Polybasic Domain Research Articles

Intrinsically Disordered Membrane Anchors of Rheb, RhoA, and DiRas3 Small GTPases: Molecular Dynamics, Membrane Organization, and Interactions.

Protein structure has been well established to play a key role in determining function; however, intrinsically disordered proteins and regions (IDPs and IDRs) defy this paradigm. IDPs and IDRs exist as an ensemble of structures rather than a stable 3D structure yet play essential roles in many cell-signaling processes. Nearly all Ras superfamily GTPases are tethered to membranes by a lipid tail at the end of a flexible IDR. The sequence of the IDR is a key determinant of membrane localization, and interaction between the IDR and the membrane has been shown to affect signaling in RAS proteins through the modulation of dynamic membrane organization. Here, we utilized atomistic molecular dynamics simulations to study the membrane interaction, conformational dynamics, and lipid sorting of three IDRs from small GTPases Rheb, RhoA, and DiRas3 in model membranes representing their physiological target membranes. We found that complementarity between the lipidated IDR sequence and target membrane lipid composition is a determinant of conformational plasticity. We also show that electrostatic interactions between anionic lipids and basic residues on IDRs are correlated with sampling of semistable conformational substates, and lack of these interactions is associated with greater conformational diversity. Finally, we show that small GTPase IDRs with a polybasic domain alter local lipid composition by segregating anionic lipids and, in some cases, excluding other lipids from their immediate vicinity in favor of anionic lipids.

Conformational ensemble-dependent lipid recognition and segregation by prenylated intrinsically disordered regions in small GTPases

We studied diverse prenylated intrinsically disordered regions (PIDRs) of Ras and Rho family small GTPases using long timescale atomistic molecular dynamics simulations in an asymmetric model membrane of phosphatidylcholine (PC) and phosphatidylserine (PS) lipids. Here we show that conformational plasticity is a key determinant of lipid sorting by polybasic PIDRs and provide evidence for lipid sorting based on both headgroup and acyl chain structures. We further show that conformational ensemble-based lipid recognition is generalizable to all polybasic PIDRs, and that the sequence outside the polybasic domain (PBD) modulates the conformational plasticity, bilayer adsorption, and interactions of PIDRs with membrane lipids. Specifically, we find that palmitoylation, the ratio of basic to acidic residues, and the hydrophobic content of the sequence outside the PBD significantly impact the diversity of conformational substates and hence the extent of conformation-dependent lipid interactions. We thus propose that the PBD is required but not sufficient for the full realization of lipid sorting by prenylated PBD-containing membrane anchors, and that the membrane anchor is not only responsible for high affinity membrane binding but also directs the protein to the right target membrane where it participates in lipid sorting.

The SOAR of STIM1 interacts with plasma membrane lipids to form ER-PM contact sites

Depletion of Ca2+ from the endoplasmic reticulum (ER) causes the ER Ca2+ sensor STIM1 to form membrane contact sites (MCSs) with the plasma membrane (PM). At the ER-PM MCS, STIM1 binds to Orai channels to induce cellular Ca2+ entry. The prevailing view of this sequential process is that STIM1 interacts with the PM and with Orai1 using two separate modules: a C-terminal polybasic domain (PBD) for the interaction with PM phosphoinositides and the STIM-Orai activation region (SOAR) for the interaction with Orai channels. Here, using electron and fluorescence microscopy and protein-lipid interaction assays, we show that oligomerization of the SOAR promotes direct interaction with PM phosphoinositides to trap STIM1 at ER-PM MCSs. The interaction depends on a cluster of conserved lysine residues within the SOAR and is co-regulated by the STIM1 coil-coiled 1 and inactivation domains. Collectively, our findings uncover a molecular mechanism for formation and regulation of ER-PM MCSs by STIM1.

The role of conformational dynamics in intrinsically disordered lipid anchors of peripheral membrane proteins in specifying interactions with membrane lipids.

Membrane binding and function of many small GTPases depends on a prenylated hypervariable region (HVR), roughly a 10-30 residue-long intrinsically disordered C-terminus that often contains a polybasic domain (PBD) adjacent to the prenyl group. A long held view was that the PBD non-specifically interacts with anionic membrane phospholipids. However, our previous work on the KRAS HVR showed that the PBD sorts anionic lipids with defined head group and acyl chain structure. Molecular dynamics (MD) simulations suggested that this sorting is realized by backbone conformational dynamics. In the current study, we asked whether this finding is generalizable to HVRs of other PBD-containing lipidated small GTPases. To answer this question, we used atomistic MD simulations to examine the dynamics and interaction patterns of PBD-containing HVRs with varying net charge, sequence length, and lipid-modification (farnesylation and geranylgeranylation). Our results show that the interaction of the PBD with anionic lipids indeed depends on the conformational dynamics and orientation of the backbone, which is primarily determined by the sequence of the PBD but less so on the length of HVR.

LIPID transfer proteins regulate store-operated calcium entry via control of plasma membrane phosphoinositides

The ER-resident proteins STIM1 together with the plasma membrane (PM)-localized Orai1 channels constitute the molecular components of the store-operated Ca2+ entry (SOCE) pathway. Prepositioning of STIM1 to the peripheral ER close to the PM ensures its efficient interaction with Orai1 upon a decrease in the ER luminal Ca2+ concentration. The C-terminal polybasic domain of STIM1 has been identified as mediating the interaction with PM phosphoinositides and hence positions the molecule to ER-PM contact sites. Here we show that STIM1 requires PM phosphatidylinositol 4-phosphate (PI4P) for efficient PM interaction. Accordingly, oxysterol binding protein related proteins (ORPs) that work at ER-PM junctions and consume PI4P gradients exert important control over the Ca2+ entry process. These studies reveal an important connection between non-vesicular lipid transport at ER-PM contact sites and regulation of ER Ca2+store refilling.

Phosphatidylserine and Phosphatidylethanolamine Asymmetry Have a Negligible Effect on the Global Structure, Dynamics, and Interactions of the KRAS Lipid Anchor.

The intrinsically disordered C-terminus of the prominent oncogenic protein KRAS-4B (KRAS) selectively interacts and clusters with phosphatidylserine (PS) lipids in the plasma membrane (PM). This 11-residue segment, called tK, contains a polybasic domain (PBD) of six contiguous lysine residues and a farnesylated cysteine. Previous molecular dynamics (MD) simulation studies of tK in phosphatidylcholine (PC)/PS bilayers have suggested that backbone conformational dynamics modulate tK-PS interactions. These simulations have been conducted in symmetric membranes whereas the PM is compositionally asymmetric, with the inner leaflet, where KRAS is localized, being enriched with PS and phosphatidylethanolamine (PE) lipids. To examine if bilayer asymmetry affects tK conformational dynamics and interaction with lipids, we conducted two 10 μs long MD simulations of tK bound to a PC/PS and a PC/PS/PE bilayer in which the PS and PE lipids are distributed in one leaflet. We found that, first, these compositional asymmetries caused differences in acyl chain dynamics between leaflets, but the equilibrium structural and dynamic properties of the two asymmetric bilayers are similar; second, in both systems tK is highly dynamic and samples at least two distinct conformational states; third, PS-tK hydrogen-bonding interactions vary with peptide backbone conformations, and lysine side chains in the PBD predominantly interact with the serine oxygens of PS. These results are in good agreement with previous observations of tK in symmetric membranes. The effects of POPS asymmetry or the presence of POPE on tK are limited to modulating the relative contribution of individual side chains to interactions with lipids and redistributing conformational substates. Additional observations include the larger flexibility of tK in the current simulations, which we attribute to the longer duration of the simulations and the use of the CHARMM36m force field, which more accurately models intrinsically disordered peptides such as tK.

Formation of self-organizing functionally distinct Rho of plants domains involves a reduced mobile population.

Rho family proteins are central to the regulation of cell polarity in eukaryotes. Rho of Plants-Guanyl nucleotide Exchange Factor (ROPGEF) can form self-organizing polar domains following co-expression with an Rho of Plants (ROP) and an ROP GTPase-Activating Protein (ROPGAP). Localization of ROPs in these domains has not been demonstrated, and the mechanisms underlying domain formation and function are not well understood. Here we show that six different ROPs form self-organizing domains when co-expressed with ROPGEF3 and GAP1 in Nicotiana benthamiana or Arabidopsis (Arabidopsis thaliana). Domain formation was associated with ROP-ROPGEF3 association, reduced ROP mobility, as revealed by time-lapse imaging and Fluorescence Recovery After Photobleaching beam size analysis, and was independent of Rho GTP Dissociation Inhibitor mediated recycling. The domain formation depended on the ROPs' activation/inactivation cycles and interaction with anionic lipids via a C-terminal polybasic domain. Coexpression with the microtubule-associated protein ROP effector INTERACTOR OF CONSTITUTIVELY ACTIVE ROP 1 (ICR1) revealed differential function of the ROP domains in the ability to recruit ICR1. Taken together, the results reveal mechanisms underlying self-organizing ROP domain formation and function.

Genetic Validation of the Palmitoylation/Depalmitoylation Cycle As a Drug Target in NRAS Mutant Hematologic Malignancies In Vivo

The C-terminal hypervariable region (HVR) of Ras proteins specifies post-translational modifications required for their subcellular localization and biologic function. The HVR of all four Ras isoforms terminates with a CAAX motif that is prenylated by farnesyltransferase, producing a weak membrane binding affinity that is further stabilized by a second signal motif. For K-Ras4b, this signal is provided by a polybasic lysine domain that allows direct localization of the protein at the plasma membrane (PM). By contrast, H-Ras, N-Ras, and K-Ras4a PM localization requires palmitoylation at cysteine(s) adjacent to the CAAX motif and is maintained through a dynamic cycle regulated by depalmitoylation and repalmitoylation. Importantly, these differences raise the provocative possibility that inhibiting palmitoylation could selectively target NRAS mutant cancer cells without toxicity to normal cells that retain intact K-Ras4b function.Indeed, Nras oncogenes with a cysteine-to-serine second site amino acid substitution at the palmitoylation site (C181S) exhibited defective transforming activity in hematopoietic stem and progenitor cells (HSPCs) ex vivo and in vivo (Cuiffo et al., Blood 2010; Xu et al., Blood 2012). However, these studies relied on retroviral promoters to express oncogenic N-RasG12D that resulted in supra-physiological protein levels in HSPCs. To overcome this limitation, we retargeted embryonic stem cells that were used to generate Lox-STOP-Lox (LSL) NrasG12D/+ knock in mice ( Haigis et al., Nat Genet 2008 ) to create a NrasG12D,C181S allele (hereafter called NrasC181S). We then performed intercrosses to generate Mx1-Cre, NrasC181S/C181S mice as well as cohorts of congenic Mx1-Cre, NrasWT/WT and Mx1-Cre, NrasG12D/G12D control animals. As expected (Li et al ., Blood 2011; Wang et al ., Cell Cycle 2011), Mx1-Cre, NrasG12D/G12D mice that were injected with poly(I:C) at weaning had a mean white blood cell (WBC) count of 58.3 x 103/µL at six months of age (Fig.1 A) and died from hematologic disease after 225 days (range 119-267 days, n= 14, Fig. 1B). By contrast, Mx1-Cre, NrasC181S/C181S mice had normal WBC counts at the same time point (mean: 11.34x103 WBC/µL) and remained well until 19-20 months of age (n=12). The difference in survival between these groups was highly significant (p=3x10-6 by Kaplan-Meier analysis). Detailed analysis of the hematopoietic compartment at six months of age (n=5 per genotype) revealed profound splenomegaly (mean weight: 1.272g) in NrasG12D/G12D mice, but normal spleen weights in the NrasWT/WT and NrasC181S/C181S animals (Fig. 1C). The expansion of granulocyte-monocyte progenitors (GMPs), megakaryocyte-erythroid progenitors (MEPs), and more primitive CD48+ Lin- c-Kit+ Sca1+ (LKS) cells previously observed in NrasG12D/G12D mice was also rescued by the C181S second site mutation. Furthermore, in vitro culture of HSPC-enriched bone marrow cells over a range of granulocyte-macrophage colony-stimulating factor (GM-CSF) concentrations revealed normal cytokine sensitivity of NrasC181S/C181S cells, while NrasG12D/G12D cells were characterized by cytokine-independent growth and GM-CSF hypersensitivity as previously described. Importantly, Ras-GTP levels were similarly elevated in bone marrow cells from both NrasG12D/G12D and NrasC181S/C181S mice compared to wild-type cells. This indicates that the C181S mutation abrogates the cytokine hypersensitivity and increased cell proliferation that results from oncogenic N-RasG12D expression without reverting the canonical biochemical consequence of the oncogenic G12D substitution. This observation, in turn, suggests that abnormal subcellular localization and trafficking of oncogenic N-Ras potently suppresses myeloid transformation. Taken together, these in vivo studies directly validate the palmitoylation/depalmitoylation cycle as a therapeutic target in hematologic malignancies and potentially in other cancers characterized by oncogenic NRAS mutations. [Display omitted] DisclosuresFirestone:Calico Life Sciences: Employment.

Electrostatic plasma membrane targeting contributes to Dlg function in cell polarity and tumorigenesis.

Discs large (Dlg) is an essential polarity protein and a tumor suppressor originally characterized in Drosophila but also well conserved in vertebrates. Like the majority of polarity proteins, plasma membrane (PM)/cortical localization of Dlg is required for its function in polarity and tumorigenesis, but the exact mechanisms targeting Dlg to the PM remain to be fully elucidated. Here, we show that, similar to recently discovered polybasic polarity proteins such as Lgl and aPKC, Dlg also contains a positively charged polybasic domain that electrostatically binds the PM phosphoinositides PI4P and PI(4,5)P2 Electrostatic targeting by the polybasic domain contributes significantly to the PM localization of Dlg in follicular and early embryonic epithelial cells, and is crucial for Dlg to regulate both polarity and tumorigenesis. The electrostatic PM targeting of Dlg is controlled by a potential phosphorylation-dependent allosteric regulation of its polybasic domain, and is specifically enhanced by the interactions between Dlg and another basolateral polarity protein and tumor suppressor, Scrib. Our studies highlight an increasingly significant role of electrostatic PM targeting of polarity proteins in regulating cell polarity.

Structural Determinants for Light-Dependent Membrane Binding of a Photoswitchable Polybasic Domain.

OptoPB is an optogenetic tool engineered by fusion of the phosphoinositide (PI)-binding polybasic domain of Rit1 (Rit-PB) to a photoreactive light-oxygen-voltage (LOV) domain. OptoPB selectively and reversibly binds the plasma membrane (PM) under blue light excitation, and in the dark, it releases back to the cytoplasm. However, the molecular mechanism of optical regulation and lipid recognition is still unclear. Here using nuclear magnetic resonance (NMR) spectroscopy, liposome pulldown assay, and surface plasmon resonance (SPR), we find that OptoPB binds to membrane mimetics containing di- or triphosphorylated phosphatidylinositols, particularly phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), an acidic phospholipid predominantly located in the eukaryotic PM. In the dark, steric hindrance prevented this protein-membrane interaction, while 470 nm blue light illumination activated it. NMR titration and site-directed mutagenesis revealed that both cationic and hydrophobic Rit-PB residues are essential to the membrane interaction, indicating that OptoPB binds the membrane via a specific PI(4,5)P2-dependent mechanism.

The KRAS and other prenylated polybasic domain membrane anchors recognize phosphatidylserine acyl chain structure

KRAS interacts with the inner leaflet of the plasma membrane (PM) using a hybrid anchor that comprises a lysine-rich polybasic domain (PBD) and a C-terminal farnesyl chain. Electrostatic interactions have been envisaged as the primary determinant of interactions between KRAS and membranes. Here, we integrated molecular dynamics (MD) simulations and superresolution spatial analysis in mammalian cells and systematically compared four equally charged KRAS anchors: the wild-type farnesyl hexa-lysine and engineered mutants comprising farnesyl hexa-arginine, geranylgeranyl hexa-lysine, and geranylgeranyl hexa-arginine. MD simulations show that these equally charged KRAS mutant anchors exhibit distinct interactions and packing patterns with different phosphatidylserine (PtdSer) species, indicating that prenylated PBD-bilayer interactions extend beyond electrostatics. Similar observations were apparent in intact cells, where each anchor exhibited binding specificities for PtdSer species with distinct acyl chain compositions. Acyl chain composition determined responsiveness of the spatial organization of different PtdSer species to diverse PM perturbations, including transmembrane potential, cholesterol depletion, and PM curvature. In consequence, the spatial organization and PM binding of each KRAS anchor precisely reflected the behavior of its preferred PtdSer ligand to these same PM perturbations. Taken together these results show that small GTPase PBD-prenyl anchors, such as that of KRAS, have the capacity to encode binding specificity for specific acyl chains as well as lipid headgroups, which allow differential responses to biophysical perturbations that may have biological and signaling consequences for the anchored GTPase.

STIM2 targets Orai1/STIM1 to the AKAP79 signaling complex and confers coupling of Ca2+ entry with NFAT1 activation

The Orai1 channel is regulated by stromal interaction molecules STIM1 and STIM2 within endoplasmic reticulum (ER)-plasma membrane (PM) contact sites. Ca2+ signals generated by Orai1 activate Ca2+-dependent gene expression. When compared with STIM1, STIM2 is a weak activator of Orai1, but it has been suggested to have a unique role in nuclear factor of activated T cells 1 (NFAT1) activation triggered by Orai1-mediated Ca2+ entry. In this study, we examined the contribution of STIM2 in NFAT1 activation. We report that STIM2 recruitment of Orai1/STIM1 to ER-PM junctions in response to depletion of ER-Ca2+ promotes assembly of the channel with AKAP79 to form a signaling complex that couples Orai1 channel function to the activation of NFAT1. Knockdown of STIM2 expression had relatively little effect on Orai1/STIM1 clustering or local and global [Ca2+]i increases but significantly attenuated NFAT1 activation and assembly of Orai1 with AKAP79. STIM1ΔK, which lacks the PIP2-binding polybasic domain, was recruited to ER-PM junctions following ER-Ca2+ depletion by binding to Orai1 and caused local and global [Ca2+]i increases comparable to those induced by STIM1 activation of Orai1. However, in contrast to STIM1, STIM1ΔK induced less NFAT1 activation and attenuated the association of Orai1 with STIM2 and AKAP79. Orai1-AKAP79 interaction and NFAT1 activation were recovered by coexpressing STIM2 with STIM1ΔK. Replacing the PIP2-binding domain of STIM1 with that of STIM2 eliminated the requirement of STIM2 for NFAT1 activation. Together, these data demonstrate an important role for STIM2 in coupling Orai1-mediated Ca2+ influx to NFAT1 activation.

A polybasic domain in aPKC mediates Par6-dependent control of membrane targeting and kinase activity.

Mechanisms coupling the atypical PKC (aPKC) kinase activity to its subcellular localization are essential for cell polarization. Unlike other members of the PKC family, aPKC has no well-defined plasma membrane (PM) or calcium binding domains, leading to the assumption that its subcellular localization relies exclusively on protein-protein interactions. Here we show that in both Drosophila and mammalian cells, the pseudosubstrate region (PSr) of aPKC acts as a polybasic domain capable of targeting aPKC to the PM via electrostatic binding to PM PI4P and PI(4,5)P2. However, physical interaction between aPKC and Par-6 is required for the PM-targeting of aPKC, likely by allosterically exposing the PSr to bind PM. Binding of Par-6 also inhibits aPKC kinase activity, and such inhibition can be relieved through Par-6 interaction with apical polarity protein Crumbs. Our data suggest a potential mechanism in which allosteric regulation of polybasic PSr by Par-6 couples the control of both aPKC subcellular localization and spatial activation of its kinase activity.

Abstract IA08: Lipid dependence of KRAS plasma membrane interactions

Abstract RAS proteins must localize to the plasma membrane (PM) in order to function. We have examined how different RAS PM anchors select and assemble highly specific phospholipids into signaling platforms called nanoclusters, how these transient lipid assemblies impact signal transmission, and in consequence how the lipid composition of the PM can operate as high-level regulator of RAS signal transmission. For example, the KRAS polybasic domain (PBD)-farnesyl membrane anchor exhibits exquisite specificity for phosphatidylserine (PtdSer) over other anionic phospholipids by virtue of defined structural dynamics of the C-terminal polybasic domain and farnesyl group. This specificity extends to PtdSer with specific combinations of lipid side chains. PtdSer binding is therefore essential for stable KRAS PM binding and the spatial organization of KRAS into nanoclusters. These and complementary results with RAC1 highlight the critical role of specific anionic PM lipids in the biologic function of small GTPases. In this context for KRAS we have now shown that the PM levels and lateral availability of PtdSer are critically dependent on sphingomyelin metabolism such that multiple perturbations of sphingomyelin content or distribution, or endolysosmal lipid trafficking, profoundly affect KRAS plasma membrane localization, nanoclustering, and function. Concordantly, directly targeting the molecular machinery that maintains PtdSer on the PM also abrogates KRAS function in multiple pancreatic cancer cell lines. These new observations suggest multiple new potential therapeutic targets to abrogate oncogenic KRAS signaling, which we are evaluating in model systems. Citation Format: John F. Hancock. Lipid dependence of KRAS plasma membrane interactions [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr IA08.

Galectin-8 binds to the Farnesylated C-terminus of K-Ras4B and Modifies Ras/ERK Signaling and Migration in Pancreatic and Lung Carcinoma Cells.

K-Ras is the most prominent driver of oncogenesis and no effective K-Ras inhibitors have been established despite decades of intensive research. Identifying new K-Ras-binding proteins and their interaction domains offers the opportunity for defining new approaches in tackling oncogenic K-Ras. We have identified Galectin-8 as a novel, direct binding protein for K-Ras4B by mass spectrometry analyses and protein interaction studies. Galectin-8 is a tandem-repeat Galectin and it is widely expressed in lung and pancreatic carcinoma cells. siRNA-mediated depletion of Galectin-8 resulted in increased K-Ras4B content and ERK1/2 activity in lung and pancreatic carcinoma cells. Moreover, cell migration and cell proliferation were inhibited by the depletion of Galectin-8. The K-Ras4B–Galectin-8 interaction is indispensably associated with the farnesylation of K-Ras4B. The lysine-rich polybasic domain (PBD), a region that is unique for K-Ras4B as compared to H- and N-Ras, stabilizes the interaction and accounts for the specificity. Binding assays with the deletion mutants of Galectin-8, comprising either of the two carbohydrate recognition domains (CRD), revealed that K-Ras4B only interacts with the N-CRD, but not with the C-CRD. Structural modeling uncovers a potential binding pocket for the hydrophobic farnesyl chain of K-Ras4B and a cluster of negatively charged amino acids for interaction with the positively charged lysine residues in the N-CRD. Our results demonstrate that Galectin-8 is a new binding partner for K-Ras4B and it interacts via the N-CRD with the farnesylated PBD of K-Ras, thereby modulating the K-Ras effector pathways as well as cell proliferation and migration.

MICU1 controls cristae junction and spatially anchors mitochondrial Ca2+ uniporter complex

Recently identified core proteins (MICU1, MCU, EMRE) forming the mitochondrial Ca2+ uniporter complex propelled investigations into its physiological workings. Here, we apply structured illumination microscopy to visualize and localize these proteins in living cells. Our data show that MICU1 localizes at the inner boundary membrane (IBM) due to electrostatic interaction of its polybasic domain. Moreover, this exclusive localization of MICU1 is important for the stability of cristae junctions (CJ), cytochrome c release and mitochondrial membrane potential. In contrast to MICU1, MCU and EMRE are homogeneously distributed at the inner mitochondrial membrane under resting conditions. However, upon Ca2+ elevation MCU and EMRE dynamically accumulate at the IBM in a MICU1-dependent manner. Eventually, our findings unveil an essential function of MICU1 in CJ stabilization and provide mechanistic insights of how sophistically MICU1 controls the MCU-Complex while maintaining the structural mitochondrial membrane framework.

SALM1 controls synapse development by promoting F‐actin/PIP2‐dependent Neurexin clustering

Synapse development requires spatiotemporally regulated recruitment of synaptic proteins. In this study, we describe a novel presynaptic mechanism of cis‐regulated oligomerization of adhesion molecules that controls synaptogenesis. We identified synaptic adhesion‐like molecule 1 (SALM1) as a constituent of the proposed presynaptic Munc18/CASK/Mint1/Lin7b organizer complex. SALM1 preferentially localized to presynaptic compartments of excitatory hippocampal neurons. SALM1 depletion in excitatory hippocampal primary neurons impaired Neurexin1β‐ and Neuroligin1‐mediated excitatory synaptogenesis and reduced synaptic vesicle clustering, synaptic transmission, and synaptic vesicle release. SALM1 promoted Neurexin1β clustering in an F‐actin‐ and PIP2‐dependent manner. Two basic residues in SALM1's juxtamembrane polybasic domain are essential for this clustering. Together, these data show that SALM1 is a presynaptic organizer of synapse development by promoting F‐actin/PIP2‐dependent clustering of Neurexin.

Dissociation of the Signaling Protein K-Ras4B from Lipid Membranes Induced by a Molecular Tweezer.

Oncogenic Ras mutations occur in more than 30 % of human cancers. K-Ras4B is the most frequently mutated isoform of Ras proteins. Development of effective K-Ras4B inhibitors has been challenging, hence new approaches to inhibit this oncogenic protein are urgently required. The polybasic domain of K-Ras4B with its stretch of lysine residues is essential for its plasma membrane targeting and localization. Employing CD and fluorescence spectroscopy, confocal fluorescence, and atomic force microscopy we show that the molecular tweezer CLR01 is able to efficiently bind to the lysine stretch in the polybasic domain of K-Ras4B, resulting in dissociation of the K-Ras4B protein from the lipid membrane and disintegration of K-Ras4B nanoclusters in the lipid bilayer. These results suggest that targeting of the polybasic domain of K-Ras4B by properly designed tweezers might represent an effective strategy for inactivation of K-Ras4B signaling.

Myristoylated alanine-rich C-kinase substrate effector domain phosphorylation regulates the growth and radiation sensitization of glioblastoma.

Glioblastoma harbors frequent alterations in receptor tyrosine kinases, phosphatidylinositol-3 kinase (PI3K) and phosphatase and tensin homolog (PTEN) that dysregulate phospholipid signaling driven tumor proliferation and therapeutic resistance. Myristoylated alanine-rich C-kinase substrate (MARCKS) is a 32 kDa intrinsically unstructured protein containing a polybasic (+13) effector domain (ED), which regulates its electrostatic sequestration of phospholipid phosphatidylinositol (4,5)-bisphosphate (PIP2), and its binding to phosphatidylserine, calcium/calmodulin, filamentous actin, while also serving as a nuclear localization sequence. MARCKS ED is phosphorylated by protein kinase C (PKC) and Rho-associated protein kinase (ROCK) kinases; however, the impact of MARCKS on glioblastoma growth and radiation sensitivity remains undetermined. In the present study, using a tetracycline-inducible system in PTEN-null U87 cells, we demonstrate that MARCKS overexpression suppresses growth and enhances radiation sensitivity in vivo. A new image cytometer, Xcyto10, was utilized to quantify differences in MARCKS ED phosphorylation on localization and its association with filamentous actin. The overexpression of the non-phosphorylatable ED mutant exerted growth-suppressive and radiation-sensitizing effects, while the pseudo-phosphorylated ED mutant exhibited an enhanced colony formation and clonogenic survival ability. The identification of MARCKS protein-protein interactions using co-immunoprecipitation coupled with tandem mass spectrometry revealed novel MARCKS-associated proteins, including importin-β and ku70. On the whole, the findings of this study suggest that the determination of the MARCKS ED phosphorylation status is essential to understanding the impact of MARCKS on cancer progression.

A polybasic motif in alternatively spliced KChIP2 isoforms prevents Ca2+ regulation of Kv4 channels

The Kv4 family of A-type voltage-gated K+ channels regulates the excitability in hippocampal pyramidal neuron dendrites and are key determinants of dendritic integration, spike timing-dependent plasticity, long-term potentiation, and learning. Kv4.2 channel expression is down-regulated following hippocampal seizures and in epilepsy, suggesting A-type currents as therapeutic targets. In addition to pore-forming Kv4 subunits, modulatory auxiliary subunits called K+ channel-interacting proteins (KChIPs) modulate Kv4 expression and activity and are required to recapitulate native hippocampal A-type currents in heterologous expression systems. KChIP mRNAs contain multiple start sites and alternative exons that generate considerable N-terminal variation and functional diversity in shaping Kv4 currents. As members of the EF-hand domain-containing neuronal Ca2+ sensor protein family, KChIP auxiliary proteins may convey Ca2+ sensitivity upon Kv4 channels; however, to what degree intracellular Ca2+ regulates KChIP-Kv4.2 complexes is unclear. To answer this question, we expressed KChIP2 with Kv4.2 in HEK293T cells, and, with whole-cell patch-clamp electrophysiology, measured an ∼1.5-fold increase in Kv4.2 current density in the presence of elevated intracellular Ca2+ Intriguingly, the Ca2+ regulation of Kv4 current was specific to KChIP2b and KChIP2c splice isoforms that lack a putative polybasic domain that is present in longer KChIP2a1 and KChIP2a isoforms. Site-directed acidification of the basic residues within the polybasic motif of KChIP2a1 rescued Ca2+-mediated regulation of Kv4 current density. These results support divergent Ca2+ regulation of Kv4 channels mediated by alternative splicing of KChIP2 isoforms. They suggest that distinct KChIP-Kv4 interactions may differentially control excitability and function of hippocampal dendrites.