Determining the ERK-regulated phosphoproteome driving KRAS-mutant cancer.

To delineate the mechanisms by which the ERK1 and ERK2 mitogen-activated protein kinases support mutant KRAS-driven cancer growth, we determined the ERK-dependent phosphoproteome in KRAS-mutant pancreatic cancer. We determined that ERK1 and ERK2 share near-identical signaling and transforming outputs and that the KRAS-regulated phosphoproteome is driven nearly completely by ERK. We identified 4666 ERK-dependent phosphosites on 2123 proteins, of which 79 and 66%, respectively, were not previously associated with ERK, substantially expanding the depth and breadth of ERK-dependent phosphorylation events and revealing a considerably more complex function for ERK in cancer. We established that ERK controls a highly dynamic and complex phosphoproteome that converges on cyclin-dependent kinase regulation and RAS homolog guanosine triphosphatase function (RHO GTPase). Our findings establish the most comprehensive molecular portrait and mechanisms by which ERK drives KRAS-dependent pancreatic cancer growth.

Recurrent RhoGAP gene fusion CLDN18-ARHGAP26 promotes RHOA activation and focal adhesion kinase and YAP-TEAD signalling in diffuse gastric cancer.

OBJECTIVE: Genomic studies of gastric cancer have identified highly recurrent genomic alterations impacting RHO signalling, especially in the diffuse gastric cancer (DGC) histological subtype. Among these alterations are interchromosomal translations leading to the fusion of the adhesion protein CLDN18 and RHO regulator ARHGAP26. It remains unclear how these fusion constructs impact the activity of the RHO pathway and what is their broader impact on gastric cancer development. Herein, we developed a model to allow us to study the function of this fusion protein in the pathogenesis of DGC and to identify potential therapeutic targets for DGC tumours with these alterations. DESIGN: We built a transgenic mouse model with LSL-CLDN18-ARHGAP26 fusion engineered into the Col1A1 locus where its expression can be induced by Cre recombinase. Using organoids generated from this model, we evaluated its oncogenic activity and the biochemical effects of the fusion protein on the RHOA pathway and its downstream cell biological effects in the pathogenesis of DGC. RESULTS: We demonstrated that induction of CLDN18-ARHGAP26 expression in gastric organoids induced the formation of signet ring cells, characteristic features of DGC and was able to cooperatively transform gastric cells when combined with the loss of the tumour suppressor geneTrp53. CLDN18-ARHGAP26 promotes the activation of RHOA and downstream effector signalling. Molecularly, the fusion promotes activation of the focal adhesion kinase (FAK) and induction of the YAP pathway. A combination of FAK and YAP/TEAD inhibition can significantly block tumour growth. CONCLUSION: These results indicate that the CLDN18-ARHGAP26 fusion is a gain-of-function DGC oncogene that leads to activation of RHOA and activation of FAK and YAP signalling. These results argue for further evaluation of emerging FAK and YAP-TEAD inhibitors for these deadly cancers.

RHOAL57V drives the development of diffuse gastric cancer through IGF1R-PAK1-YAP1 signaling.

Cancer-associated mutations in the guanosine triphosphatase (GTPase) RHOA are found at different locations from the mutational hotspots in the structurally and biochemically related RAS. Tyr42-to-Cys (Y42C) and Leu57-to-Val (L57V) substitutions are the two most prevalent RHOA mutations in diffuse gastric cancer (DGC). RHOAY42C exhibits a gain-of-function phenotype and is an oncogenic driver in DGC. Here, we determined how RHOAL57V promotes DGC growth. In mouse gastric organoids with deletion of Cdh1, which encodes the cell adhesion protein E-cadherin, the expression of RHOAL57V, but not of wild-type RHOA, induced an abnormal morphology similar to that of patient-derived DGC organoids. RHOAL57V also exhibited a gain-of-function phenotype and promoted F-actin stress fiber formation and cell migration. RHOAL57V retained interaction with effectors but exhibited impaired RHOA-intrinsic and GAP-catalyzed GTP hydrolysis, which favored formation of the active GTP-bound state. Introduction of missense mutations at KRAS residues analogous to Tyr42 and Leu57 in RHOA did not activate KRAS oncogenic potential, indicating distinct functional effects in otherwise highly related GTPases. Both RHOA mutants stimulated the transcriptional co-activator YAP1 through actin dynamics to promote DGC progression; however, RHOAL57V additionally did so by activating the kinases IGF1R and PAK1, distinct from the FAK-mediated mechanism induced by RHOAY42C. Our results reveal that RHOAL57V and RHOAY42C drive the development of DGC through distinct biochemical and signaling mechanisms.

Functional and biological heterogeneity of KRASQ61 mutations.

Missense mutations at the three hotspots in the guanosine triphosphatase (GTPase) RAS-Gly12, Gly13, and Gln61 (commonly known as G12, G13, and Q61, respectively)-occur differentially among the three RAS isoforms. Q61 mutations in KRAS are infrequent and differ markedly in occurrence. Q61H is the predominant mutant (at 57%), followed by Q61R/L/K (collectively 40%), and Q61P and Q61E are the rarest (2 and 1%, respectively). Probability analysis suggested that mutational susceptibility to different DNA base changes cannot account for this distribution. Therefore, we investigated whether these frequencies might be explained by differences in the biochemical, structural, and biological properties of KRASQ61 mutants. Expression of KRASQ61 mutants in NIH 3T3 fibroblasts and RIE-1 epithelial cells caused various alterations in morphology, growth transformation, effector signaling, and metabolism. The relatively rare KRASQ61E mutant stimulated actin stress fiber formation, a phenotype distinct from that of KRASQ61H/R/L/P, which disrupted actin cytoskeletal organization. The crystal structure of KRASQ61E was unexpectedly similar to that of wild-type KRAS, a potential basis for its weak oncogenicity. KRASQ61H/L/R-mutant pancreatic ductal adenocarcinoma (PDAC) cell lines exhibited KRAS-dependent growth and, as observed with KRASG12-mutant PDAC, were susceptible to concurrent inhibition of ERK-MAPK signaling and of autophagy. Our results uncover phenotypic heterogeneity among KRASQ61 mutants and support the potential utility of therapeutic strategies that target KRASQ61 mutant-specific signaling and cellular output.

RHOA takes the RHOad less traveled to cancer.

RAS and RHO GTPases function as signaling nodes that regulate diverse cellular processes. Whereas RAS mutations were identified in human cancers nearly four decades ago, only recently have mutations in two RHO GTPases, RAC1 and RHOA, been identified in cancer. RAS mutations are found in a diverse spectrum of human cancer types. By contrast, RAC1 and RHOA mutations are associated with distinct and restricted cancer types. Despite a conservation of RAS and RAC1 residues that comprise mutational hotspots, RHOA mutations comprise highly divergent hotspots. Whereas RAS and RAC1 act as oncogenes, RHOA may act as both an oncogene and a tumor suppressor. Thus, while RAS and RHO each take different mutational paths, they arrive at the same biological destination as cancer drivers.

Concurrent Inhibition of ERK and Farnesyltransferase Suppresses the Growth of HRAS Mutant Head and Neck Squamous Cell Carcinoma.

Human papilloma virus (HPV)-negative head and neck squamous cell carcinoma (HNSCC) is a common cancer worldwide with an unmet need for more effective, less toxic treatments. Currently, both the disease and the treatment of HNSCC cause significant mortality and morbidity. Targeted therapies hold new promise for patients with HPV-negative status whose tumors harbor oncogenic HRAS mutations. Recent promising clinical results have renewed interest in the development of farnesyltransferase inhibitors (FTIs) as a therapeutic strategy for HRAS-mutant cancers. With the advent of clinical evaluation of the FTI tipifarnib for the treatment of HRAS-mutant HNSCC, we investigated the activity of tipifarnib and inhibitors of HRAS effector signaling in HRAS-mutant HNSCC cell lines. First, we validated that HRAS is a cancer driver in HRAS-mutant HNSCC lines. Second, we showed that treatment with the FTI tipifarnib largely phenocopied HRAS silencing, supporting HRAS as a key target of FTI antitumor activity. Third, we performed reverse-phase protein array analyses to profile FTI treatment-induced changes in global signaling, and conducted CRISPR/Cas9 genetic loss-of-function screens to identify previously unreported genes and pathways that modulate sensitivity to tipifarnib. Fourth, we determined that concurrent inhibition of HRAS effector signaling (ERK, PI3K, mTORC1) increased sensitivity to tipifarnib treatment, in part by overcoming tipifarnib-induced compensatory signaling. We also determined that ERK inhibition could block tipifarnib-induced epithelial-to-mesenchymal transition, providing a potential basis for the effectiveness of this combination. Our results support future investigations of these and other combination treatments for HRAS mutant HNSCC.

Aberrant Expression and Subcellular Localization of ECT2 Drives Colorectal Cancer Progression and Growth.

ECT2 is an activator of RHO GTPases that is essential for cytokinesis. In addition, ECT2 was identified as an oncoprotein when expressed ectopically in NIH/3T3 fibroblasts. However, oncogenic activation of ECT2 resulted from N-terminal truncation, and such truncated ECT2 proteins have not been found in patients with cancer. In this study, we observed elevated expression of full-length ECT2 protein in preneoplastic colon adenomas, driven by increased ECT2 mRNA abundance and associated with APC tumor-suppressor loss. Elevated ECT2 levels were detected in the cytoplasm and nucleus of colorectal cancer tissue, suggesting cytoplasmic mislocalization as one mechanism of early oncogenic ECT2 activation. Importantly, elevated nuclear ECT2 correlated with poorly differentiated tumors, and a low cytoplasmic:nuclear ratio of ECT2 protein correlated with poor patient survival, suggesting that nuclear and cytoplasmic ECT2 play distinct roles in colorectal cancer. Depletion of ECT2 reduced anchorage-independent cancer cell growth and invasion independent of its function in cytokinesis, and loss of Ect2 extended survival in a Kras G12D Apc-null colon cancer mouse model. Expression of ECT2 variants with impaired nuclear localization or guanine nucleotide exchange catalytic activity failed to restore cancer cell growth or invasion, indicating that active, nuclear ECT2 is required to support tumor progression. Nuclear ECT2 promoted ribosomal DNA transcription and ribosome biogenesis in colorectal cancer. These results support a driver role for both cytoplasmic and nuclear ECT2 overexpression in colorectal cancer and emphasize the critical role of precise subcellular localization in dictating ECT2 function in neoplastic cells. SIGNIFICANCE: ECT2 overexpression and mislocalization support its role as a driver in colon cancer that is independent from its function in normal cell cytokinesis.

Targeting p130Cas- and microtubule-dependent MYC regulation sensitizes pancreatic cancer to ERK MAPK inhibition.

To identify therapeutic targets for KRAS mutant pancreatic cancer, we conduct a druggable genome small interfering RNA (siRNA) screen and determine that suppression of BCAR1 sensitizes pancreatic cancer cells to ERK inhibition. Integrative analysis of genome-scale CRISPR-Cas9 screens also identify BCAR1 as a top synthetic lethal interactor with mutant KRAS. BCAR1 encodes the SRC substrate p130Cas. We determine that SRC-inhibitor-mediated suppression of p130Cas phosphorylation impairs MYC transcription through a DOCK1-RAC1-ß-catenin-dependent mechanism. Additionally, genetic suppression of TUBB3, encoding the ßIII-tubulin subunit of microtubules, or pharmacological inhibition of microtubule function decreases levels of MYC protein in a calpain-dependent manner and potently sensitizes pancreatic cancer cells to ERK inhibition. Accordingly, the combination of a dual SRC/tubulin inhibitor with an ERK inhibitor cooperates to reduce MYC protein and synergistically suppress the growth of KRAS mutant pancreatic cancer. Thus, we demonstrate that mechanistically diverse combinations with ERK inhibition suppress MYC to impair pancreatic cancer proliferation.

RAS and RHO family GTPase mutations in cancer: twin sons of different mothers?

The RAS and RHO family comprise two major branches of the RAS superfamily of small GTPases. These proteins function as regulated molecular switches and control cytoplasmic signaling networks that regulate a diversity of cellular processes, including cell proliferation and cell migration. In the early 1980s, mutationally activated RAS genes encoding KRAS, HRAS and NRAS were discovered in human cancer and now comprise the most frequently mutated oncogene family in cancer. Only recently, exome sequencing studies identified cancer-associated alterations in two RHO family GTPases, RAC1 and RHOA. RAS and RHO proteins share significant identity in their amino acid sequences, protein structure and biochemistry. Cancer-associated RAS mutant proteins harbor missense mutations that are found primarily at one of three mutational hotspots (G12, G13 and Q61) and have been identified as gain-of-function oncogenic alterations. Although these residues are conserved in RHO family proteins, the gain-of-function mutations found in RAC1 are found primarily at a distinct hotspot. Unexpectedly, the cancer-associated mutations found with RHOA are located at different hotspots than those found with RAS. Furthermore, since the RHOA mutations suggested a loss-of-function phenotype, it has been unclear whether RHOA functions as an oncogene or tumor suppressor in cancer development. Finally, whereas RAS mutations are found in a broad spectrum of cancer types, RHOA and RAC1 mutations occur in a highly restricted range of cancer types. In this review, we focus on RHOA missense mutations found in cancer and their role in driving tumorigenesis, with comparisons to cancer-associated mutations in RAC1 and RAS GTPases.

The molecular basis for immune dysregulation by the hyperactivated E62K mutant of the GTPase RAC2.

The RAS-related C3 botulinum toxin substrate 2 (RAC2) is a member of the RHO subclass of RAS superfamily GTPases required for proper immune function. An activating mutation in a key switch II region of RAC2 (RAC2E62K) involved in recognizing modulatory factors and effectors has been identified in patients with common variable immune deficiency. To better understand how the mutation dysregulates RAC2 function, we evaluated the structure and stability, guanine nucleotide exchange factor (GEF) and GTPase-activating protein (GAP) activity, and effector binding of RAC2E62K Our findings indicate the E62K mutation does not alter RAC2 structure or stability. However, it does alter GEF specificity, as RAC2E62K is activated by the DOCK GEF, DOCK2, but not by the Dbl homology GEF, TIAM1, both of which activate the parent protein. Our previous data further showed that the E62K mutation impairs GAP activity for RAC2E62K As this disease mutation is also found in RAS GTPases, we assessed GAP-stimulated GTP hydrolysis for KRAS and observed a similar impairment, suggesting that the mutation plays a conserved role in GAP activation. We also investigated whether the E62K mutation alters effector binding, as activated RAC2 binds effectors to transmit signaling through effector pathways. We find that RAC2E62K retains binding to an NADPH oxidase (NOX2) subunit, p67phox, and to the RAC-binding domain of p21-activated kinase, consistent with our earlier findings. Taken together, our findings indicate that the RAC2E62K mutation promotes immune dysfunction by promoting RAC2 hyperactivation, altering GEF specificity, and impairing GAP function yet retaining key effector interactions.

Low-Dose Vertical Inhibition of the RAF-MEK-ERK Cascade Causes Apoptotic Death of KRAS Mutant Cancers.

We address whether combinations with a pan-RAF inhibitor (RAFi) would be effective in KRAS mutant pancreatic ductal adenocarcinoma (PDAC). Chemical library and CRISPR genetic screens identify combinations causing apoptotic anti-tumor activity. The most potent combination, concurrent inhibition of RAF (RAFi) and ERK (ERKi), is highly synergistic at low doses in cell line, organoid, and rat models of PDAC, whereas each inhibitor alone is only cytostatic. Comprehensive mechanistic signaling studies using reverse phase protein array (RPPA) pathway mapping and RNA sequencing (RNA-seq) show that RAFi/ERKi induced insensitivity to loss of negative feedback and system failures including loss of ERK signaling, FOSL1, and MYC; shutdown of the MYC transcriptome; and induction of mesenchymal-to-epithelial transition. We conclude that low-dose vertical inhibition of the RAF-MEK-ERK cascade is an effective therapeutic strategy for KRAS mutant PDAC.

Gain-of-Function RHOA Mutations Promote Focal Adhesion Kinase Activation and Dependency in Diffuse Gastric Cancer.

Diffuse gastric cancer (DGC) is a lethal malignancy lacking effective systemic therapy. Among the most provocative recent results in DGC has been that of highly recurrent missense mutations in the GTPase RHOA. The function of these mutations has remained unresolved. We demonstrate that RHOAY42C, the most common RHOA mutation in DGC, is a gain-of-function oncogenic mutant, and that expression of RHOAY42C with inactivation of the canonical tumor suppressor Cdh1 induces metastatic DGC in a mouse model. Biochemically, RHOAY42C exhibits impaired GTP hydrolysis and enhances interaction with its effector ROCK. RHOA Y42C mutation and Cdh1 loss induce actin/cytoskeletal rearrangements and activity of focal adhesion kinase (FAK), which activates YAP-TAZ, PI3K-AKT, and ß-catenin. RHOAY42C murine models were sensitive to FAK inhibition and to combined YAP and PI3K pathway blockade. These results, coupled with sensitivity to FAK inhibition in patient-derived DGC cell lines, nominate FAK as a novel target for these cancers. SIGNIFICANCE: The functional significance of recurrent RHOA mutations in DGC has remained unresolved. Through biochemical studies and mouse modeling of the hotspot RHOAY42C mutation, we establish that these mutations are activating, detail their effects upon cell signaling, and define how RHOA-mediated FAK activation imparts sensitivity to pharmacologic FAK inhibitors.See related commentary by Benton and Chernoff, p. 182.This article is highlighted in the In This Issue feature, p. 161.

Inflammation-Sensitive Myosin-X Functionally Supports Leukocyte Extravasation by Cdc42-Mediated ICAM-1-Rich Endothelial Filopodia Formation.

Leukocyte transendothelial migration is key to inflammation. Leukocytes first start rolling over the inflamed endothelium, followed by firmly adhering to it. Under inflammatory conditions, endothelial cells express small finger-like protrusions that stick out into the lumen. The function and regulation of these structures are unclear. We present evidence that these ICAM-1- and F-actin-rich endothelial finger-like protrusions are filopodia and function as adhesive structures for leukocytes to transit from rolling to crawling but are dispensable for diapedesis. Mechanistically, these structures require the motor function of myosin-X, activity of the small GTPase Cdc42, and p21-activated kinase 4. Moreover, myosin-X expression is under control of TNF-α-mediated c-Jun N-terminal kinase activity and is upregulated in human atherosclerotic regions. To our knowledge, this is the first study to identify that regulation of endothelial filopodia is crucial for leukocyte extravasation, in particular for the initiation of leukocyte adhesion under flow conditions.

Endothelial CD2AP Binds the Receptor ICAM-1 To Control Mechanosignaling, Leukocyte Adhesion, and the Route of Leukocyte Diapedesis In Vitro.

Inflammation is driven by excessive transmigration (diapedesis) of leukocytes from the blood to the tissue across the endothelial cell monolayer that lines blood vessels. Leukocyte adhesion, crawling, and transmigration are regulated by clustering of the endothelial mechanosensitive receptor intercellular adhesion molecule-1 (ICAM-1). Whereas several proteins are known to promote ICAM-1 function, the molecular mechanisms that limit ICAM-1-mediated adhesion to prevent excessive leukocyte transmigration remain unknown. We identify the endothelial actin-binding protein CD2-associated protein (CD2AP) as a novel interaction partner of ICAM-1. Loss of CD2AP stimulates the dynamics of ICAM-1 clustering, which facilitates the formation of ICAM-1 complexes on the endothelial cell surface. Consequently, neutrophil adhesion is increased, but crawling is decreased. In turn, this promotes the neutrophil preference for the transcellular over the paracellular transmigration route. Mechanistically, CD2AP is required for mechanosensitive ICAM-1 downstream signaling toward activation of the PI3K, and recruitment of F-actin and of the actin-branching protein cortactin. Moreover, CD2AP is necessary for ICAM-1-induced Rac1 recruitment and activation. Mechanical force applied on ICAM-1 impairs CD2AP binding to ICAM-1, suggesting that a tension-induced negative feedback loop promotes ICAM-1-mediated neutrophil crawling and paracellular transmigration. To our knowledge, these data show for the first time that the mechanoreceptor ICAM-1 is negatively regulated by an actin-binding adaptor protein, i.e., CD2AP, to allow a balanced and spatiotemporal control of its adhesive function. CD2AP is important in kidney dysfunction that is accompanied by inflammation. Our findings provide a mechanistic basis for the role of CD2AP in inflamed vessels, identifying this adaptor protein as a potential therapeutic target.

A local VE-cadherin and Trio-based signaling complex stabilizes endothelial junctions through Rac1.

Endothelial cell-cell junctions maintain a restrictive barrier that is tightly regulated to allow dynamic responses to permeability-inducing angiogenic factors, as well as to inflammatory agents and adherent leukocytes. The ability of these stimuli to transiently remodel adherens junctions depends on Rho-GTPase-controlled cytoskeletal rearrangements. How the activity of Rho-GTPases is spatio-temporally controlled at endothelial adherens junctions by guanine-nucleotide exchange factors (GEFs) is incompletely understood. Here, we identify a crucial role for the Rho-GEF Trio in stabilizing junctions based around vascular endothelial (VE)-cadherin (also known as CDH5). Trio interacts with VE-cadherin and locally activates Rac1 at adherens junctions during the formation of nascent contacts, as assessed using a novel FRET-based Rac1 biosensor and biochemical assays. The Rac-GEF domain of Trio is responsible for the remodeling of junctional actin from radial into cortical actin bundles, a crucial step for junction stabilization. This promotes the formation of linear adherens junctions and increases endothelial monolayer resistance. Collectively, our data show the importance of spatio-temporal regulation of the actin cytoskeleton through Trio and Rac1 at VE-cadherin-based cell-cell junctions in the maintenance of the endothelial barrier.

Cell-stiffness-induced mechanosignaling - a key driver of leukocyte transendothelial migration.

The breaching of cellular and structural barriers by migrating cells is a driving factor in development, inflammation and tumor cell metastasis. One of the most extensively studied examples is the extravasation of activated leukocytes across the vascular endothelium, the inner lining of blood vessels. Each step of this leukocyte transendothelial migration (TEM) process is regulated by distinct endothelial adhesion receptors such as the intercellular adhesion molecule 1 (ICAM1). Adherent leukocytes exert force on these receptors, which sense mechanical cues and transform them into localized mechanosignaling in endothelial cells. In turn, the function of the mechanoreceptors is controlled by the stiffness of the endothelial cells and of the underlying substrate representing a positive-feedback loop. In this Commentary, we focus on the mechanotransduction in leukocytes and endothelial cells, which is induced in response to variations in substrate stiffness. Recent studies have described the first key proteins involved in these mechanosensitive events, allowing us to identify common regulatory mechanisms in both cell types. Finally, we discuss how endothelial cell stiffness controls the individual steps in the leukocyte TEM process. We identify endothelial cell stiffness as an important component, in addition to locally presented chemokines and adhesion receptors, which guides leukocytes to sites that permit TEM.

Toward understanding RhoGTPase specificity: structure, function and local activation.

Cell adhesion and migration are regulated through the concerted action of cytoskeletal dynamics and adhesion proteins, the activity of which is governed by RhoGTPases. Specific RhoGTPase signaling requires spatio-temporal activation and coordination of subsequent protein-protein and protein-lipid interactions. The nature, location and duration of these interactions are dependent on polarized extracellular triggers, such as cell-cell contact, and intracellular modifying events, such as phosphorylation. RhoA, RhoB, and RhoC are highly homologous GTPases that, however, succeed in generating specific intracellular responses. Here, we discuss the key features that contribute to this specificity. These not only include the well-studied switch regions, the conformation of which is nucleotide-dependent, but also additional regions and seemingly small differences in primary sequence that also contribute to specific interactions. These differences translate into differential surface charge distribution, local exposure of amino acid side-chains and isoform-specific post-translational modifications. The available evidence supports the notion that multiple regions in RhoA/B/C cooperate to provide specificity in binding to regulators and effectors. These specific interactions are highly regulated in time and space. We therefore subsequently discuss current approaches means to visualize and analyze localized GTPase activation using biosensors that allow imaging of isoform-specific, localized regulation.

Actin-binding proteins differentially regulate endothelial cell stiffness, ICAM-1 function and neutrophil transmigration.

Chronic vascular inflammation is driven by interactions between activated leukocytes and the endothelium. Leukocyte ß2-integrins bind to endothelial intercellular adhesion molecule 1 (ICAM-1), which allows leukocyte spreading, crawling and transendothelial migration. Leukocytes scan the vascular endothelium for permissive sites to transmigrate, which suggests that there is apical membrane heterogeneity within the endothelium. However, the molecular basis for this heterogeneity is unknown. Leukocyte adhesion induces ICAM-1 clustering, which promotes its association to the actin-binding proteins filamin B, α-actinin-4 and cortactin. We show that these endothelial proteins differentially control adhesion, spreading and transmigration of neutrophils. Loss of filamin B, α-actinin-4 and cortactin revealed adaptor-specific effects on a nuclear-to-peripheral gradient of endothelial cell stiffness. By contrast, increasing endothelial cell stiffness stimulates ICAM-1 function. We identify endothelial α-actinin-4 as a key regulator of endothelial cell stiffness and of ICAM-1-mediated neutrophil transmigration. Finally, we found that the endothelial lining of human and murine atherosclerotic plaques shows elevated levels of α-actinin-4. These results identify endothelial cell stiffness as an important regulator of endothelial surface heterogeneity and of ICAM-1 function, which in turn controls the adhesion and transmigration of neutrophils.

The human minor histocompatibility antigen 1 is a RhoGAP.

The human minor Histocompatibility Antigen HMHA-1 is a major target of immune responses after allogeneic stem cell transplantation applied for the treatment of leukemia and solid tumors. The restriction of its expression to hematopoietic cells and many solid tumors raised questions regarding its cellular functions. Sequence analysis of the HMHA-1 encoding HMHA1 protein revealed the presence of a possible C-terminal RhoGTPase Activating Protein (GAP) domain and an N-terminal BAR domain. Rho-family GTPases, including Rac1, Cdc42, and RhoA are key regulators of the actin cytoskeleton and control cell spreading and migration. RhoGTPase activity is under tight control as aberrant signaling can lead to pathology, including inflammation and cancer. Whereas Guanine nucleotide Exchange Factors (GEFs) mediate the exchange of GDP for GTP resulting in RhoGTPase activation, GAPs catalyze the low intrinsic GTPase activity of active RhoGTPases, resulting in inactivation. Here we identify the HMHA1 protein as a novel RhoGAP. We show that HMHA1 constructs, lacking the N-terminal region, negatively regulate the actin cytoskeleton as well as cell spreading. Furthermore, we show that HMHA1 regulates RhoGTPase activity in vitro and in vivo. Finally, we demonstrate that the HMHA1 N-terminal BAR domain is auto-inhibitory as HMHA1 mutants lacking this region, but not full-length HMHA1, showed GAP activity towards RhoGTPases. In conclusion, this study shows that HMHA1 acts as a RhoGAP to regulate GTPase activity, cytoskeletal remodeling and cell spreading, which are crucial functions in normal hematopoietic and cancer cells.