Fig 2: Simplified schemes showing the organization of the junctional complexes of polarized epithelial cells in vertebrates.The sub-apical complex includes a structure recently described126 as well the tight junction (TJ) and region ‘below’ this typically described adherens junction (AJ), which includes both Afadin and cadherin complexes. The smaller cadherin punctate junctions along the lateral contacts are not explicitly indicated. Typical non-transmembrane components (for example, p120ctn and ß-catenin) are often used as markers and these are indicative of well-studied components. The Afadin/nectin compartment in vertebrates is often spatially segregated from cadherin as described in the text.

Fig 3: 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.