Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1984 Mar 1;98(3):1144–1149. doi: 10.1083/jcb.98.3.1144

Dual regulation of intermediate filament phosphorylation

PMCID: PMC2113137  PMID: 6199363

Abstract

Intermediate filament proteins have been isolated from ME-180, cells of a human cervical carcinoma. Eight of these proteins have been identified as keratins by immunologic cross-reactivity to antibodies raised against authentic human epidermal keratins. The ME-180 keratin proteins consist of two major subunits designated MEK-1 and MEK-2 with approximate molecular weights of 58,000 and 53,000, respectively, and six minor subunits of 59, 57, 52.5, 50.5, 45, and 40 kilodaltons. When ME-180 cells were incubated for 2-24 h in the presence of [32P]orthophosphate, MEK-1 and MEK-2 as well as the 52.5- and 40- kilodalton keratins were phosphorylated at their serine residues. V8 protease digests revealed that phosphorylation of MEK-2 is restricted to one peptide representing approximately half the molecule. Regulation of MEK-1 and MEK-2 phosphorylation has been studied by prelabeling the cells for 2 h in 32P-labeled medium. This was followed by up to 2 h of continued incubation in the same medium after the addition of a variety of perturbing agents. The phosphorylation of MEK-2 increased in the presence of 10(-4) M dibutyryl cyclic AMP (twofold), 1 mM methylisobutylxanthine (2.5-fold), 10(-5) M isoproterenol (fivefold), and 10(-9) M cholera toxin (sevenfold). In contrast, MEK-1 phosphorylation was unaffected by these agents. Neither cyclic GMP, Ca++, hydrocortisone, nor epidermal growth factor had any effect on the phosphorylation of MEK-1 or MEK-2. The results indicate that the phosphorylation of these two keratins is independently controlled by cyclic AMP-dependent kinase for MEK-2 and by cyclic nucleotide- independent kinase for MEK-1. The observed differences in control suggest distinct functions for MEK-1 and MEK-2 within the cytoskeletal network.

Full Text

The Full Text of this article is available as a PDF (756.3 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Anthony R. S., Spector L. B. Phosphorylated acetate kinase. Its isolation and reactivity. J Biol Chem. 1972 Apr 10;247(7):2120–2125. [PubMed] [Google Scholar]
  2. Bitte L., Kabat D. Isotopic labeling and analysis of phosphoproteins from mammalian ribosomes. Methods Enzymol. 1974;30:563–590. doi: 10.1016/0076-6879(74)30056-0. [DOI] [PubMed] [Google Scholar]
  3. Bladon P. T., Bowden P. E., Cunliffe W. J., Wood E. J. Prekeratin biosynthesis in human scalp epidermis. Biochem J. 1982 Oct 15;208(1):179–187. doi: 10.1042/bj2080179. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cleveland D. W., Fischer S. G., Kirschner M. W., Laemmli U. K. Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis. J Biol Chem. 1977 Feb 10;252(3):1102–1106. [PubMed] [Google Scholar]
  5. Cohen S., Carpenter G., King L., Jr Epidermal growth factor-receptor-protein kinase interactions. Co-purification of receptor and epidermal growth factor-enhanced phosphorylation activity. J Biol Chem. 1980 May 25;255(10):4834–4842. [PubMed] [Google Scholar]
  6. Dale B. A., Vadlamudi B., DeLap L. W., Bernstein I. A. Similarities between stratum corneum basic protein and histidine-rich protein II from newborn rat epidermis. Biochim Biophys Acta. 1981 Mar 27;668(1):98–106. doi: 10.1016/0005-2795(81)90153-7. [DOI] [PubMed] [Google Scholar]
  7. Franke W. W., Schiller D. L., Moll R., Winter S., Schmid E., Engelbrecht I., Denk H., Krepler R., Platzer B. Diversity of cytokeratins. Differentiation specific expression of cytokeratin polypeptides in epithelial cells and tissues. J Mol Biol. 1981 Dec 25;153(4):933–959. doi: 10.1016/0022-2836(81)90460-5. [DOI] [PubMed] [Google Scholar]
  8. Franke W. W., Schmid E., Winter S., Osborn M., Weber K. Widespread occurrence of intermediate-sized filaments of the vimentin-type in cultured cells from diverse vertebrates. Exp Cell Res. 1979 Oct 1;123(1):25–46. doi: 10.1016/0014-4827(79)90418-x. [DOI] [PubMed] [Google Scholar]
  9. Geisler N., Plessmann U., Weber K. Related amino acid sequences in neurofilaments and non-neural intermediate filaments. Nature. 1982 Apr 1;296(5856):448–450. doi: 10.1038/296448a0. [DOI] [PubMed] [Google Scholar]
  10. Gilmartin M. E., Culbertson V. B., Freedberg I. M. Phosphorylation of epidermal keratins. J Invest Dermatol. 1980 Sep;75(3):211–216. doi: 10.1111/1523-1747.ep12522887. [DOI] [PubMed] [Google Scholar]
  11. Glass W. F., 2nd, Briggs R. C., Hnilica L. S. Identification of tissue-specific nuclear antigens transferred to nitrocellulose from polyacrylamide gels. Science. 1981 Jan 2;211(4477):70–72. doi: 10.1126/science.7003713. [DOI] [PubMed] [Google Scholar]
  12. Henderson D., Weber K. Immuno-electron microscopical identification of the two types of intermediate filaments in established epithelial cells. Exp Cell Res. 1981 Apr;132(2):297–311. doi: 10.1016/0014-4827(81)90106-3. [DOI] [PubMed] [Google Scholar]
  13. Hennings H., Michael D., Cheng C., Steinert P., Holbrook K., Yuspa S. H. Calcium regulation of growth and differentiation of mouse epidermal cells in culture. Cell. 1980 Jan;19(1):245–254. doi: 10.1016/0092-8674(80)90406-7. [DOI] [PubMed] [Google Scholar]
  14. Keski-Oja J., Heine U. I., Rapp U. R., Wetzel B. Epidermal growth factor-induced alterations in proliferating mouse epithelial cells. Exp Cell Res. 1980 Aug;128(2):279–290. doi: 10.1016/0014-4827(80)90064-6. [DOI] [PubMed] [Google Scholar]
  15. Kessler S. W. Cell membrane antigen isolation with the staphylococcal protein A-antibody adsorbent. J Immunol. 1976 Nov;117(5 Pt 1):1482–1490. [PubMed] [Google Scholar]
  16. 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]
  17. Lazarides E. Intermediate filaments: a chemically heterogeneous, developmentally regulated class of proteins. Annu Rev Biochem. 1982;51:219–250. doi: 10.1146/annurev.bi.51.070182.001251. [DOI] [PubMed] [Google Scholar]
  18. Lonsdale-Eccles J. D., Haugen J. A., Dale B. A. A phosphorylated keratohyalin-derived precursor of epidermal stratum corneum basic protein. J Biol Chem. 1980 Mar 25;255(6):2235–2238. [PubMed] [Google Scholar]
  19. 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]
  20. O'Connor C. M., Gard D. L., Lazarides E. Phosphorylation of intermediate filament proteins by cAMP-dependent protein kinases. Cell. 1981 Jan;23(1):135–143. doi: 10.1016/0092-8674(81)90278-6. [DOI] [PubMed] [Google Scholar]
  21. O'Farrell P. H. High resolution two-dimensional electrophoresis of proteins. J Biol Chem. 1975 May 25;250(10):4007–4021. [PMC free article] [PubMed] [Google Scholar]
  22. Okada N., Kitano Y., Ichihara K. Effects of cholera toxin on proliferation of cultured human keratinocytes in relation to intracellular cyclic AMP levels. J Invest Dermatol. 1982 Jul;79(1):42–47. doi: 10.1111/1523-1747.ep12510580. [DOI] [PubMed] [Google Scholar]
  23. Pruss R. M., Mirsky R., Raff M. C., Thorpe R., Dowding A. J., Anderton B. H. All classes of intermediate filaments share a common antigenic determinant defined by a monoclonal antibody. Cell. 1981 Dec;27(3 Pt 2):419–428. doi: 10.1016/0092-8674(81)90383-4. [DOI] [PubMed] [Google Scholar]
  24. Steinert P. M., Cantieri J. S., Teller D. C., Lonsdale-Eccles J. D., Dale B. A. Characterization of a class of cationic proteins that specifically interact with intermediate filaments. Proc Natl Acad Sci U S A. 1981 Jul;78(7):4097–4101. doi: 10.1073/pnas.78.7.4097. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Steinert P. M., Idler W. W. The polypeptide composition of bovine epidermal alpha-keratin. Biochem J. 1975 Dec;151(3):603–614. doi: 10.1042/bj1510603. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Steinert P. M., Rice R. H., Roop D. R., Trus B. L., Steven A. C. Complete amino acid sequence of a mouse epidermal keratin subunit and implications for the structure of intermediate filaments. Nature. 1983 Apr 28;302(5911):794–800. doi: 10.1038/302794a0. [DOI] [PubMed] [Google Scholar]
  27. Steinert P. M., Wantz M. L., Idler W. W. O-phosphoserine content of intermediate filament subunits. Biochemistry. 1982 Jan 5;21(1):177–183. doi: 10.1021/bi00530a030. [DOI] [PubMed] [Google Scholar]
  28. Sun T. T., Green H. Cultured epithelial cells of cornea, conjunctiva and skin: absence of marked intrinsic divergence of their differentiated states. Nature. 1977 Oct 6;269(5628):489–493. doi: 10.1038/269489a0. [DOI] [PubMed] [Google Scholar]
  29. Sun T. T., Green H. Keratin filaments of cultured human epidermal cells. Formation of intermolecular disulfide bonds during terminal differentiation. J Biol Chem. 1978 Mar 25;253(6):2053–2060. [PubMed] [Google Scholar]
  30. Sykes J. A., Whitescarver J., Jernstrom P., Nolan J. F., Byatt P. Some properties of a new epithelial cell line of human origin. J Natl Cancer Inst. 1970 Jul;45(1):107–122. [PubMed] [Google Scholar]
  31. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Woodcock-Mitchell J., Eichner R., Nelson W. G., Sun T. T. Immunolocalization of keratin polypeptides in human epidermis using monoclonal antibodies. J Cell Biol. 1982 Nov;95(2 Pt 1):580–588. doi: 10.1083/jcb.95.2.580. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Wu Y. J., Parker L. M., Binder N. E., Beckett M. A., Sinard J. H., Griffiths C. T., Rheinwald J. G. The mesothelial keratins: a new family of cytoskeletal proteins identified in cultured mesothelial cells and nonkeratinizing epithelia. Cell. 1982 Dec;31(3 Pt 2):693–703. doi: 10.1016/0092-8674(82)90324-5. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES