The first step of RAF activation involves binding to active RAS, resulting in the recruitment of RAF to the plasma membrane. To understand the molecular details of RAS-RAF interaction, we present crystal structures of wild-type and oncogenic mutants of KRAS complexed with the RAS-binding domain (RBD) and the membrane-interacting cysteine-rich domain (CRD) from the N-terminal regulatory region of RAF1. Our structures reveal that RBD and CRD interact with each other to form one structural entity in which both RBD and CRD interact extensively with KRAS. Mutations at the KRAS-CRD interface result in a significant reduction in RAF1 activation despite only a modest decrease in binding affinity. Combining our structures and published data, we provide a model of RAS-RAF complexation at the membrane, and molecular insights into RAS-RAF interaction during the process of RAS-mediated RAF activation.

Allele-specific signaling by different KRAS alleles remains poorly understood. The KRAS^G12R mutation displays uneven prevalence among cancers that harbor the highest occurrence of KRAS mutations: It is rare (∼1%) in lung and colorectal cancers, yet relatively common (∼20%) in pancreatic ductal adenocarcinoma (PDAC), suggesting context-specific properties. We evaluated whether KRAS^G12R is functionally distinct from the more common KRAS^G12D- or KRAS^G12V-mutant proteins (KRAS^G12D/V). We found that KRAS^G12D/V but not KRAS^G12R drives macropinocytosis and that MYC is essential for macropinocytosis in KRAS^G12D/V- but not KRAS^G12R-mutant PDAC. Surprisingly, we found that KRAS^G12R is defective for interaction with a key effector, p110α PI3K (PI3Kα), due to structural perturbations in switch II. Instead, upregulated KRAS-independent PI3Kγ activity was able to support macropinocytosis in KRAS^G12R-mutant PDAC. Finally, we determined that KRAS^G12R-mutant PDAC displayed a distinct drug sensitivity profile compared with KRAS^G12D-mutant PDAC but is still responsive to the combined inhibition of ERK and autophagy. Significance: We determined that KRAS^G12R is impaired in activating a key effector, p110α PI3K. As such, KRAS^G12R is impaired in driving macropinocytosis. However, overexpression of PI3Kγ in PDAC compensates for this deficiency, providing one basis for the prevalence of this otherwise rare KRAS mutant in pancreatic cancer but not other cancers. See related commentary by Falcomatà et al., p. 23. This article is highlighted in the In This Issue feature, p. 1

A human lipid transfer protein (GLTPD1, named here CPTP) is shown to regulate eicosanoid production by mediating the intermembrane transfer of the phosphorylated sphingolipid ceramide-1-phosphate through a non-vesicular transport mechanism elucidated by structural, functional and biological data. The bioactive signalling lipid ceramide-1-phosphate (C1P) regulates diverse processes ranging from growth and survival to pro-inflammatory responses. In this study, Dinshaw Patel and colleagues address how C1P is transported to specific sites in the cell. A novel lipid transfer protein called ceramide-1-phosphate transfer protein (CPTP) was identified, and structural and functional studies reveal the mechanism by which C1P is transported from its site of synthesis in the Golgi complex to the plasma membrane. Phosphorylated sphingolipids ceramide-1-phosphate (C1P) and sphingosine-1-phosphate (S1P) have emerged as key regulators of cell growth, survival, migration and inflammation1,2,3,4,5. C1P produced by ceramide kinase is an activator of group IVA cytosolic phospholipase A2α (cPLA2α), the rate-limiting releaser of arachidonic acid used for pro-inflammatory eicosanoid production3,6,7,8,9, which contributes to disease pathogenesis in asthma or airway hyper-responsiveness, cancer, atherosclerosis and thrombosis. To modulate eicosanoid action and avoid the damaging effects of chronic inflammation, cells require efficient targeting, trafficking and presentation of C1P to specific cellular sites. Vesicular trafficking is likely10 but non-vesicular mechanisms for C1P sensing, transfer and presentation remain unexplored11,12. Moreover, the molecular basis for selective recognition and binding among signalling lipids with phosphate headgroups, namely C1P, phosphatidic acid or their lyso-derivatives, remains unclear. Here, a ubiquitously expressed lipid transfer protein, human GLTPD1, named here CPTP, is shown to specifically transfer C1P between membranes. Crystal structures establish C1P binding through a novel surface-localized, phosphate headgroup recognition centre connected to an interior hydrophobic pocket that adaptively expands to ensheath differing-length lipid chains using a cleft-like gating mechanism. The two-layer, α-helically-dominated ‘sandwich’ topology identifies CPTP as the prototype for a new glycolipid transfer protein fold13 subfamily. CPTP resides in the cell cytosol but associates with the trans-Golgi network, nucleus and plasma membrane. RNA interference-induced CPTP depletion elevates C1P steady-state levels and alters Golgi cisternae stack morphology. The resulting C1P decrease in plasma membranes and increase in the Golgi complex stimulates cPLA2α release of arachidonic acid, triggering pro-inflammatory eicosanoid generation.