Identification of phosphorylation sites in keratinocyte transglutaminase

• Published in: Biochemical journal Vol. 320; no. 2; pp. 547 - 550
• Main Authors: RICE, Robert H., MEHRPOUYAN, Majid, QIN, Qin, PHILLIPS, Marjorie A., LEE, Young M.
• Format: Journal Article
• Language: English
• Published: England 01.12.1996
• Subjects: 3T3 Cells; Amino Acid Sequence; Animals; Cells, Cultured; Chromatography, High Pressure Liquid; Humans; Keratinocytes - enzymology; Mice; Molecular Sequence Data; Peptide Fragments - chemistry; Peptide Fragments - isolation & purification; Peptide Mapping; Phosphopeptides - chemistry; Phosphopeptides - isolation & purification; Phosphoproteins - chemistry; Phosphoproteins - isolation & purification; Phosphorylation; Skin; Transglutaminases - chemistry; Transglutaminases - isolation & purification; Trypsin
• ISSN: 0264-6021; 1470-8728
• DOI: 10.1042/bj3200547

Phosphorylation of keratinocyte transglutaminase occurs in its N-terminal extension and is stimulated several-fold by the protein kinase C agonist phorbol myristate acetate. In the present work, this stimulation was prevented by simultaneous treatment of the cells with the protein kinase C-selective inhibitor GF109203X. In contrast, phosphorylation occurring in the absence of phorbol ester was essentially unaffected by GF109203X, although it was decreased dramatically by the non-selective kinase inhibitor staurosporine. Four serine residues that are subject to phosphorylation have been identified by sequencing of radioactive tryptic peptides. Serines at positions 24 and 92, each containing 2–8% of the total radioactivity with or without phorbol ester stimulation, were minor sites of phosphorylation. Serine-82 was by far the dominant site of phosphorylation in the absence of phorbol ester treatment, and was also the major site in its presence. Serine-85 was phosphorylated to a high degree in the presence but not in the absence of phorbol ester stimulation. Thus the data indicate the influence of at least two different kinase activities in transglutaminase phosphorylation.