Extended Data Fig. 5: H1149P patient mutation impairs TIAM1 activity and PLCE1 patient mutation E623Q leads to diminished GTP-bound RhoA.

a, H1149P mutation is located within the Dbl homology (DH) domain responsible for GEF activity. TIAM1 contains an N-terminal pleckstrin homology (PH), coiled-coiled (CC), extension (Ex), RAS binding (RBD), PDZ, Dbl-homology (DH) and PH domains, with the patient mutation falling within the DH domain. b, Schematic of PLCE1 protein with domains annotated. Patient E623Q mutation is located in the Ras GEF domain. PLCE1 contains a Guanine nucleotide exchange factor for Ras-like small GTPases (RAS GEF), Pleckstrin Homology (PH), Phospholipase C catalytic domain X (PLCX), Phospholipase C catalytic domain Y (PLCY), Protein Kinase C conserved region 2 (C2), RAS association domain 1 (RA1), and RAS association domain 2 (RA2). c, Construct expression H1149P (n = 76) is equivalent to wildtype (n = 85) in Phalloidin quantification. d, Construct expression H1149P in constitutive active (C.A.) (n = 53). Src Rac1 Förster resonance energy transfer (FRET) is equivalent to wildtype. P value adjusted with Bonferroni. Kruskal-Wallis followed by a two-sided pairwise Wilcoxon test, P value adjusted with Bonferroni. Data shown with Hampel filter. Error bar: standard error of the mean. P values: ns: not significant. e, Active GTP-bound form of RhoA precipitated from HEK293 expressing Myc-tagged PLCE1 using a GST-rhotekin pulldown assay. Overexpression of WT PLCE1 resulted in a substantial decrease in relative RhoA activity compared with mock cells. Compared to WT, cells transfected with variant forms of PLCE1 exhibited marked differences in GTP-bound RhoA.