Characterization of the Interactions of Estrogen Receptor and MNAR in the Activation of cSrc

• Published in: Molecular endocrinology (Baltimore, Md.) Md.), 2004-05, Vol.18 (5), p.1096-1108
• Main Authors: Barletta, Frank, Wong, Chi-Wai, McNally, Chris, Komm, Barry S, Katzenellenbogen, Benita, Cheskis, Boris J
• Format: Article
• Language: English
• Subjects: Co-Repressor Proteins; Estrogen Receptor alpha - metabolism; Humans; MAP Kinase Signaling System - physiology; Mutation - genetics; Phosphorylation; Protein Binding; Protein Structure, Tertiary - physiology; src Homology Domains - physiology; src-Family Kinases - metabolism; Trans-Activators - metabolism; Transcription Factors; Tumor Cells, Cultured
• ISSN: 0888-8809; 1944-9917
• DOI: 10.1210/me.2003-0335

In this study, we have evaluated the molecular mechanism of Src activation after its interaction with estrogen receptor α (ERα) and a newly identified scaffold protein, called MNAR (modulator of nongenomic activity of ER). Under basal condition, Src enzymatic activity is inhibited by intramolecular interactions. The enzyme can be activated by interaction between the SH2 domain of Src and phosphotyrosine-containing sequences and/or by interaction between the SH3 domain of Src and proteins containing PXXP motifs. Mutational analysis and functional evaluation of MNAR and the use of ERα and cSrc mutants revealed that MNAR interacts with Src’s SH3 domain via its N-terminal PXXP motif. Mutation of this motif abolished both the MNAR-induced activation of Src and the stimulation of ER transcriptional activity. ER interacts with Src’s SH2 domain using phosphotyrosine 537, and this complex was further stabilized by MNAR-ER interaction. Mapping studies reveal that both the A/B domain and Y537 of ERα are required for MNAR-induced activation of ER transcriptional activity. The region responsible for MNAR interaction with ER maps to two N-terminal LXXLL motifs of MNAR. Mutation of these motifs prevented ER-MNAR complex formation and eliminated activation of the Src/MAPK pathway. These data explicate how the coordinate interactions between MNAR, ER, and Src lead to Src activation. Our findings also demonstrate that MNAR is a scaffold protein that mediates ER-Src interaction and plays an important role in the integration of ER action in Src-mediated signaling.