Supplemental Material

This article contains the following supporting material:

• Figure 1 - Dvl-phosphorylation by PAR1b-immunoprecipitates depends on the kinase activity of PAR1b
  Protein A-tagged wildtype PAR1b (WT-PAR1b, upper panels,A) and kinase-dead PAR1b (PAR1b-K49A, lower panels,A) were isolated on IgG-sepharose from whole cell lysates and utilized for in vitro kinase assays with soluble Dvl-peptides as substrates as described in Materials and Methods (see also Figure 1B). The Dvl-mutants with point-mutations were incubated at a tenfold higher concentration than the Dvl-peptide with the wildtype sequence. Similar amounts of substrate (see Commassie stained gels in A) and kinase (see immunoblot in B) were utilized for the assays with wildtype and mutant PAR1b. PAR1b-K49A yielded only 10-15% of the γ³²P-ATP-incorporation mediated by wildtype PAR1b (compare autoradiographs in A).

• Figure 2 - A Par1b-binding peptide of Dvl does not disrupt polarization

  A) MDCK polarization
  Control MDCK cells transiently transfected with either vector DNA or cDNA encoding the Par1b-interacting Dvl-peptide Dvl(aa200-250) were subjected to cell-cell-adhesion assays (upper panels) collagen overlay (middle panels) or Ca-switch assays (lower panels). Cells were labeled for the indicated markers. Presented are 4x wide-field images in the upper panel, confocal x-z views in the two middle panels and a confocal x-y section taken through the center of the cells in the MT (lower)-panels. Note, the cross-section through vertical MTs in the control cells that are absent in the Dvl-samples.
  Table: Results from a Hanging Drop assay as described in Material and methods and in Fig.1 that is representative of 3 independent experiments. Scale bars= 300μm (cell-cell adhesion panels), 10μm (all other panels).
  B)Dvl-peptide expression
  Cells expressing either Flag-tagged Dvl(aa200-250) (left) or Flag-tagged full length Dvl (right) were analyzed with a Flag antibody by indirect immunofluorescence

• Figure 3 - Par1b and Dvl do not regulate the total Tx100-resistant actin pool
  Total SDS-lysates (#1,2) or SDS-lysates of the Tx100-resistant cell fraction (#3,4) were prepared from monolayers treated as described in Fig. 5 and probed in quantitative immunoblots for actin. #3,4 contain twice as much lysate as #1,2. The Graph represents 2 independent experiments with standard error.