Myotubularin-related proteins regulate KRAS function by controlling plasma membrane levels of polyphosphoinositides and phosphatidylserine

KRAS is a small GTPase, ubiquitously expressed in mammalian cells, that functions as a molecular switch to regulate cell proliferation and differentiation. Oncogenic mutations that render KRAS constitutively active occur frequently in human cancers. KRAS must localize to the plasma membrane (PM) for biological activity. KRAS PM binding is mediated by interactions of the KRAS membrane anchor with phosphatidylserine (PtdSer), therefore, depleting PM PtdSer content abrogates KRAS PM binding and oncogenic function. From a genome-wide siRNA screen to search for genes that regulate KRAS PM localization, we identified a set of phosphatidylinositol (PI) 3-phosphatase family members: myotubularin-related (MTMR) proteins 2, 3, 4 and 7. Here we show that knockdown of MTMR 2/3/4/7 expression disrupts KRAS PM interactions. The molecular mechanism involves depletion of PM PI 4-phosphate (PI4P) levels, which in turn disrupts the subcellular localization and operation of oxysterol-binding protein related protein (ORP) 5, a PtdSer lipid transfer protein that maintains PM PtdSer content. Concomitantly, silencing MTMR 2/3/4/7 expression elevates PM levels of PI3P and reduces PM and total cellular levels of PtdSer. In summary we propose that the PI 3-phosphatase activity provided by MTMR proteins is required to generate PM PI for the synthesis of PM PI4P, which in turn, promotes the PM localization of PtdSer and KRAS.


Introduction
H-, N-and KRAS proteins are small GTPases that oscillate between inactive GDP-bound and active GTP-bound conformational states and operate in signaling cascades that control cell growth and proliferation (Gorfe & Cho, 2019;Henkels et al, 2021).Consistent with this key regulatory role, activating mutations of RAS are present in ~20% of human cancers, with the majority occurring in the KRAS isoform (Prior et al, 2020).All RAS isoforms must be localized to the inner leaflet of the plasma membrane (PM) and be spatially organized into nanoclusters for biological activity (Gorfe & Cho, 2019;Henkels et al., 2021).Membrane interactions are mediated by the RAS C-terminal membrane anchor (Zhou et al, 2017).In the case of KRAS4B, the major expressed splice variant of KRAS (Hood et al, 2023) (hereafter KRAS), this anchor comprises a C-terminal farnesyl cysteine methyl ester, generated by posttranslational modification, and an adjacent polybasic domain of six continuous lysines (Gutierrez et al, 1989;Hancock et al, 1991;Hancock et al, 1989;Hancock et al, 1990).Recent work has shown that the KRAS membrane anchor encodes exquisite binding specificity for phosphatidylserine (PtdSer) lipids with one saturated and one desaturated acyl chain (Zhou et al., 2017).This lipid binding specificity is effectively hard wired into the anchor structure and renders KRAS PM binding, nanoclustering and hence biological function absolutely dependent on PM PtdSer content (Cho et al, 2012b;Cho et al, 2013;van der Hoeven et al, 2013;van der Hoeven et al, 2018).
PtdSer is synthesized in the ER and delivered to the PM against a concentration gradient by the lipid transfer proteins (LTPs), oxysterol-binding protein-related protein (ORP) 5 and 8, operating at ER-PM membrane contact sites.One molecule of phosphatidylinositol (PI) 4-phosphate (P), generated by PM localized PI 4-kinase III (PI4KA) from PI, is exchanged by ORP5/8 for one molecule of ER localized PtdSer (Chung et al, 2015;Moser von Filseck et al, 2015).PI4P delivered to the ER is converted to PI by the SAC1 phosphatase, which operating together with PI4KA, maintains the PI4P gradient that in turn, drives PtdSer transport to the PM (Chung et al., 2015;Moser von Filseck et al., 2015).Concordantly genetic and pharmacological studies have shown that inhibition or loss of any component of the PtdSer transport machinery, including PI4KA, EFR3A (the PM anchor protein for the kinase), ORP5, ORP8 and SAC1 results in a reduction in PM PtdSer content, displacement of KRAS from the PM and abrogation of KRAS signaling (Gulyas et al, 2017;Kattan et al, 2019;Kattan et al, 2021;Sohn et al, 2018).
Other pharmacological approaches to deplete PM PtdSer also dissociate KRAS from the PM (Cho et al., 2012b;Garrido et al, 2020;Miller et al, 2019;Tan et al, 2019;Tan et al, 2018;van der Hoeven et al., 2018).The PM is a dynamic organelle, thus KRAS on endomembranes is sequestered by a chaperone protein, phosphodiesterase 6, and released to perinuclear membranes in the ARL2/3-dependent manner.The negative charge on the recycling endosome membranes then electrostatically traps KRAS, from where vesicular transport returns KRAS to the PM (Chandra et al, 2012;Schmick et al, 2014).KRAS phosphorylation at the Ser181 residue can disrupt this electrostatic interaction, depleting KRAS from the PM (Cho et al, 2016a;Kovar et al, 2020).
In this context we carried out a cell-based high content screen using a human genomic short interfering (siRNA) library to identify additional potential regulators of KRAS PM localization.Among the hits from the assay were multiple genes that encode proteins with phosphatase activity.Four of these genes encode myotubularin-related (MTMR) proteins 2, 3, 4 and 7, which have 3-phosphatase activity towards phosphatidylinositol (PI) 3-phosphate (P) and PI(3,5)-bisphosphate (PI(3,5)P 2 ) (Robinson & Dixon, 2006).In this study, we examined the role of MTMR proteins in the PM localization of KRAS.Our data demonstrate that knockdown (KD) of MTMR 2, 3, 4 or 7 dissociates KRAS, but not HRAS from the PM.We show inter alia that the molecular mechanism involves depletion of the PI4P and PtdSer content of the PM, extending the repertoire of proteins required to support the operation of the ORP5/8 LTPs and hence sustain KRAS PM localization.
Preparing T47D cells for imaging.5 x 10 5 T47D cells were seeded on a coverslip on day 1 in RPMI-1640 medium containing 10% FBS, 2mM L-glutamine and 10 g/mL insulin (complete growth medium).On day 2, fresh complete growth medium was supplemented.On day 3, cells were fixed with 4% PFA for 30 min, quenched with 50 mM NH 4 Cl for 10 min and mounted with Vectashield (H-1000, Vector Laboratories).For CellMask staining, cells were incubated with CellMask stain (1:5,000) for 1 h in complete growth medium and fixed with 4% PFA.Lipidomic analysis.T47D cells stably expressing mGFP-KRASG12V with MTMR 2, 3, 4 or 7 knockdown were cultured in complete growth medium containing 0.5 µg/ml puromycin.Triplicate samples of 1x10 6 cells were prepared in 333 L Dulbecco's PBS (DPBS without Ca 2+ and Mg 2+ ,     14190144, Invitrogen).Lipid extraction and analysis using electron spray ionization and MS/ MS were performed at Lipotype GmbH (Dresden, Germany), as described previously (Gerl et al, 2012;Sampaio et al, 2011).Automated processing of acquired mass spectra, identification, and quantification of detected lipid species were done by LipidXplorer software.Only lipid identifications with a signal-to-noise ratio >5, an absolute abundance of at least 1 pmol, and a signal intensity 5-fold higher than in corresponding blank samples were considered for further data analysis.The abundance of lipids is represented as a heat map with log 2 scale relative to control (scrambled shRNA) cells.
Western blotting.Preparation of cell lysates and immunoblotting were performed as described previously (Cho et al, 2011).Briefly, cells were washed twice with ice-cold 1x phosphatebuffered saline (PBS).Cells were harvested in lysis buffer B containing 50 mM Tris-Cl (pH 7.5), plus protease and phosphatase inhibitors.SDS-PAGE and immunoblotting were generally performed using 20-25 μg of lysate from each sample group.Signals were detected by enhanced chemiluminescence (Cat# 34578 and 34075, Thermo Fisher Scientific) and imaged using an Amersham Imager 600 (GE Healthcare).ImageJ software (v1.52) was used to quantify band intensity.
C. elegans vulva quantification assay.let-60 (n1046) worms were kindly provided by Swathi Arur (MD Anderson Cancer Center, Houston, TX).RNAi was induced by feeding let-60 worms through adult stage with E. coli HT115, producing double-stranded RNA to target genes.The presence of the multivulva phenotype was scored using a DIC/Nomarski microscope.All RNAi clones were from the C. elegans RNAi (Ahringer) collection (Source BioScience) and were sequenced.
Lysenin staining.A maltose binding protein (MBP)-GFP-lysenin fragment (amino acid residues 161 to 297) was purified as previously described (Maekawa et al, 2016).WT T47D cells were fixed with 4% PFA, and permeabilized with 0.05% saponin for 30 min at room temperature (RT), followed by labeling with (15 g/mL) MBP-GFP-lysenin for 15 min at RT. Cells were quenched with 50 mM NH 4 Cl in the dark for 10 min, and further labeled with DAPI (2.5 g/mL) in the dark for 10 min.
Proliferation assay.T47D cells stably expressing mGFP-KRASG12V and MTMR 2, 3, 4 or 7 knockdown were seeded at 1 ×10 4 in triplicate onto a 96-well plate in complete growth medium containing 0.5 g/mL puromycin.Cells were cultured for 4 days.Fresh complete growth medium containing 0.5 g/mL puromycin was supplemented every 24 h.On day 5, cell proliferation was assayed by counting cells using Countess II cell counter (Invitrogen).
Electron microscopy.Plasma membrane sheets were prepared from Caco-2 or T47D cells expressing GFP-tagged proteins of interest and fixed as previously described (Hancock & Prior, 2005;Prior et al, 2003a;Prior et al, 2003b).For univariate analysis, plasma membrane sheets were labeled with anti-GFP antibody conjugated to 4.5-nm gold particles.Digital images of the immunogold-labeled plasma membrane sheets were taken in a transmission electron microscope.Intact 1 m 2 areas of the plasma membrane sheet were identified using ImageJ software, and the (x, y) coordinates of the gold particles were determined (Hancock & Prior, 2005;Prior et al., 2003a;Prior et al., 2003b).K-functions (Ripley, 1977) were calculated and standardized on the 99% confidence interval (CI) for univariate functions (Diggle, 2000;Hancock & Prior, 2005;Prior et al., 2003a;Prior et al., 2003b).In the case of univariate functions, a value of L(r)-r greater than the CI indicates significant clustering, and the maximum value of the function (L max ) estimates the extent of clustering.Differences between replicated point patterns were analyzed by constructing bootstrap tests as described previously (Diggle, 2000;Plowman & Hancock, 2005), and the statistical significance against the results obtained with 1,000 bootstrap samples was evaluated.

Results
Genome-wide siRNA screening identifies novel genes regulating KRAS PM interaction.
To identify novel regulators of KRAS PM interaction, we performed an image-based high content screen of a human genomic siRNA library.A Caco-2 (human colorectal adenocarcinoma) cell line stably expressing monomeric green fluorescent protein (GFP)-tagged oncogenic mutant KRAS (KRASG12V) was generated and transfected with siRNA pools comprising four different oligonucleotides against each gene.Four days after transfection, cells were imaged using an automated confocal microscope to analyze the extent of KRAS mislocalization from the PM.We then narrowed the list of hits using a counter screen against HRASG12V to identify KRASG12V-specific mediators.Bioinformatics analysis revealed that 8 of these putative KRASG12V-specific mediators encode proteins with phosphatase activity (Table 1), 4 of which are members of the myotubularin-related (MTMR) protein family.There are 14 human myotubularins, which exhibit 3-phosphatase activity towards PI3P and PI(3,5)P 2 , generating PI and PI5P, respectively (Robinson & Dixon, 2006).MTMR proteins have previously been shown to regulate many cellular processes including endocytosis, membrane trafficking, cell proliferation, autophagy, cytokinesis and cytoskeletal dynamics (Hnia et al, 2012).We selected this MTMR gene set for further analysis.
T47D (human mammary gland ductal carcinoma) epithelial cells stably expressing GFP-KRASG12V were infected with lentiviruses expressing shRNA targeting MTMR2, 3, 4 and 7, followed by puromycin selection.For each target gene, we tested 4 different shRNAs and selected the two most effective KD for further experiments.Immunoblotting verified that endogenous expression levels of MTMR2, 3 and 7 were reduced by the cognate shRNAs (Fig. 1A and S1).MTMR4 mRNA levels were also reduced by shRNA expression; we evaluated mRNA because the anti-MTMR4 antibodies we tested were not able to detect endogenous MTMR4 (Fig. 1A).To study KRAS cellular localization cells were incubated with CellMask, a dye staining cellular membranes (Hu et al, 2013;Maechler et al, 2019;Monkemoller et al, 2015;Park et al, 2019), and imaged by confocal microscopy.To quantify the extent of KRAS distribution to endomembranes, we calculated Manders' coefficient, which measures the fraction of CellMask co-localized with GFP-KRASG12V (Cho et al., 2012b;Manders et al, 1993;Rehl et al, 2023).Our data show that while GFP-KRASG12V is predominantly localized to the PM in control cells, it is distributed intracellularly in the KD cell lines (Fig. 1B).Manders' coefficients for CellMask are correspondingly higher in the KD cell lines, indicating that KRASG12V is redistributed to endomembranes after silencing MTMR 2/3/4/7.To directly quantify the extent of KRAS dissociation from the PM, intact basal PM sheets from T47D stably co-expressing GFP-KRASG12V and shRNA for MTMR 2/3/4/7 were prepared and labeled with gold-conjugated anti-GFP antibodies and analyzed by electron microscopy (EM).We observed significant reductions in anti-GFP immunogold labeling after the KD, indicating loss of KRASG12V from the inner PM leaflet (Fig. 1C and S2).Spatial mapping of GFP-KRASG12V on the PM in MTMR 2/3/4/7 KD cells revealed significant decreases in L max , the peak value of the L(r)-r clustering statistic that measures the extent of KRAS nanoclustering, which is essential for high-fidelity RAS signal transduction (Cho & Hancock, 2013;Cho et al, 2012a;Tian et al, 2007) (Fig. 1D).
The loss of inner leaflet PtdSer was not due to flipping to the outer leaflet since Annexin V binding to the outer PM leaflet, as measured by flow cytometry, did not increase in MTMR 2/3/4/7 KD cells (Fig. S5).Finally, whole cell lipidomics revealed that MTMR 2/3/4/7 KD resulted in a significant reduction in total cellular PtdSer content (Figs.2C and D).Together, these results show that MTMR 2/3/4/7 potently regulates the PM and total levels of PtdSer; consistent with a mechanistic role for MTMR 2/3/4/7 in controlling PtdSer cellular distribution.
Given early endosomes (Fig. 4A and B).Moreover, our data show some PM decoration with GFP-2xFYVE in all cells, indicative of PI3P at the PM (arrowheads in Fig. 4A).To formally quantify PM PI3P content, we performed EM analysis on intact basal PM sheets prepared from MTMR 2/3/4/7 KD T47D cells expressing GFP-2xFYVE.The data reveal that silencing MTMR 2/3/4/7 significantly elevates PI3P levels in the PM (Fig. 4C).Intriguingly, confocal and EM imaging further show GFP-MTMR 2/3/4/7 localization to the PM (Fig. 4D and E).Together, our data suggest that MTMR 2/3/4/7 localize to the PM, such that genetic depletion disrupts PI3P conversion to PI at the PM, which increases PI3P content at the PM and early endosomes.

Discussion
We PtdSer transport by the ORP5/8 machinery (Fig. 5C).In addition to the PM, MTMR 2/3/4/7 localize to endosomes and promotes their trafficking by regulating endosomal PI3P and PI(3,5)P 2 levels (Bonneick et al, 2005;Franklin et al., 2011;Mochizuki & Majerus, 2003;Naughtin et al., 2010;Previtali et al, 2007;Walker et al., 2001) have identified new roles for the phosphatases, MTMR 2/3/4/7, in maintaining the PM localization of PI4P, PtdSer and KRAS.We show that KD of MTMR 2/3/4/7 dissociates PtdSer and KRAS from the PM and also reduces total cellular PtdSer level.Concomitantly, MTMR 2/3/4/7 KD elevates PM PI3P content, reduces PM PI4P content and dissociates ORP5 from the ER-PM MCS.Collecting these observations together, we propose that the PM-localized MTMR phosphatases are required to help maintain PM levels of PI for generating PM PI4P by PI4KA, which in turn maintains total PtdSer levels and drives ER to PM transport of PtdSer.PI is synthesized from phosphatidic acid at the cytosolic face of ER, where a fraction of PI is used for the synthesis of glycosylphosphatidylinositol-anchored proteins(Blunsom & Cockcroft, 2020), with the remaining PI being widely distributed across intracellular cytosolic membranes(Ashlin et al, 2021;Pemberton et al, 2020; Zewe et al, 2020).PI delivered from the ER directly to the PM by the LTPs, Nir2/3 and TMEM24(Posor et al, 2022), is a crucial substrate for PM polyphosphoinositides (PPIn).Simple overexpression of Nir2/3 and TMEM24 elevates the synthesis and total amount of PM PPIn(Chang et al, 2013; Kim et al, 2015; Lees et al, 2017), suggesting that PM PI is constantly converted to PPIn and the PI supply to the PM is ratelimiting.In this context, the pathway to PI generation from PI3P by MTMR 2/3/4/7 at the PM, emerges as a critical alternative supplier of PI to allow sufficient PM PI4P generation hence Figure 1.MTMR 2/3/4/7 regulate the PM localization of KRASG12V.(A) T47D cells stably expressing GFP-KRASG12V were infected with lentivirus expressing scrambled shRNA (SCR) or shRNA targeting MTMR2, 3, 4 or 7, followed by 1 ug/mL puromycin selection.Cell lysates were prepared and immunoblotted with anti-MTMR2, 3 or 7 antibodies.Actin blots are shown as loading controls.For MTMR4, cDNA was amplified with primers specific for MTMR4 exons 1 and 5, or GAPDH exons 2 and 3 as a loading control.(B) These cells were incubated with CellMask for 1 h at 37 o C incubator, fixed with 4% PFA and imaged by confocal microscopy.Representative GFP-KRASG12V images are shown.Their corresponding CellMask and merged images are shown in Fig. S2.The inserted values represent a mean fraction  S.E.M. of CellMask colocalizing with GFP-KRASG12V calculated by Manders' coefficient from three independent experiments.Scale bar -10 m.(C) Intact basal PM sheets prepared from T47D cells co-expressing GFP-KRASG12V and shRNA targeting MTMR2, 3, 4 or 7 were labeled with anti-GFP-conjugated gold particles and visualized by EM.Representative EM images are shown in Fig. S3.The graphs show a mean number of gold particles ± SEM (n ≥ 15).(D) Spatial mapping of the same gold-labeled PM sheets was performed.The peak values, L max , representing the extent of KRAS spatial organization, are shown as bar graphs (n ≥ 15).Significant differences between control (SCR-expressing) and MTMR-silenced cells were assessed by one-way ANOVA test for (B and C) and bootstrap tests for (D) (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Figure 4 .
Figure 4. MTMR 2/3/4/7 regulate cellular PI3P contents.(A) T47D cells expressing scrambled shRNA (SCR) or shRNA targeting MTMR2, 3, 4 or 7, followed by 1 ug/mL puromycin selection, were overexpressed with GFP-2xFYVE.Cells were incubated with CellMask for 1 h at 37 o C incubator, fixed with 4% PFA and imaged by confocal microscopy.Representative GFP-2xFYVE images are shown.Their corresponding CellMask and merged images are shown in Fig. S7.A selected region (the white square) is shown at a higher magnification.Arrowheads indicate the PM-staining of GFP-2xFYVE.(B) The graph represents a mean fraction  S.E.M (n

Figure 5 .
Figure 5. MTMR 2/3/4/7 regulate cell growth and C. elegans transformed by oncogenic KRAS.(A) T47D cells stably expressing GFP-KRASG12V were infected with lentivirusexpressing scrambled shRNA (SCR) or shRNA targeting MTMR2, 3, 4 or 7, followed by 1 ug/mL puromycin selection.Cells were seeded on a 96-well plate and cultured for 4 days.Complete growth medium was replaced every 24 h.Cell numbers were counted to measure cell proliferation.The graph shows the mean cell proliferation  S.E.M. from three independent experiments relative to that for the control cells (SCR-expressing).Significant differences between control and MTMR knockdown cells were assessed using one-way ANOVA test (* p<0.05, ** p<0.01, *** p<0.001).(B) RNAi was induced by feeding let-60 (n1046) L1 worms through adult stage with E. coli strain HT115, producing dsRNA to target genes.The presence of the multi-vulva phenotype was scored using DIC/Nomarski microscopy.100 -200 worms were assayed per RNAi knockdown.Significant differences were evaluated in Student's t-tests (** p < 0.01 and *** p < 0.001).

Figure 6 .
Figure 6.A working model of our study.PITPs deliver newly synthesized PI from the ER to PM, and MTMR at the PM concomitantly converts PI3P to PI, which are which are crucial substrate for PM PPIn like PI4P.ORP5 and 8, the lipid transfer proteins for PtdSer, exchanges ER PtdSer with PM PI4P at ER-PM membrane-contacting sites, which enriches PM PtdSer contents.This, in turn, allows stable PM binding of KRAS, and thereby KRAS signal output.When MTMR is depleted, PM PI3P conversion to PI is blocked, providing insufficient PM PI contents for PI4P synthesis.This dissociates ORP5/8 from ER-PM membrane-contacting sites, resulting in reduced PM localization of PtdSer and thereby KRAS.PITPs -PI transfer proteins, Figure 1

Table 1 . 8 novel genes, which encode proteins with phosphatase activity are identified to regulate KRAS/PM localization.
(Alvarez-Prats et al, 2018)itali et al., 2007)the demyelination of nerve cells(Bonneick et al., 2005;Previtali et al., 2007).Intriguingly, PtdSer is the main component of myelin sheath(Ma et al, 2022), and inhibition of PI4KA in Schwann cells reduces PM and total levels of PtdSer and PI4P, and induces aberrant myelination(Alvarez-Prats et al, 2018).Thus, in addition to disrupted endosomal trafficking by MTMR2 inactivation, it is plausible that the reduced PM levels of PtdSer and PI4P further contributes to the demyelination observed in Charcot-Marie-Tooth disease type 4B1.In sum, our study proposes a new role for the MTMR phosphatases in maintaining PM PI4P and PtdSer contents by maintaining an adequate supply of PM PI from PI3P.Depleting MTMR 2/3/4/7 in turn, reduces PM and total cellular PtdSer levels, blocking KRAS PM localization and KRAS signaling.Thus, the mechanisms that maintain PM PI content may be contain useful targets to abrogate oncogenic KRAS signaling.