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Figure 4


Fig. 4. 14-3-3 proteins bind to ADAM22 at tandem consensus binding sites with differing affinity. (A) Immunoprecipitation (IP) from HEK 293T cell lysates co-expressing Myc-tagged full length 14-3-3ß and FLAG-tagged full length ADAM22v4 wild type, ADAM22v4 substitution mutants of the first and/or second 14-3-3 consensus binding motifs or the ADAM22v4 C-terminal deletion mutant. Proteins were immunoprecipitated with anti-Myc antibodies and immunoblots probed with anti-FLAG. As controls, immunoblots were re-probed with anti-Myc antibodies and the HEK 293T cell lysates expressing the FLAG-tagged ADAM22v4 wild-type and mutant proteins were immunoblotted and probed with anti-FLAG. (B) Purified GST-14-3-3{zeta} fusion proteins bound to glutathione-Sepharose were incubated with HEK 293T cell lysates expressing FLAG-tagged full length ADAM22v4 wild-type, ADAM22v4 substitution mutants of the first and/or second 14-3-3 consensus binding motifs or ADAM22v4 C-terminal deletion mutant. Immunoblots from subsequent GST pull-down assays were probed with anti-FLAG antibodies and then re-probed with anti-GST antibodies as control for GST-14-3-3{zeta} fusion protein levels. The HEK 293T cell lysates expressing the FLAG-tagged ADAM22v4 wild-type and mutant proteins were immunoblotted and probed with anti-FLAG as control for ADAM22 precursor and mature form protein levels. (C) MaV203 yeast strain containing pDBLeu constructs for ADAM22v1 cytoplasmic domain or ADAM22v4 cytoplasmic domain interacting with a pPC86 clone expressing 14-3-3ß and yeast containing a pDBLeu construct for the ADAM22v4 substitution mutant of the first and second 14-3-3 consensus binding motifs which no longer interacts with pPC86 library clones expressing 14-3-3 protein family members ß, {zeta} and {tau}. Growth on SD/-Leu/-Trp master plates for bait/library plasmid maintenance, SD/-Leu/-Trp/-His/+30 mM3AT for HIS3 reporter expression, SD/-Leu/-Trp/-Ura for URA3 reporter expression; and X-gal assays on nitrocellulose membrane for lacZ reporter expression.





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