Phorbol ester and hydrogen peroxide synergistically induce the interaction of diacylglycerol kinase gamma with the Src homology 2 and C1 domains of beta2-chimaerin.
DGKgamma (diacylglycerol kinase gamma) was reported to interact with beta2-chimaerin, a GAP (GTPase-activating protein) for Rac, in response to epidermal growth factor. Here we found that PMA and H2O2 also induced the interaction of DGKgamma with beta2-chimaerin. It is noteworthy that simultaneous addition of PMA and H2O2 synergistically enhanced the interaction. In this case, PMA was replaceable by DAG (diacylglycerol). The beta2-chimaerin translocation from the cytoplasm to the plasma membrane caused by PMA plus H2O2 was further enhanced by the expression of DGKgamma. Moreover, DGKgamma apparently enhanced the beta2-chimaerin GAP activity upon cell stimulation with PMA. PMA was found to be mainly required for a conversion of beta2-chimaerin into an active form. On the other hand, H2O2 was suggested to induce a release of Zn2+ from the C1 domain of beta2-chimaerin. By stepwise deletion analysis, we demonstrated that the SH2 (Src homology 2) and C1 domains of beta2-chimaerin interacted with the N-terminal half of catalytic region of DGKgamma. Unexpectedly, the SH2 domain of beta2-chimaerin contributes to the interaction independently of phosphotyrosine. Taken together, these results suggest that the functional link between DGKgamma and beta2-chimaerin has a broad significance in response to a wide range of cell stimuli. Our work offers a novel mechanism of protein-protein interaction, that is, the phosphotyrosine-independent interaction of the SH2 domain acting in co-operation with the C1 domain.
Yasuda
Satoshi
S
Department of Biochemistry, Sapporo Medical University School of Medicine, South-1, West-17, Chuo-ku, Sapporo, 060-8556, Japan.
Kai
Masahiro
M
Imai
Shin-ichi
S
Kanoh
Hideo
H
Sakane
Fumio
F
D000818:Animals; D019556:COS Cells; D002384:Catalysis; D002522:Chlorocebus aethiops; D019852:Diacylglycerol Kinase / Q000378:metabolism*; D017353:Gene Deletion; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D009363:Neoplasm Proteins / Q000737:chemistry*; D010703:Phorbol Esters / Q000378:metabolism*; D011485:Protein Binding; D011493:Protein Kinase C / Q000378:metabolism; D017434:Protein Structure, Tertiary; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D015032:Zinc / Q000737:chemistry; D018909:src Homology Domains