Fig. 5: Monoubiquitylation of PAK1 restricts CDC42 signaling.

a KLHL4 depletion increases CDC42 activity in cells undergoing early stages of neural conversion. Control or KLHL4-depleted CRISPRi iPSCs were subjected to neural conversion for 3d, lysed, and endogenous active (GTP-bound) small GTPases were affinity-purified using GST-PBD (CDC42/RAC1) or GST-RBD (RhoA) followed by immunoblotting with indicated antibodies. GST was used as a specificity control for binding. b Quantification of the experiment depicted in panel (a). Relative small GTPase activity was calculated by dividing the GTP-bound state to the total levels of each small GTPase followed by normalization to control cells. mean of n = 3 biological replicates, error bars = s.d., CDC42: P = 0.0202, RAC1: P = 0.9888, RhoA: P = 0.864, unpaired t test. c Ubiquitylated PAK1 can restore normal levels of CDC42 activity in KLHL4-deficient cells undergoing early stages of neural conversion. Control or KLHL4-depleted CRISPRi iPSCs were reconstituted with dox-inducible ^HAPAK1, ^HAPAK1-Ub, or ^HAPAK1-Ub^I44A, treated with dox and subjected to neural conversion for 3d. GTP-bound CDC42 was affinity-purified and relative CDC42 activity was calculated as described above. mean of n = 3 biological replicates, error bars denote s.d., PAK1: P = 0.4227, PAK1-Ub: P < 0.0001, PAK1-Ub^I44A: P < 0.0001, PAK1-Ub vs. PAK1-Ub^I44A: P = 0.0002, one-way ANOVA. d Model of how CRL3-KLHL4 restricts CDC42 signaling during ectodermal differentiation. PAK1 canonically acts as a CDC42 effector kinase but can be converted into a CDC42 inhibitor. This requires (i) recruitment of PAK1 to CRL3-KLHL4 by the CDC42 GEF GIT1-β-PIX and (ii) monoubiquitylation.