Phosphorylation sites of protein kinase C delta in H2O2-treated cells and its activation by tyrosine kinase in vitro.
Protein kinase C delta (PKC delta) is normally activated by diacylglycerol produced from receptor-mediated hydrolysis of inositol phospholipids. On stimulation of cells with H(2)O(2), the enzyme is tyrosine phosphorylated, with a concomitant increase in enzymatic activity. This activation does not appear to accompany its translocation to membranes. In the present study, the tyrosine phosphorylation sites of PKC delta in the H(2)O(2)-treated cells were identified as Tyr-311, Tyr-332, and Tyr-512 by mass spectrometric analysis with the use of the precursor-scan method and by immunoblot analysis with the use of phosphorylation site-specific antibodies. Tyr-311 was the predominant modification site among them. In an in vitro study, phosphorylation at this site by Lck, a non-receptor-type tyrosine kinase, enhanced the basal enzymatic activity and elevated its maximal velocity in the presence of diacylglycerol. The mutation of Tyr-311 to phenylalanine prevented the increase in this maximal activity, but replacement of the other two tyrosine residues did not block such an effect. The results indicate that phosphorylation at Tyr-311 between the regulatory and catalytic domains is a critical step for generation of the active PKC delta in response to H(2)O(2).
Konishi
H
H
Biosignal Research Center, Kobe University, Kobe 657-8501, Japan.
Yamauchi
E
E
Taniguchi
H
H
Yamamoto
T
T
Matsuzaki
H
H
Takemura
Y
Y
Ohmae
K
K
Kikkawa
U
U
Nishizuka
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D004789:Enzyme Activation; D006861:Hydrogen Peroxide / Q000494:pharmacology; D015151:Immunoblotting; D007527:Isoenzymes / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011493:Protein Kinase C / Q000737:chemistry* / Q000378:metabolism; D051745:Protein Kinase C-delta; D011505:Protein-Tyrosine Kinases / Q000502:physiology*; D014443:Tyrosine / Q000378:metabolism
D007527:Isoenzymes; D014443:Tyrosine; D006861:Hydrogen Peroxide; D011505:Protein-Tyrosine Kinases; D011493:Protein Kinase C; D051745:Protein Kinase C-delta