KRas is a major proto-oncogene product whose signaling activity depends on its level of enrichment on the plasma membrane (PM). This PM localization relies on posttranslational prenylation for membrane affinity, while PM specificity has been attributed to electrostatic interactions between negatively charged phospholipids in the PM and basic amino-acids in the C terminus of KRas. By measuring kinetic parameters of KRas dynamics in living cells with a cellular-automata-based data-fitting approach in realistic cell-geometries, we show that charge-based specificity is not sufficient to generate PM enrichment in light of the total surface area of endomembranes. Instead, mislocalized KRas is continuously sequestered from endomembranes by cytosolic PDEδ to be unloaded in an Arl2-dependent manner to perinuclear membranes. Electrostatic interactions then trap KRas at the recycling endosome (RE), from where vesicular transport restores enrichment on the PM. This energy driven reaction-diffusion cycle explains how small molecule targeting of PDEδ affects the spatial organization of KRas.

SummaryThe proto-oncogenic epidermal growth factor receptor (EGFR) is a tyrosine kinase whose sensitivity to growth factors and signal duration determines cellular behavior. We resolve how EGFR's response to epidermal growth factor (EGF) originates from dynamically established recursive interactions with spatially organized protein tyrosine phosphatases (PTPs). Reciprocal genetic PTP perturbations enabled identification of receptor-like PTPRG/J at the plasma membrane and ER-associated PTPN2 as the major EGFR dephosphorylating activities. Imaging spatial-temporal PTP reactivity revealed that vesicular trafficking establishes a spatially distributed negative feedback with PTPN2 that determines signal duration. On the other hand, single-cell dose-response analysis uncovered a reactive oxygen species-mediated toggle switch between autocatalytically activated monomeric EGFR and the tumor suppressor PTPRG that governs EGFR's sensitivity to EGF. Vesicular recycling of monomeric EGFR unifies the interactions with these PTPs on distinct membrane systems, dynamically generating a network architecture that can sense and respond to time-varying growth factor signals.

The localization and signaling of S-palmitoylated peripheral membrane proteins is sustained by an acylation cycle in which acyl protein thioesterases (APTs) depalmitoylate mislocalized palmitoylated proteins on endomembranes. However, the APTs are themselves reversibly S-palmitoylated, which localizes thioesterase activity to the site of the antagonistc palmitoylation activity on the Golgi. Here, we resolve this conundrum by showing that palmitoylation of APTs is labile due to autodepalmitoylation, creating two interconverting thioesterase pools: palmitoylated APT on the Golgi and depalmitoylated APT in the cytoplasm, with distinct functionality. By imaging APT-substrate catalytic intermediates, we show that it is the depalmitoylated soluble APT pool that depalmitoylates substrates on all membranes in the cell, thereby establishing its function as release factor of mislocalized palmitoylated proteins in the acylation cycle. The autodepalmitoylating activity on the Golgi constitutes a homeostatic regulation mechanism of APT levels at the Golgi that ensures robust partitioning of APT substrates between the plasma membrane and the Golgi.

The peripheral membrane proto-oncogene Src family protein tyrosine kinases relay growth factor signals to the cytoplasm of mammalian cells. We unravel the spatial cycles of solubilisation, trapping on perinuclear membrane compartments and vesicular transport that counter entropic equilibration to endomembranes for maintaining the enrichment and activity of Src family protein tyrosine kinases at the plasma membrane. The solubilising factor UNC119 sequesters myristoylated Src family protein tyrosine kinases from the cytoplasm, enhancing their diffusion to effectively release Src family protein tyrosine kinases on the recycling endosome by localised Arl2/3 activity. Src is then trapped on the recycling endosome via electrostatic interactions, whereas Fyn is quickly released to be kinetically trapped on the Golgi by palmitoyl acyl-transferase activity. Vesicular trafficking from these compartments restores enrichment of the Src family protein tyrosine kinases to the plasma membrane. Interference with these spatial cycles by UNC119 knockdown disrupts Src family protein tyrosine kinase localisation and signalling activity, indicating that UNC119 could be a drug target to affect oncogenic Src family protein tyrosine kinase signalling.

19Growth factors initiate anabolism by activating mechanistic target of rapamycin complex 1 20 (mTORC1) via the small GTPase Rheb. We show that the GTPase cycle of Rheb is spatially 21 regulated by the interaction with its GDI-like solubilizing factor (GSF) -PDEδ. Arl2-GTP 22 mediated localized release of cytosolic Rheb-GTP from PDEδ deposits it onto perinuclear 23 membranes where it forms a complex with mTORC1. The membrane associated GTPase 24 activating protein (GAP) TSC2 hydrolyzes Rheb-GTP, weakening the interaction with mTOR. 25Rheb-GDP is readily released into the cytosol where it is maintained soluble by interaction 26 with PDEδ. This solubilized Rheb is re-activated by nucleotide exchange to be re-deposited 27 by Arl2-mediated release onto perinuclear membranes. This spatial GTPase cycle thereby 28 enables mTORC1 activation to be solely controlled by growth factor induced inactivation of 29 TSC2. The coupling between mTOR activation and spatially regulated Rheb nucleotide 30 exchange makes growth factor induced proliferation critically dependent on PDEδ 31 expression. 32 33 102 6 different time points after insulin stimulation of serum-starved TSC2+/+ MEFs also displayed 103 a transient recruitment of endogenous Rheb to mTOR containing membranes (Fig. 1d). 104 Enrichment of endogenous Rheb on mTOR-rich membranes was slightly delayed compared 105 to ectopically expressed mCitrine-Rheb, (30 min in the IF versus 12 min in the live-cell time 106 course), reflecting a shift in the reaction rate upon increasing the concentration of a reactant 107 according to the law of mass action. This transient increase in Rheb-enrichment on mTOR 108 containing membrane indicates that the interchange between the soluble and membrane-109 bound fraction of Rheb is dynamic, likely mediated and maintained by the solubilizing factor 110 PDEδ. 111 Arl2-mediated localized release from PDEδ deposits Rheb on perinuclear membranes 112 We examined how the interaction of Rheb with PDEδ influences its cytosol-membrane 113 partitioning by inhibiting PDEδ function using the small-molecule inhibitor Deltarasin that 114 targets PDEδ's prenyl-binding pocket (29). For this, we monitored the localization of Rheb, 115 N-terminally tagged with mCherry (mCherry-Rheb) as well as the hypervariable region of 116 Rheb, N-terminally tagged with mCitrine (mCitrine-Rheb HVR) in TSC2+/+ MEFs after 117 treatment with 3 µM Deltarasin. This resulted in a gradual loss of the perinuclear enrichment 118 for both proteins, demonstrating that the interaction of PDEδ via the farnesyl group in the 119 HVR is necessary for generating the perinuclear concentration of Rheb (Fig. 2a). We also 120 directly monitored the effect of Deltarasin on the interaction between Rheb and PDEδ by 121 fluorescence lifetime imaging microscopy of Förster resonance energy transfer (FLIM-FRET), 122 with mCitrine-Rheb as the donor and PDEδ, C-terminally tagged with mCherry (mCherry-PDEδ) as FRET acceptor (29, 32, 33) (Fig. 2b). The interaction between mCitrine-Rheb and 124 mCherry-PDEδ w...