Fig 2: PAK1 at K420 acetylation promotes the binding of PAK1 to ATG5 and autophagy initiation in GBM cells upon hypoxia. (A) LN229 and U251 cells were treated with 30 µM anacardic acid (AA) and subjected to hypoxia or normoxia for one day. Then the indicated antibodies were probed to analyze the cell lysates. (B) MS identified the site of acetylation of PAK1 in hypoxia-treated GBM cells. (C) Ni-NTA agarose beads were used to immobilize bacterially purified WT His-PAK1 or His-PAK1K420R proteins, then incubated with HA-ELP3 and Ac-CoA. Then these beads were incubated with purified GST or GST-ATG5 to perform His bead pull-down assay. (D) The endogenous PAK1 in LN229 cells was knocked down. Then these cells were reintroduced with WT Flag-PAK1 or Flag-PAK1K420R mutant and were subjected to hypoxia or not. Then immunoprecipitation analysis with indicated antibodies was performed. (E) The endogenous PAK1 in LN229 cells was knocked down. Then these cells were reintroduced with WT Flag-PAK1, or Flag-PAK1K420R, and were subjected to hypoxia or not. Western blots were performed as indicated. (F) GFP-LC3 was transiently expressed in PAK1-depleted LN229 cells which were reintroduced with WT Flag-PAK1 or Flag-PAK1K420R or Flag Flag-PAK1K420Q. Representative pictures were presented and quantitation of GFP-LC3 puncta from 10 different images was performed. Scale bar: 0.01 mm. (G) The endogenous PAK1 in LN229 cells was knocked down. Then these cells were reintroduced with WT Flag-PAK1 or Flag-PAK1K420Q mutant were subjected to hypoxia or not. Then immunoprecipitation analysis with indicated antibodies was performed. (H) The endogenous PAK1 in LN229 cells was knocked down. Then these cells were reintroduced with WT Flag-PAK1 or Flag-PAK1K420Q and were subjected to hypoxia or not. Western blots were performed as indicated

Fig 3: Bioinformatic analysis in GBM patients reveals an important relationship between PAK1 and autophagy. The cellular component (A) and KEGG pathway (B) for PAK1-positively-associated genes. (C) The annotation indicated that PAK1 was positively associated with several biological processes mainly involved in immune-related processes, protein modification, metabolic processes, cell death, and particularly autophagy. (D) The heatmap of autophagy-related genes (blue-red), pathway, and relevant clinical features

Fig 4: PGE 2 activates MEF2 through PAK2 in NRVMs After serum starvation, NRVMs were pretreated with or without 50 µM NSC23766 for 2 h and stimulated with 1 µM PGE2 for 1 h. Total and phosphorylated PKD levels were analyzed by immunoblot. PKD was activated by PGE2, but it was not affected by NSC.After infection with Flag-HDAC5 and serum starvation, NRVMs were treated with 50 µM NSC23766 for 2 h prior to stimulation. The cells were stimulated with 1 µM PGE2 for 1 h. HDAC5 phosphorylation at Ser-498 was detected by immunoblot. The phosphorylation of HDAC5 at this site was not prevented by NSC.NRVMs and neonatal cardiac fibroblasts (CFs) were serum starved, pretreated with vehicle or 200 nM L798106 (EP3 receptor antagonist) for 20 min, and were stimulated with 1 µM PGE2 for 10 min. PAK2 and PKD phosphorylation were assessed by immunoblot. Calsequestrin was used as a myocyte marker.MEF2 activity was determined in cells infected with the 3xMEF2-Luc reporter after serum starvation. Cells were stimulated with vehicle or 1 µM PGE2 for 24 h after 1-h of 30 µM IPA-3 pretreatment. IPA-3 inhibited the MEF2 activation by PGE2.Data information: In (A–C), representative images from three independent experiments are shown. In (D), values are mean ± s.e.m., n = 3, biological replicates, *P < 0.05 (two-way ANOVA, P < 0.0001). The exact n and P-values can also be found in the Source Data Excel file for Fig 5.Source data are available online for this figure.

Fig 5: Addition of PAK1 inhibitor to KRAS prenylation inhibitors dramatically alters cell morphology and motility. Photomicrographs of standard wound healing assays in H157 and A549 cells (KRAS mutant). Cells were exposed to (i) farnesyltransferase inhibitor (BMS-225975 at 2 µM) and geranylgeranyltransferase inhibitor (P61A6 at 2 µM) [F + G]; (ii) PAK1 inhibitor (IPA-3 at 5 µM); (iii) combination of the three inhibitors or (iv) the inhibitor vehicles for 24-48 hours.