Source was yeast cell line; bait produced as transgenic fusion protein; prey produced as transgenic fusion protein (prey from unspecified library).

Two-hybrid system: yeast GAL4-BD/GAL4-AD

Co-expression of ksr and Dsor1 increase cnk-ave interaction with cnk aa 2-549 and 2-384 constructs, but not aa 2-209 which lacks the PDZ domain.

Source was cell extract of S2 cell line; bait produced from transfected construct; prey produced from transfected construct.

Purified ave-cnk heterodimer was mixed with an equimolar ratio of ksr-Dsor1 heterodimer, which eluted as a higher order complex captured as series of binary interactions that do not necessarily indicate direct protein-protein contact. Mutations in cnk or ksr interfere with the higher order cnk-ave-ksr-Dsor1 complex.

Interaction in vitro; proteins produced as a recombinant fusion proteins in bacterial cell system.

cryoEM structure of ave-cnk-ksr-Dsor1 complex captured as a series of binary interactions that are not all direct. cryoEM indicates direct interaction between ave-cnk, ksr-Dsor1, cnk-ksr and cnk-Dsor1.

Interaction in vitro; proteins produced as recombinant fusion proteins in bacterial system.

Co-expression with Ras85D, Raf, Dsor1 and ksr ameliorates cnk SAM and PDZ mutations that decrease or disrupt cnk-ave interaction. Individual mutations in aa 81 and 283 to disrupt a salt interaction within cnk reduce interaction with ave, while mutation of both, restore the interaction to wild type levels. Disruption of cnk-ave interaction with cnk N82E, N82W or Q84E mutations only occur with co-expression of ksr and Dsor1.

Source was cell extract of S2 cell line; bait produced from transfected construct; prey produced from transfected construct.