Tyrosine phosphorylation of protein kinase D in the pleckstrin homology domain leads to activation.
Protein kinase D (PKD) is a member of the AGC family of Ser/Thr kinases and is distantly related to protein kinase C (PKC). Formerly known as PKCmu, PKD contains protein domains not found in conventional PKC isoforms. A functional pleckstrin homology (PH) domain is critical for the regulation of PKD activity. Here we report that PKD is tyrosine-phosphorylated within the PH domain, leading to activation. This phosphorylation is mediated by a pathway that consists of the Src and Abl tyrosine kinases and occurs in response to stimulation with pervanadate and oxidative stress. Mutational analysis revealed three tyrosine phosphorylation sites (Tyr(432), Tyr(463), and Tyr(502)), which are regulated by the Src-Abl pathway, and phosphorylation of only one of these (Tyr(463)) leads to PKD activation. By using a phospho-specific antibody, we show that Abl directly phosphorylates PKD at Tyr(463) in vitro, and in cells phosphorylation of this site is sufficient to mediate full activation of PKD. Mutation of the other two sites, Tyr(432) and Tyr(502), had no significant influence on PKD activity. These data reveal a tyrosine phosphorylation-dependent activation mechanism for PKD and suggest that this event contributes to the release of the autoinhibitory PKD PH domain leading to kinase activation and downstream responses.
Storz
Peter
P
Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215, USA.
Döppler
Heike
H
Johannes
Franz-Josef
FJ
Toker
Alex
A
D000595:Amino Acid Sequence; D001798:Blood Proteins / Q000737:chemistry* / Q000378:metabolism; D015153:Blotting, Western; D004247:DNA / Q000378:metabolism; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D015151:Immunoblotting; D008969:Molecular Sequence Data; D009154:Mutation; D010750:Phosphoproteins / Q000737:chemistry* / Q000378:metabolism; D010766:Phosphorylation; D011233:Precipitin Tests; D011493:Protein Kinase C / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction; D013997:Time Factors; D014162:Transfection; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism*; D014638:Vanadates / Q000494:pharmacology
D001798:Blood Proteins; D004791:Enzyme Inhibitors; D010750:Phosphoproteins; D011994:Recombinant Proteins; C076715:pervanadate; C039077:platelet protein P47; D014638:Vanadates; D014443:Tyrosine; D004247:DNA; D006861:Hydrogen Peroxide; C089212:protein kinase D; D011493:Protein Kinase C