Structural analysis of the PTEN:P-Rex2 signalling node reveals how cancer-associated mutations coordinate to hyperactivate Rac1

Abstract

The PTEN:P-Rex2 complex is one of the most commonly mutated signaling nodes in metastatic cancer. Assembly of the PTEN:P-Rex2 complex inhibits the activity of both proteins, and its dysregulation can drive PI3K-AKT signaling and cell proliferation. Here, using extensive crosslinking mass spectrometry and functional studies, we provide crucial mechanistic insights into PTEN:P-Rex2 complex assembly and co-inhibition. PTEN is anchored to P-Rex2 by interactions between the PTEN PDZ-BM tail and the second PDZ domain of P-Rex2. This interaction bridges PTEN across the P-Rex2 surface, occluding PTEN membrane-binding and PI(3,4,5)P3 hydrolysis. Conversely, PTEN both allosterically promotes an autoinhibited P-Rex2 conformation and occludes Gβγ binding and GPCR activation. These insights allow us to define a new gain-of-function class of cancer mutations within the PTEN:P-Rex2 interface that uncouples PTEN inhibition of Rac1 signaling. These findings provide a mechanistic framework to understand the dysregulation of the PTEN:P-Rex2 signaling node in metastatic cancer.3 PTEN:P-Rex2 complex assembly occurs independently of both PTEN phosphatase activity 13 and P-Rex2 GEF activity 14 . However, the flexibility of both PTEN and P-Rex2 has limited high-resolution insights into the assembly of the complex. Domain deletion studies suggest that the P-Rex2 DH-PH domains are necessary to inhibit PTEN activity, mediated by an interaction between the P-Rex2 PH domain and the PTEN DUSP and C2 domains 14 . A second binding site between the PTEN PDZ-BM and the P-Rex2 IP4P domain was sufficient to suppress P-Rex2 GEF-mediated breast cancer cell invasion 4 . However, studies diverge on the requirement for the PDZ-BM for complex assembly 3 . P-Rex2 is mutated frequently in cancer. In melanoma, the incidence of P-Rex2 mutations has been reported as high as 27% within the TCGA cohort and 14% in a separate cohort of 107 melanomas 15-17 . High-incidence of P-Rex2 mutations has also been reported in pancreatic, breast, and lung cancers 18-20 . More broadly, P-Rex2 is the fourth most commonly mutated oncogene in the MET500 cohort of metastatic cancer, underlining a potential role as a metastatic driver 21 . Strikingly, within the same MET500 cohort, PTEN is the third most commonly mutated tumour-suppressor protein 21 . This combined incidence highlights the PTEN:P-Rex2 complex as one of the most commonly mutated signaling nodes in metastatic cancer.PTEN cancer-associated mutations are generally clustered to the catalytic domain (e.g. C124S) and frequently result in PTEN suppression and enhanced PI3K-AKT signaling 5 . P-Rex2 cancerassociated somatic mutations are not clustered in hot-spots, but range throughout the primary sequence 15,19,21 . As we know little about the structure of P-Rex2 or its interaction with PTEN, it has proven difficult to predict the importance of individual mutations to the dysregulation of PI3K-AKT and RhoGTPase signaling. For example, melanoma-associated missense mutations have proven challenging to predict in terms of f...