Identification of tyrosine 187 as a protein kinase C-delta phosphorylation site.
Protein kinase C-delta (PKC-delta) has been demonstrated to be phosphorylated on tyrosine residue(s) in many different biological systems (Li, W., Yu, J.-C., Michieli, P., Beeler, J. F., Ellmore, N., Heidaran, M. A., and Pierce, J. H. (1994) Mol. Cell. Biol. 14, 6727-6735; Li, W., Mischak, H., Yu, J.-C., Wang, L.-M., Mushinski, J. F., Heidaran, M. A., and Pierce, J. H. (1994) J. Biol. Chem. 269, 2349-2352; Denning, M. F., Dlugosz, A. A., Howett, M. A., and Yuspa, S. H. (1993) J. Biol. Chem. 268, 26079-26081). Tyrosine phosphorylation of PKC-delta has also been shown to occur in vitro when purified PKC-delta is coincubated with different tyrosine kinase sources. However, the tyrosine phosphorylation site(s) is currently unknown and the exact effect of this phosphorylation on its serine/threonine kinase activity and biological functions is still controversial. To directly investigate the potential role of PKC-delta tyrosine phosphorylation, tyrosine 187 was converted to phenylalanine (PKC-deltaY187F) by site-directed mutagenesis, and expression vectors containing PKC-deltaY187F cDNAs were transfected into both 32D myeloid progenitor cells and NIH 3T3 fibroblasts. The results showed that tyrosine 187 of PKC-delta became phosphorylated in vivo in response to 12-O-tetradecanoylphorbol-13-acetate stimulation or platelet-derived growth factor receptor activation. In vivo labeling and subsequent two-dimensional phosphopeptide analysis demonstrated that one phosphopeptide was absent in PKC-deltaY187F when compared to wild type PKC-delta, further substantiating that tyrosine 187 of PKC-delta is phosphorylated in vivo. Although the phosphotyrosine content of PKC-deltaY187F was reduced compared with PKC-deltaWT, the kinase activity of PKC-deltaY187F toward a PKC-delta substrate was not altered. Moreover, 12-O-tetradecanoylphorbol-13-acetate-mediated monocytic differentiation of 32D cells was not affected by expression of the PKC-deltaY187F mutant. Taken together, these results suggest that tyrosine phosphorylation of PKC-delta on 187 may not influence PKC-delta activation and known functions.
Li
W
W
Laboratory of Cellular and Molecular Biology, National Cancer Institute, Bethesda, Maryland 20892, USA.
Chen
X H
XH
Kelley
C A
CA
Alimandi
M
M
Zhang
J
J
Chen
Q
Q
Bottaro
D P
DP
Pierce
J H
JH
D016475:3T3 Cells; D000818:Animals; D018076:DNA, Complementary / Q000737:chemistry; D015180:Electrophoresis, Gel, Two-Dimensional; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000378:metabolism; D010766:Phosphorylation; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism*; D051745:Protein Kinase C-delta; D013379:Substrate Specificity; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection; D014443:Tyrosine / Q000378:metabolism*
D018076:DNA, Complementary; D007527:Isoenzymes; D010982:Platelet-Derived Growth Factor; D014443:Tyrosine; D010649:Phenylalanine; C497259:Prkcd protein, mouse; D011493:Protein Kinase C; D051745:Protein Kinase C-delta; D013755:Tetradecanoylphorbol Acetate