Phorbol ester-stimulated phosphorylation of keratinocyte transglutaminase in the membrane anchorage region.
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.
Chakravarty
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Charles A. Dana Laboratory of Toxicology, Harvard School of Public Health, Boston, MA 02115.
Rong
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XH
Rice
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RH
D001665:Binding Sites; D002462:Cell Membrane / Q000201:enzymology*; D002478:Cells, Cultured; D006801:Humans; D007163:Immunosorbent Techniques; D015603:Keratinocytes / Q000201:enzymology*; D010446:Peptide Fragments / Q000302:isolation & purification; D010710:Phosphates / Q000378:metabolism; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D011503:Transglutaminases / Q000378:metabolism*; D014357:Trypsin / Q000378:metabolism
D010446:Peptide Fragments; D010710:Phosphates; D010768:Phosphoserine; D011503:Transglutaminases; D011493:Protein Kinase C; D014357:Trypsin; D013755:Tetradecanoylphorbol Acetate