Biochemical and Biophysical Research Communications
CIN85 regulates the ability of MEKK4 to activate the p38 MAP kinase pathway
Section snippets
Materials and methods
Antibodies and reagents. EGF was purchased from Intergen. Mouse anti-HA antibody (12CA5) was from Roche Diagnostic, rabbit anti-FLAG antibody was from Sigma–Aldrich. Anti-MEKK4 and anti-p38 rabbit sera were kindly provided by P. Gerwins. Anti-MKK3/6 rabbit antibody (sc-13069) and goat anti-MEKK4 antibody (sc-6846) were from Santa Cruz. Anti-Cbl (RF) and anti-CIN85 (CT) antibodies were previously described [6], [8]. Anti-phospho-p38Thr180/Tyr182 rabbit polyclonal antibody, anti-phospho-JNK
CIN85 interacts with MEKK4
The search for PxxxPR motif containing proteins led to the identification of MEKK4, which contains three of the CIN85 putative binding motifs (Fig. 1A). In order to confirm that CIN85 really binds to MEKK4, we first performed GST pull-down analysis with the SH3 domains fused individually or in tandem to GST. As shown in Fig. 1B, each SH3 domain of CIN85 binds individually to MEKK4 and all three SH3s expressed in tandem (GST-SH3ABC) bind MEKK4 even more potently. These interactions are specific
Discussion
Since its identification as a Cbl-interacting protein [20], CIN85 has been found to interact with many molecular partners through its SH3 domains and proline-rich region. The identification of the particular proline motif recognized by CIN85’s SH3 domains led to the discovery of numerous CIN85-interacting proteins [9], [10], [11]. Many of these proteins are involved in the formation and trafficking of endocytic vesicles, an essential mechanism for the down-modulation of RTKs by CIN85, as well
Acknowledgments
We thank K. Kowanetz for the initial identification of MEKK4 as a putative CIN85 binding partner. We are also thankful to P. Gerwins, A. Fornace, and M. Landstrom for providing us with expression vectors. This work was partially supported by the Swedish Strategic Foundation, Boehringer Ingelheim Foundation and Deutsche Forschungsgemeinschaft to I.D.). Philippe Soubeyran is a research fellow from the ARC (Association pour la Recherche sur le Cancer).
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