Phosphatidylinositol 4-phosphate at the Golgi complex: novel roles in regulating plasma membrane lipid composition and organization. - ABSTRACT KRAS proteins are membrane-associated GTPases that regulate cell proliferation, differentiation, and apoptosis by cycling between an active, GTP-bound state and an inactive, GDP-bound state. Serving as a key component of growth factor receptor signaling pathways, KRAS must localize to the plasma membrane (PM) and assemble into transient, lipid-based platforms known as nanoclusters. These nanoclusters facilitate the recruitment and activation of the RAF/MAPK pathway, ensuring precise and efficient signal transmission. KRAS is anchored to the PM via a C-terminal membrane domain consisting of a farnesyl-cysteine-methyl- ester and a polybasic region with six consecutive Lys residues. This membrane anchor exhibits a highly specific binding affinity for phosphatidylserine (PS) species containing one saturated and one unsaturated acyl chain. Since KRAS PM localization and nanoclustering are intrinsically linked to PM levels of mixed-chain PS, any reduction in PS availability, whether due to the loss of ORP5 or ORP8, the PS transfer proteins localized at ER- PM membrane contact sites (MCSs), or by lowering PM levels of phosphatidylinositol 4-phosphate (PI4P), which drives PS transport, results in KRAS PM mislocalization, thereby disrupting signal transmission and oncogenic activity. We conducted high-content imaging-based screening of a human genomic siRNA library and identified 157 candidates that regulate KRAS PM localization, including 32 genes affecting PM PS distribution. One is PI4- kinase type IIIb (PI4KB), which converts PI to PI4P primarily at the Golgi complex. We previously reported that loss or inhibition of PI4KB transports PM PS to mitochondria, resulting in KRAS mislocalization to mitochondria and perturbed KRAS PM nanoclustering. Also, cellular glucose starvation disrupts PI4KB Golgi localization and its activity, leading to the transport of PM PS and KRAS to mitochondria. Together, these findings suggest the premise that KRAS PM binding, spatial organization and function rely on Golgi PI4P content. This leads to our core hypothesis: Golgi PI4P plays a crucial role in regulating lipid composition and organization of the inner PM leaflet. In this grant, we will investigate the mechanisms by which Golgi PI4P controls PM lipid composition and organization, using KRAS membrane interactions and signaling as a model system. In Specific Aim 1, we will explore the extent to which Golgi PI4P operates as a spatial organizer of the inner PM and ER- PM MCSs. Specific Aim 2 will determine how glucose maintains Golgi PI4P levels by regulating protein glycosylation. This proposal will provide new mechanistic insights into how Golgi PI4P regulates the composition and organization of the PM, while linking glucose metabolism to Golgi PI4P regulation. Understanding these molecular mechanisms may reveal a novel vulnerability in KRAS-driven oncogenesis with potential therapeutic implications.
