Abstract
The small GTPase RAB35 positively regulates PI3K signaling.
Major finding: The small GTPase RAB35 positively regulates PI3K signaling.
Mechanism: Mutant RAB35 drives PDGFR-mediated activation of PI3K–AKT signaling by regulating PDGFR localization.
Impact: Somatic RAB35 mutations may promote tumorigenesis via dysregulated endosomal trafficking.
Aberrant activation of the intracellular PI3K–AKT–mTOR signaling pathway, which regulates critical processes such as cell cycle and survival, is one of the most common occurrences in human cancers and has been the focus of targeted therapy development. However, inhibitors targeting PI3K, AKT, or mTOR have shown limited clinical benefit. To identify new regulators of the PI3K–AKT pathway, Wheeler and colleagues screened 7,450 shRNAs for kinases or GTPases that affect AKT phosphorylation at serine 473 (S473), and identified 29 genes that had not been previously implicated in PI3K–AKT signaling, as well as genes known to regulate AKT phosphorylation. Of the 29 genes, the most-represented functional group was the RAB GTPases, which regulate endomembrane trafficking. Knockdown of RAB35, one of the five RAB GTPases identified in the screen, in multiple cell lines resulted in decreased AKT phosphorylation at S473 as well as diminished phosphorylation of phosphoinositide-dependent kinase 1 (PDK1) and PI3K-dependent phosphorylation of FOXO1/3A, suggesting that RAB35 is a critical regulator of PI3K–AKT signaling that acts upstream of PDK1 and mTORC2. Consistent with these findings, RAB35 functioned downstream of growth factor receptors and interacted with PI3K in a nucleotide-dependent manner. Furthermore, expression of two known cancer-related RAB35 missense mutations, RAB35A151T or RAB35F161L, resulted in increased AKT phosphorylation, enhanced cell viability, resistance to apoptosis under growth-factor deprivation, and PI3K-dependent transformation, indicative of a gain-of-function effect. Cells expressing the constitutively active mutant RAB35Q67L exhibited increased phosphorylation of platelet-derived growth factor receptor α/β (PDGFRα/β), AKT, and FOXO1/3A, which was abrogated by treatment with a PDGFR inhibitor. In addition, expression of RAB35Q67L induced PDGFRα trafficking through lysosomal-associated membrane protein 2 (LAMP2)–positive endomembranes independent of ligand stimulation, suggesting that dysregulated endosomal trafficking may drive AKT activation. Together, these results characterize the role of RAB35 in regulating PI3K–AKT signaling and the potential role of gain-of-function RAB35 missense mutations in cancer.