Fig 1: Schematic illustration of PAK group I structure. The numbers within the individual PAK1?15 and PAK2 domains indicate the sequence homology with the corresponding domains of the full-length PAK1. The dimerization sequence (dimer, AA 79–86) is identical for all three isoforms. The figure depicts the position of some important elements: the autoinhibitory domain (AID), the kinase domain (kinase), the autophosphorylation site S144/S141, the phosphorylation site T212, the cleavage site for caspase-3 (casp3) and the myristoylation region (myr). The C-terminal domains of PAK1?15 and PAK2 are similar to each other (79% homology), but different from that of PAK1-full. The scheme was created by the manuscript authors (KK).
Fig 2: Band positions for the exogenous PAK1 forms. HEK293T cells were transfected with plasmids coding for PAK1-full or PAK1?15, incubated for 48 h and lysed. The proteins were resolved on 18 × 18 cm gels and blotted to nitrocellulose membranes, which were incubated with different antibodies as indicated. Lanes: 1 – untransfected cells, 2 – exogenous PAK1-full, 3 – exogenous PAK1?15.
Fig 3: (a) Effect of siRNA PAK1/PAK2 on western-blot band intensity (antibody specificity control). HEK293T cells were transfected with siRNA targeting PAK1 or PAK2 and incubated for 48 h. The cell lysates were resolved on 18 × 18 cm gels and PAK expression was detected using different antibodies as indicated. Lanes: 1 – untransfected control, 2 – siRNA PAK1, 3 – siRNA PAK2. The band intensities for siRNA-treated samples were corrected using the loading controls (actin) and given as relative to the corresponding untransfected controls. (b) Direct comparison of signals from total PAK (PAK1 + PAK2, left) and pPAK Ser144/141 (right) antibodies. HEK293T cell lysate was resolved on a large gel, the proteins were transfered to a nitrocellulose membrane. The membrane was vertically cut, the individual parts were incubated with the indicated primary antibodies, then with the corresponding secondary antibody. The membrane was reassembled, covered with the chemiluminiscence substrate and the signal was recorded from both parts at once.
Fig 4: Model of the Intersection among Receptor-Mediated Endocytosis of Wnt, Macropinocytosis, and LysosomesDuring Wnt signaling, micropinocytosis of the LRP6/Fz receptor results in the sequestration of GSK3 (blue) inside the intraluminal vesicles of MVBs. As cytosolic levels of GSK3 decrease, Pak1 induces the submembraneous actin machinery to form macropinocytic cups that close and engulf macromolecules (red) from the surrounding medium. The axin/GSK3 complex represses macropinocytosis. When Axin1 is mutated, GSK3 is unable to repress the actin machinery, resulting in a prodigious amount of nutrient uptake by macropinocytosis. When GSK3 is inhibited pharmacologically or with DN-GSK3, macropinocytosis is stimulated to a similar degree than that caused by Wnt3a ligand treatment. The results presented in this study point to a hitherto-unappreciated role for GSK3 and axin in the suppression of macropinocytosis in basal conditions.
Fig 5: Effect of alkaline phosphatase treatment on western-blot bands of PAK. Lysates from HEK293T cells, control or transfected with plasmids coding for PAK1-full or PAK1?15, were incubated overnight with alkaline phosphatase (AP). The proteins were resolved on 18 × 18 cm gels and blotted to nitrocellulose membranes, which were incubated with different antibodies as indicated. The total protein load was five times lower in the lanes containing transfected cells than in those containing the control cells. The numbers indicate the band intensities in AP-treated samples as relative values compared to the corresponding untreated samples. In case of multiple bands, the signal from the sample was evaluated as a whole.
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