamics of O-linked glycosylation of human cytokeratin 8 and 18. J Biol Chem. 1992 Feb 25;267(6):3901–3906. [PubMed] [Google Scholar]
  13. Conklin D. S., Galaktionov K., Beach D. 14-3-3 proteins associate with cdc25 phosphatases. Proc Natl Acad Sci U S A. 1995 Aug 15;92(17):7892–7896. doi: 10.1073/pnas.92.17.7892. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Dubois T., Rommel C., Howell S., Steinhussen U., Soneji Y., Morrice N., Moelling K., Aitken A. 14-3-3 is phosphorylated by casein kinase I on residue 233. Phosphorylation at this site in vivo regulates Raf/14-3-3 interaction. J Biol Chem. 1997 Nov 14;272(46):28882–28888. doi: 10.1074/jbc.272.46.28882. [DOI] [PubMed] [Google Scholar]
  15. Freed E., Symons M., Macdonald S. G., McCormick F., Ruggieri R. Binding of 14-3-3 proteins to the protein kinase Raf and effects on its activation. Science. 1994 Sep 16;265(5179):1713–1716. doi: 10.1126/science.8085158. [DOI] [PubMed] [Google Scholar]
  16. Fu H., Xia K., Pallas D. C., Cui C., Conroy K., Narsimhan R. P., Mamon H., Collier R. J., Roberts T. M. Interaction of the protein kinase Raf-1 with 14-3-3 proteins. Science. 1994 Oct 7;266(5182):126–129. doi: 10.1126/science.7939632. [DOI] [PubMed] [Google Scholar]
  17. Fuchs E. The cytoskeleton and disease: genetic disorders of intermediate filaments. Annu Rev Genet. 1996;30:197–231. doi: 10.1146/annurev.genet.30.1.197. [DOI] [PubMed] [Google Scholar]
  18. Fuchs E., Weber K. Intermediate filaments: structure, dynamics, function, and disease. Annu Rev Biochem. 1994;63:345–382. doi: 10.1146/annurev.bi.63.070194.002021. [DOI] [PubMed] [Google Scholar]
  19. Furukawa Y., Ikuta N., Omata S., Yamauchi T., Isobe T., Ichimura T. Demonstration of the phosphorylation-dependent interaction of tryptophan hydroxylase with the 14-3-3 protein. Biochem Biophys Res Commun. 1993 Jul 15;194(1):144–149. doi: 10.1006/bbrc.1993.1796. [DOI] [PubMed] [Google Scholar]
  20. Ichimura T., Isobe T., Okuyama T., Takahashi N., Araki K., Kuwano R., Takahashi Y. Molecular cloning of cDNA coding for brain-specific 14-3-3 protein, a protein kinase-dependent activator of tyrosine and tryptophan hydroxylases. Proc Natl Acad Sci U S A. 1988 Oct;85(19):7084–7088. doi: 10.1073/pnas.85.19.7084. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Inagaki M., Inagaki N., Takahashi T., Takai Y. Phosphorylation-dependent control of structures of intermediate filaments: a novel approach using site- and phosphorylation state-specific antibodies. J Biochem. 1997 Mar;121(3):407–414. doi: 10.1093/oxfordjournals.jbchem.a021603. [DOI] [PubMed] [Google Scholar]
  22. Irvine A. D., Corden L. D., Swensson O., Swensson B., Moore J. E., Frazer D. G., Smith F. J., Knowlton R. G., Christophers E., Rochels R. Mutations in cornea-specific keratin K3 or K12 genes cause Meesmann's corneal dystrophy. Nat Genet. 1997 Jun;16(2):184–187. doi: 10.1038/ng0697-184. [DOI] [PubMed] [Google Scholar]
  23. Kartenbeck J., Leuschner U., Mayer R., Keppler D. Absence of the canalicular isoform of the MRP gene-encoded conjugate export pump from the hepatocytes in Dubin-Johnson syndrome. Hepatology. 1996 May;23(5):1061–1066. doi: 10.1053/jhep.1996.v23.pm0008621134. [DOI] [PubMed] [Google Scholar]
  24. Ku N. O., Liao J., Chou C. F., Omary M. B. Implications of intermediate filament protein phosphorylation. Cancer Metastasis Rev. 1996 Dec;15(4):429–444. doi: 10.1007/BF00054011. [DOI] [PubMed] [Google Scholar]
  25. Ku N. O., Michie S., Oshima R. G., Omary M. B. Chronic hepatitis, hepatocyte fragility, and increased soluble phosphoglycokeratins in transgenic mice expressing a keratin 18 conserved arginine mutant. J Cell Biol. 1995 Dec;131(5):1303–1314. doi: 10.1083/jcb.131.5.1303. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Ku N. O., Omary M. B. Identification and mutational analysis of the glycosylation sites of human keratin 18. J Biol Chem. 1995 May 19;270(20):11820–11827. doi: 10.1074/jbc.270.20.11820. [DOI] [PubMed] [Google Scholar]
  27. Ku N. O., Omary M. B. Identification of the major physiologic phosphorylation site of human keratin 18: potential kinases and a role in filament reorganization. J Cell Biol. 1994 Oct;127(1):161–171. doi: 10.1083/jcb.127.1.161. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Ku N. O., Omary M. B. Phosphorylation of human keratin 8 in vivo at conserved head domain serine 23 and at epidermal growth factor-stimulated tail domain serine 431. J Biol Chem. 1997 Mar 14;272(11):7556–7564. doi: 10.1074/jbc.272.11.7556. [DOI] [PubMed] [Google Scholar]
  29. Ku N. O., Wright T. L., Terrault N. A., Gish R., Omary M. B. Mutation of human keratin 18 in association with cryptogenic cirrhosis. J Clin Invest. 1997 Jan 1;99(1):19–23. doi: 10.1172/JCI119127. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Köhler G., Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature. 1975 Aug 7;256(5517):495–497. doi: 10.1038/256495a0. [DOI] [PubMed] [Google Scholar]
  31. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  32. Leube R. E., Bosch F. X., Romano V., Zimbelmann R., Höfler H., Franke W. W. Cytokeratin expression in simple epithelia. III. Detection of mRNAs encoding human cytokeratins nos. 8 and 18 in normal and tumor cells by hybridization with cDNA sequences in vitro and in situ. Differentiation. 1986;33(1):69–85. doi: 10.1111/j.1432-0436.1986.tb00412.x. [DOI] [PubMed] [Google Scholar]
  33. Liao J., Ku N. O., Omary M. B. Stress, apoptosis, and mitosis induce phosphorylation of human keratin 8 at Ser-73 in tissues and cultured cells. J Biol Chem. 1997 Jul 11;272(28):17565–17573. doi: 10.1074/jbc.272.28.17565. [DOI] [PubMed] [Google Scholar]
  34. Liao J., Ku N. O., Omary M. B. Two-dimensional gel analysis of glandular keratin intermediate filament phosphorylation. Electrophoresis. 1996 Nov;17(11):1671–1676. doi: 10.1002/elps.1150171104. [DOI] [PubMed] [Google Scholar]
  35. Liao J., Lowthert L. A., Ghori N., Omary M. B. The 70-kDa heat shock proteins associate with glandular intermediate filaments in an ATP-dependent manner. J Biol Chem. 1995 Jan 13;270(2):915–922. doi: 10.1074/jbc.270.2.915. [DOI] [PubMed] [Google Scholar]
  36. Liao J., Lowthert L. A., Ku N. O., Fernandez R., Omary M. B. Dynamics of human keratin 18 phosphorylation: polarized distribution of phosphorylated keratins in simple epithelial tissues. J Cell Biol. 1995 Dec;131(5):1291–1301. doi: 10.1083/jcb.131.5.1291. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Liao J., Lowthert L. A., Omary M. B. Heat stress or rotavirus infection of human epithelial cells generates a distinct hyperphosphorylated form of keratin 8. Exp Cell Res. 1995 Aug;219(2):348–357. doi: 10.1006/excr.1995.1238. [DOI] [PubMed] [Google Scholar]
  38. Liao J., Omary M. B. 14-3-3 proteins associate with phosphorylated simple epithelial keratins during cell cycle progression and act as a solubility cofactor. J Cell Biol. 1996 Apr;133(2):345–357. doi: 10.1083/jcb.133.2.345. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Liu Y. C., Elly C., Yoshida H., Bonnefoy-Berard N., Altman A. Activation-modulated association of 14-3-3 proteins with Cbl in T cells. J Biol Chem. 1996 Jun 14;271(24):14591–14595. doi: 10.1074/jbc.271.24.14591. [DOI] [PubMed] [Google Scholar]
  40. Liu Y. C., Liu Y., Elly C., Yoshida H., Lipkowitz S., Altman A. Serine phosphorylation of Cbl induced by phorbol ester enhances its association with 14-3-3 proteins in T cells via a novel serine-rich 14-3-3-binding motif. J Biol Chem. 1997 Apr 11;272(15):9979–9985. doi: 10.1074/jbc.272.15.9979. [DOI] [PubMed] [Google Scholar]
  41. Lowthert L. A., Ku N. O., Liao J., Coulombe P. A., Omary M. B. Empigen BB: a useful detergent for solubilization and biochemical analysis of keratins. Biochem Biophys Res Commun. 1995 Jan 5;206(1):370–379. doi: 10.1006/bbrc.1995.1051. [DOI] [PubMed] [Google Scholar]
  42. McLean W. H., Lane E. B. Intermediate filaments in disease. Curr Opin Cell Biol. 1995 Feb;7(1):118–125. doi: 10.1016/0955-0674(95)80053-0. [DOI] [PubMed] [Google Scholar]
  43. Michaud N. R., Fabian J. R., Mathes K. D., Morrison D. K. 14-3-3 is not essential for Raf-1 function: identification of Raf-1 proteins that are biologically activated in a 14-3-3- and Ras-independent manner. Mol Cell Biol. 1995 Jun;15(6):3390–3397. doi: 10.1128/mcb.15.6.3390. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Moll R., Franke W. W., Schiller D. L., Geiger B., Krepler R. The catalog of human cytokeratins: patterns of expression in normal epithelia, tumors and cultured cells. Cell. 1982 Nov;31(1):11–24. doi: 10.1016/0092-8674(82)90400-7. [DOI] [PubMed] [Google Scholar]
  45. Muslin A. J., Tanner J. W., Allen P. M., Shaw A. S. Interaction of 14-3-3 with signaling proteins is mediated by the recognition of phosphoserine. Cell. 1996 Mar 22;84(6):889–897. doi: 10.1016/s0092-8674(00)81067-3. [DOI] [PubMed] [Google Scholar]
  46. Omary M. B., Ku N. O. Intermediate filament proteins of the liver: emerging disease association and functions. Hepatology. 1997 May;25(5):1043–1048. doi: 10.1002/hep.510250537. [DOI] [PubMed] [Google Scholar]
  47. Oshima R. G., Millán J. L., Ceceña G. Comparison of mouse and human keratin 18: a component of intermediate filaments expressed prior to implantation. Differentiation. 1986;33(1):61–68. doi: 10.1111/j.1432-0436.1986.tb00411.x. [DOI] [PubMed] [Google Scholar]
  48. Pallas D. C., Fu H., Haehnel L. C., Weller W., Collier R. J., Roberts T. M. Association of polyomavirus middle tumor antigen with 14-3-3 proteins. Science. 1994 Jul 22;265(5171):535–537. doi: 10.1126/science.8036498. [DOI] [PubMed] [Google Scholar]
  49. Peng C. Y., Graves P. R., Thoma R. S., Wu Z., Shaw A. S., Piwnica-Worms H. Mitotic and G2 checkpoint control: regulation of 14-3-3 protein binding by phosphorylation of Cdc25C on serine-216. Science. 1997 Sep 5;277(5331):1501–1505. doi: 10.1126/science.277.5331.1501. [DOI] [PubMed] [Google Scholar]
  50. Reuther G. W., Fu H., Cripe L. D., Collier R. J., Pendergast A. M. Association of the protein kinases c-Bcr and Bcr-Abl with proteins of the 14-3-3 family. Science. 1994 Oct 7;266(5182):129–133. doi: 10.1126/science.7939633. [DOI] [PubMed] [Google Scholar]
  51. Soellner P., Quinlan R. A., Franke W. W. Identification of a distinct soluble subunit of an intermediate filament protein: tetrameric vimentin from living cells. Proc Natl Acad Sci U S A. 1985 Dec;82(23):7929–7933. doi: 10.1073/pnas.82.23.7929. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Steinert P. M., Roop D. R. Molecular and cellular biology of intermediate filaments. Annu Rev Biochem. 1988;57:593–625. doi: 10.1146/annurev.bi.57.070188.003113. [DOI] [PubMed] [Google Scholar]
  53. Zha J., Harada H., Yang E., Jockel J., Korsmeyer S. J. Serine phosphorylation of death agonist BAD in response to survival factor results in binding to 14-3-3 not BCL-X(L) Cell. 1996 Nov 15;87(4):619–628. doi: 10.1016/s0092-8674(00)81382-3. [DOI] [PubMed] [Google Scholar]
  54. Zhang S. H., Kobayashi R., Graves P. R., Piwnica-Worms H., Tonks N. K. Serine phosphorylation-dependent association of the band 4.1-related protein-tyrosine phosphatase PTPH1 with 14-3-3beta protein. J Biol Chem. 1997 Oct 24;272(43):27281–27287. doi: 10.1074/jbc.272.43.27281. [DOI] [PubMed] [Google Scholar]

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