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Fig. 2. Interaction of Shark and Ddok in yeast and S2 cells. (A) Dependence of the interaction between Shark and Ddok on tyrosine phosphorylation. LexA, the DNA-binding domain in the pGilda vector was fused to Shark in pGilda without activated Src (Lex A-Shark), to Shark in pGilda with activated Src (LexA-Shark/Src) or, as a control, to nothing (LexA). AD, the activation domain in the pB42AD vector was fused to either Ddok (AD-Ddok), DdokYF mutations (AD-Ddok Y---F) or nothing (AD). Transformed yeast were plated on replica plates containing X-gal for screening ß-galactosidase-positive blue colonies (upper panel) and Leu^- plates for screening Leu-positive colonies (lower panel). (B) Y427, Y499, Y515 and Y537 are involved in the tyrosine phosphorylation-dependent interaction of Ddok with Shark. The constructs used in A were used to perform the more sensitive ß-galactosidase assays of yeast cell lysates (±s.e.m.; n=3; ^*P<0.05, Student's t-test, significantly different from LexA-Shark/Src+AD-Ddok). (C) Ddok antibody detects a single protein in a western blot of Drosophila embryo extract. (D) Endogenous Shark and Ddok are associated in S2 cells. (E) Dependence of Shark and Ddok association on Ddok tyrosine phosphorylation in co-transfected S2 cells. S2 cells were transfected with pMT vector alone, Myc-Shark alone, Ddok-Flag alone, both Myc-Shark and Ddok-Flag, or Myc-Shark with each of the following Ddok-Flag mutants individually: Ddok-M1 (Ddok Y499F,Y515F), Ddok-M2 (Ddok Y499F,Y515F,Y427F), or Ddok-M3 (Ddok Y499F,Y515F,Y427F,Y537F). Cell lysates were immunoprecipitated with Flag antibody and subjected to SDS-PAGE and western blotting with the indicated antibodies. (F) Inhibition of Ddok tyrosine phosphorylation in S2 cells by the Src inhibitor, PP2. Ddok-Flag or Ddok-Flag+Shark transfected cells were incubated in medium containing 10 µm PP2 or solvent (DMSO) for 1 hour prior to the immunoprecipitation of cell lysates with Flag antibody. (G) Ddok and Shark are colocalized at the cell cortex. S2 cells were transfected with Myc-Shark alone (a,b), Ddok-Flag alone (c,d), or co-transfected with Myc-Shark and Ddok-Flag (e-h), immunofluorescently stained for Myc (red; b,g,h) and Flag (green; d,f,h), and examined by confocal microscopy. The merged panel (h) shows the colocalization (yellow).

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