Fig 2: Peptide spot analysis of putative target sites in PAK4-proximal proteins.a To assess the effect of single amino-acid substitution on a selected optimal PAK4 substrate, we tested 13aa synthetic peptides (Pepspots, Jerini) derived from PAK4 pseudosubstrate motif (SARRPKPLVDPAD) in which the proline in bold is replaced by Ser(0). This is similar to an optimal substrate for PAKs (RKRRNSLAYKK) termed PAKtide145 but optimal for kinase binding. Based on structural considerations the Arg side chain at position -2 or -3 occupies a pocket that mediates interactions found in PAKs and other S/T kinases, including PKA146. The contribution of each side-chain to peptide phosphorylation was assessed by sequential alanine substitution. b We selected in vivo basic-directed phosphorylation sites identified in Afadin, scribble, ZO-1, DLG5 and p120ctn as compiled in the Phosphosite database (V6.5.9.3). The corresponding synthetic peptides were synthesized and subjected to in situ phosphorylation. The extent of phosphorylation (32P signal) ranges from detectable (-/+) to very strong (+++), with no signal shown as ns. c Schematic of the domain structure and relative positions of the p120-catenin phosphorylation sites as indicated in the table. d Western blot showing the inhibition of p120ctn Ser320 phosphorylation by PF-3758309 U2OS cells113.

Fig 3: p120 isoform localization in the skin. (a) Histological section of epidermis, containing a hair follicle and sebaceous glands. (b) p120-1 (6H11 mAb) and p120-3 expression (anti-p120-3 pAb) in the epidermis, the root sheath of a hair follicle and a sebaceous gland. Panels on the right represent a magnification of the area in the dashed boxes. Open and filled arrowheads indicate p120-1-positive cells in the basal layer (filled) and intermediate layers (open).

Fig 4: P120 isoform association with adherens junctions in 4T1 cells. (A) Membrane–cytosol fractionation. (B) Co-immunoprecipitation with isoform-specific antibodies for p120-1 (6H11 mAb) and p120-3 (anti-p120-3 pAb). IP antibodies are indicated above the blots (* p < 0.05). (C) Cytosol extraction. All membranes were incubated with the antibodies indicated adjacent to the blots. In the blots stained for p120, the upper band represents p120-1 and the lower band represents p120-3. Quantifications are shown in bar graphs below the blots.

Fig 5: Disposition of PAK4, Afadin and ZO1 in MDCK cells.a Confluent MDCK cells grown on glass coverslips (4 days) were fixed with methanol and co-stained using rabbit anti-PAK4 and mouse anti-Afadin/anti-ß-catenin or anti-ZO1. Images were collected on an Olympus Fluoview confocal microscope with ×100 oil objective. Lower panels show MDCK cell grown in Matrigel to form 3D-cultured acini (8 days) fixed in methanol and immuno-stained with anti-PAK4, anti-p120ctn (×60 objective). b Sub-confluent (2 day) cells were stained using rabbit antibodies specific for PAK4 and mouse antibody specific for p120 (×60 objective). Repeated in three independent experiments. Scale bars: 10 µm.