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. 2007 Sep 17;27(23):8113–8126. doi: 10.1128/MCB.00794-07

FIG. 1.

FIG. 1.

Yeast library screening identified AIDA-1 as an EphA8-interacting protein. (A) The JM amino acid sequences of the indicated Eph family members were aligned using the ClustalW program. Gaps are represented by dashes. The numbers above the two sets of sequences mark the locations of amino acids within the EphA8 JM region. The dotted underline indicates the specific amino acid residues of the EphA8 JM region that are critical for interaction with the AIDA-1b PTB domain. (B) Domain structures of EphA8, AIDA-1b, and the interacting clone of AIDA identified in the yeast two-hybrid screen. G, immunoglobulin-like domain; C, cysteine-rich motif; F, fibronectin type III repeat domain; T, transmembrane domain; JM, juxtamembrane domain; K, kinase domain; S, SAM domain; A, ankyrin repeat domain; P, PTB domain. (C) X-Gal staining analysis indicated that the PTB domain of AIDA-1 binds to the JM domain of EphA8. Yeast transformants were cultured on Trp− Leu− selective plates in the absence of 3-AT prior to X-Gal staining using a filter-lifting method. Yeast cells transformed with NR-2A and PSD-95 served as positive controls for these yeast two-hybrid systems. Yeast cells transformed with the tyrosine kinase domain (TKD) of EphA8 as bait served as a negative control. Yeast transformants showing negative X-Gal staining on Trp− Leu− selective plates did not grow on His− Trp− Leu− selective plates in the presence of 1 mM 3-AT (data not shown). (D) AIDA-1 PTB domain interacts only with the JM domain of EphA8, not with those of EphA2, EphA4, and EphB2, in yeast. The JM sequences of the Eph family members shown in panel A were expressed as bait proteins fused to the LexA DNA binding domain in yeast. Yeast transformants were cultured on Trp− Leu− selective plates, and X-Gal staining was performed essentially as described for panel C. (E) Schematic diagram showing the various EphA8-JM deletion constructs that were subcloned into the bait construct described for panels C and D. (F) The AIDA PTB domain was coexpressed in the yeast two-hybrid assay with various deletion mutants of EphA8-JM, and X-Gal staining was performed essentially as described for panel C.