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. 1990 Oct 1;271(1):25–30. doi: 10.1042/bj2710025

Phorbol ester-stimulated phosphorylation of keratinocyte transglutaminase in the membrane anchorage region.

R Chakravarty 1, X H Rong 1, R H Rice 1
PMCID: PMC1149509  PMID: 1977383

Abstract

The membrane-bound transglutaminase of cultured keratinocytes became radioactively labelled upon addition of [32P]Pi to the medium. Transglutaminase phosphorylation was also demonstrable using particulate material isolated from cell homogenates. Compatible with mediation of the labelling by protein kinase C, the degree of phosphorylation in intact cells was stimulated approx. 5-fold in 4 h on treatment with the tumour-promoting phorbol ester phorbol 12-myristate 13-acetate, but not by phorbol. The extent of labelling was virtually unaffected by cycloheximide inhibition of protein synthesis, indicating that it arose primarily through turnover of phosphate in the membrane-bound enzyme. Phosphoamino acid analysis detected labelling only of serine residues. Most of the label was removed by trypsin release of the enzyme from the particulate fraction of cell homogenates, which deletes a membrane anchorage region of approximately 10 kDa. Upon trypsin treatment of the enzyme after immunoprecipitation, the phosphate label was recovered in soluble peptide material with a size of several thousand Da or less. Indicative of fragmentation of the membrane anchorage region, this material was separable by h.p.l.c. into two equally labelled peptides. Moreover, when the enzyme was labelled with [3H]palmitate or [3H]myristate, the fatty-acid-labelled peptide material required non-ionic detergent for solubilization and was separable from the phosphate-labelled material by gel filtration. Phorbol ester treatment of cultured keratinocytes in high- or low- Ca2(+)-containing medium was not accompanied by an appreciable protein-synthesis-independent change in transglutaminase activity. Independent of possible alteration of the intrinsic catalytic activity of the enzyme, phosphorylation may well modulate its interaction with substrate proteins, a potential site for physiological regulation.

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Selected References

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

  1. Allen-Hoffmann B. L., Rheinwald J. G. Polycyclic aromatic hydrocarbon mutagenesis of human epidermal keratinocytes in culture. Proc Natl Acad Sci U S A. 1984 Dec;81(24):7802–7806. doi: 10.1073/pnas.81.24.7802. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Chakravarty R., Rice R. H. Acylation of keratinocyte transglutaminase by palmitic and myristic acids in the membrane Anchorage region. J Biol Chem. 1989 Jan 5;264(1):625–629. [PubMed] [Google Scholar]
  3. Choi E. J., Toscano W. A., Jr Modulation of adenylate cyclase in human keratinocytes by protein kinase C. J Biol Chem. 1988 Nov 15;263(32):17167–17172. [PubMed] [Google Scholar]
  4. Cooper J. A., Sefton B. M., Hunter T. Detection and quantification of phosphotyrosine in proteins. Methods Enzymol. 1983;99:387–402. doi: 10.1016/0076-6879(83)99075-4. [DOI] [PubMed] [Google Scholar]
  5. Dixon H. B., Perham R. N. Reversible blocking of amino groups with citraconic anhydride. Biochem J. 1968 Sep;109(2):312–314. doi: 10.1042/bj1090312. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Floyd E. E., Jetten A. M. Regulation of type I (epidermal) transglutaminase mRNA levels during squamous differentiation: down regulation by retinoids. Mol Cell Biol. 1989 Nov;9(11):4846–4851. doi: 10.1128/mcb.9.11.4846. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Gabrielson E. W., Rosen G. M., Grafstrom R. C., Strauss K. E., Miyashita M., Harris C. C. Role of oxygen radicals in 12-O-tetradecanoylphorbol-13-acetate-induced squamous differentiation of cultured normal human bronchial epithelial cells. Cancer Res. 1988 Feb 15;48(4):822–825. [PubMed] [Google Scholar]
  8. Gordeladze J. O., Björo T., Torjesen P. A., Ostberg B. C., Haug E., Gautvik K. M. Protein kinase C stimulates adenylate cyclase activity in prolactin-secreting rat adenoma (GH4C1) pituicytes by inactivating the inhibitory GTP-binding protein Gi. Eur J Biochem. 1989 Aug 1;183(2):397–406. doi: 10.1111/j.1432-1033.1989.tb14941.x. [DOI] [PubMed] [Google Scholar]
  9. Hashimoto Y., Soderling T. R. Regulation of calcineurin by phosphorylation. Identification of the regulatory site phosphorylated by Ca2+/calmodulin-dependent protein kinase II and protein kinase C. J Biol Chem. 1989 Oct 5;264(28):16524–16529. [PubMed] [Google Scholar]
  10. 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]
  11. Iizuka H., Sakai H., Tamura T. Effects of the tumor promoter, phorbol 12-myristate, 13-acetate, on the epidermal adenylate cyclase system: evidence for adenylate cyclase-regulation by protein kinase C. J Invest Dermatol. 1989 Sep;93(3):387–391. [PubMed] [Google Scholar]
  12. Isseroff R. R., Stephens L. E., Gross J. L. Subcellular distribution of protein kinase C/phorbol ester receptors in differentiating mouse keratinocytes. J Cell Physiol. 1989 Nov;141(2):235–242. doi: 10.1002/jcp.1041410202. [DOI] [PubMed] [Google Scholar]
  13. Jeng A. Y., Lichti U., Strickland J. E., Blumberg P. M. Similar effects of phospholipase C and phorbol ester tumor promoters on primary mouse epidermal cells. Cancer Res. 1985 Nov;45(11 Pt 2):5714–5721. [PubMed] [Google Scholar]
  14. 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]
  15. Lichti U., Ben T., Yuspa S. H. Retinoic acid-induced transglutaminase in mouse epidermal cells is distinct from epidermal transglutaminase. J Biol Chem. 1985 Feb 10;260(3):1422–1426. [PubMed] [Google Scholar]
  16. MATOLTSY A. G., BALSAMO C. A. A study of the components of the cornified epithelium of human skin. J Biophys Biochem Cytol. 1955 Jul 25;1(4):339–360. doi: 10.1083/jcb.1.4.339. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Michel S., Schmidt R., Robinson S. M., Shroot B., Reichert U. Identification and subcellular distribution of cornified envelope precursor proteins in the transformed human keratinocyte line SV-K14. J Invest Dermatol. 1987 Mar;88(3):301–305. doi: 10.1111/1523-1747.ep12466177. [DOI] [PubMed] [Google Scholar]
  18. Nagae S., Lichti U., De Luca L. M., Yuspa S. H. Effect of retinoic acid on cornified envelope formation: difference between spontaneous envelope formation in vivo or in vitro and expression of envelope competence. J Invest Dermatol. 1987 Jul;89(1):51–58. doi: 10.1111/1523-1747.ep12580383. [DOI] [PubMed] [Google Scholar]
  19. Palfrey H. C., Waseem A. Protein kinase C in the human erythrocyte. Translocation to the plasma membrane and phosphorylation of bands 4.1 and 4.9 and other membrane proteins. J Biol Chem. 1985 Dec 15;260(29):16021–16029. [PubMed] [Google Scholar]
  20. Parkinson E. K., Grabham P., Emmerson A. A subpopulation of cultured human keratinocytes which is resistant to the induction of terminal differentiation-related changes by phorbol, 12-myristate, 13-acetate: evidence for an increase in the resistant population following transformation. Carcinogenesis. 1983;4(7):857–861. doi: 10.1093/carcin/4.7.857. [DOI] [PubMed] [Google Scholar]
  21. Ramwani J. J., Epand R. M., Moscarello M. A. Secondary structure of charge isomers of myelin basic protein before and after phosphorylation. Biochemistry. 1989 Aug 8;28(16):6538–6543. doi: 10.1021/bi00442a002. [DOI] [PubMed] [Google Scholar]
  22. Resing K. A., Dale B. A., Walsh K. A. Multiple copies of phosphorylated filaggrin in epidermal profilaggrin demonstrated by analysis of tryptic peptides. Biochemistry. 1985 Jul 16;24(15):4167–4175. doi: 10.1021/bi00336a053. [DOI] [PubMed] [Google Scholar]
  23. Rice R. H., Green H. Presence in human epidermal cells of a soluble protein precursor of the cross-linked envelope: activation of the cross-linking by calcium ions. Cell. 1979 Nov;18(3):681–694. doi: 10.1016/0092-8674(79)90123-5. [DOI] [PubMed] [Google Scholar]
  24. Rice R. H., Green H. Relation of protein synthesis and transglutaminase activity to formation of the cross-linked envelope during terminal differentiation of the cultured human epidermal keratinocyte. J Cell Biol. 1978 Mar;76(3):705–711. doi: 10.1083/jcb.76.3.705. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Rice R. H., Green H. The cornified envelope of terminally differentiated human epidermal keratinocytes consists of cross-linked protein. Cell. 1977 Jun;11(2):417–422. doi: 10.1016/0092-8674(77)90059-9. [DOI] [PubMed] [Google Scholar]
  26. Rice R. H., Rong X. H., Chakravarty R. Proteolytic release of keratinocyte transglutaminase. Biochem J. 1990 Jan 15;265(2):351–357. doi: 10.1042/bj2650351. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Rice R. H., Rong X. H., Chakravarty R. Suppression of keratinocyte differentiation in SSC-9 human squamous carcinoma cells by benzo[a]pyrene, 12-O-tetradecanoylphorbol-13-acetate and hydroxyurea. Carcinogenesis. 1988 Oct;9(10):1885–1890. doi: 10.1093/carcin/9.10.1885. [DOI] [PubMed] [Google Scholar]
  28. Rubin A. L., Rice R. H. Characterization of the calcium sensitivity of differentiation in SCC-13 human squamous carcinoma cells. In Vitro Cell Dev Biol. 1988 Sep;24(9):857–861. doi: 10.1007/BF02623894. [DOI] [PubMed] [Google Scholar]
  29. Rubin A. L., Rice R. H. Differential regulation by retinoic acid and calcium of transglutaminases in cultured neoplastic and normal human keratinocytes. Cancer Res. 1986 May;46(5):2356–2361. [PubMed] [Google Scholar]
  30. Simon M., Green H. Enzymatic cross-linking of involucrin and other proteins by keratinocyte particulates in vitro. Cell. 1985 Mar;40(3):677–683. doi: 10.1016/0092-8674(85)90216-8. [DOI] [PubMed] [Google Scholar]
  31. Simon M., Green H. Participation of membrane-associated proteins in the formation of the cross-linked envelope of the keratinocyte. Cell. 1984 Apr;36(4):827–834. doi: 10.1016/0092-8674(84)90032-1. [DOI] [PubMed] [Google Scholar]
  32. Smith P. K., Krohn R. I., Hermanson G. T., Mallia A. K., Gartner F. H., Provenzano M. D., Fujimoto E. K., Goeke N. M., Olson B. J., Klenk D. C. Measurement of protein using bicinchoninic acid. Anal Biochem. 1985 Oct;150(1):76–85. doi: 10.1016/0003-2697(85)90442-7. [DOI] [PubMed] [Google Scholar]
  33. Steinert P. M. The dynamic phosphorylation of the human intermediate filament keratin 1 chain. J Biol Chem. 1988 Sep 15;263(26):13333–13339. [PubMed] [Google Scholar]
  34. Takayama S., White M. F., Kahn C. R. Phorbol ester-induced serine phosphorylation of the insulin receptor decreases its tyrosine kinase activity. J Biol Chem. 1988 Mar 5;263(7):3440–3447. [PubMed] [Google Scholar]
  35. Thacher S. M., Coe E. L., Rice R. H. Retinoid suppression of transglutaminase activity and envelope competence in cultured human epidermal carcinoma cells. Hydrocortisone is a potent antagonist or retinyl acetate but not retinoic acid. Differentiation. 1985;29(1):82–87. doi: 10.1111/j.1432-0436.1985.tb00296.x. [DOI] [PubMed] [Google Scholar]
  36. Thacher S. M. Purification of keratinocyte transglutaminase and its expression during squamous differentiation. J Invest Dermatol. 1989 Apr;92(4):578–584. [PubMed] [Google Scholar]
  37. Thacher S. M., Rice R. H. Keratinocyte-specific transglutaminase of cultured human epidermal cells: relation to cross-linked envelope formation and terminal differentiation. Cell. 1985 Mar;40(3):685–695. doi: 10.1016/0092-8674(85)90217-x. [DOI] [PubMed] [Google Scholar]
  38. Warhol M. J., Roth J., Lucocq J. M., Pinkus G. S., Rice R. H. Immuno-ultrastructural localization of involucrin in squamous epithelium and cultured keratinocytes. J Histochem Cytochem. 1985 Feb;33(2):141–149. doi: 10.1177/33.2.2578499. [DOI] [PubMed] [Google Scholar]
  39. Watt F. M., Green H. Stratification and terminal differentiation of cultured epidermal cells. Nature. 1982 Feb 4;295(5848):434–436. doi: 10.1038/295434a0. [DOI] [PubMed] [Google Scholar]
  40. Wille J. J., Jr, Pittelkow M. R., Scott R. E. Normal and transformed human prokeratinocytes express divergent effects of a tumor promoter on cell cycle-mediated control of proliferation and differentiation. Carcinogenesis. 1985 Aug;6(8):1181–1187. doi: 10.1093/carcin/6.8.1181. [DOI] [PubMed] [Google Scholar]
  41. Yuspa S. H., Ben T., Hennings H., Lichti U. Divergent responses in epidermal basal cells exposed to the tumor promoter 12-O-tetradecanoylphorbol-13-acetate. Cancer Res. 1982 Jun;42(6):2344–2349. [PubMed] [Google Scholar]

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