Figure 8

PP2A associates directly with Dvl2 and dephosphorylates Dvl2. Panel A, probing interaction of Dvl2 and PP2A in vivo. Whole-cell extracts (2 mg) prepared from F9 cells expressing Rfz1 were incubated with immobilized GST gel alone or with GST-PP2A C-subunit-immobilized gel. Bound proteins were released and resolved by SDS-PAGE. The resolved proteins were subjected to blotting and stained with either with anti-PP2A C subunit antibodies (top panel) or anti-Dvl2 antibodies (bottom panel). Panel B, pull-downs of Dvl2 from F9 cells reveal associated PP2A. F9 cells lysates were subjected to analysis by pull-downs of either Dvl2 or of mouse IgG (as a control). The immune precipitates were subjected to SDS-PAGE, immunoblotting, and staining with either anti-Dvl2 antibodies (top panel) or anti-PP2A C-subunit antibodies (bottom panel). Panel C, Dvl2 domains structure. Panel D, direct association of Dvl2 and PP2A in vitro. Purified mouse PP2A enzyme (PP2A, AC subunit dimers) was incubated with one of four domains of Dvl2 engineered as a fusion protein with GST and then immobilized: immobilized GST-PDZ, GST-DIX, GST-DEP, GST-SH3 and GST itself (as a control) at 4°C for 1 hr. The bound proteins were separated by SDS-PAGE, blotted, and stained with either anti-PP2A C-subunit antibodies or anti-GST antibodies. Panel E, PP2A dephosphorylates phospho-rDvl2. 6-Histidinyl-tagged phosphorylated Dvl2 was expressed in Sf9 cell and purified by Ni-NTA column chromatography as described in Methods. Purified phospho-rDvl2 protein was incubated with either purified calf alkaline phosphatase or with purified PP2A for 1.5 hr. The incubation was terminated by addition of SDS-PAGE sample buffer. The samples were subjected to SDS-PAGE, blotted, and stained with either anti-phospho-Ser antibodies (top of panel) or with anti-Dvl2 antibodies (bottom of panel). The immune complexes were made visible by use of an alkaline phosphatase-conjugated, second antibody and BCIP/NBT as substrates. The results shown are from a single experiment, duplicated with essentially similar results.