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The mitochondrial antiviral signaling protein (MAVS) orchestrates host antiviral innate immune response to RNA virus infection. However, how MAVS signaling is controlled to eradicate virus while preventing self-destructive inflammation remains obscure. Here, we show that protein geranylgeranylation, a posttranslational lipid modification of proteins, limits MAVS-mediated immune signaling by targeting Rho family small guanosine triphosphatase Rac1 into the mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs) at the mitochondria-ER junction. Protein geranylgeranylation and subsequent palmitoylation promote Rac1 translocation into MAMs upon viral infection. MAM-localized Rac1 limits MAVS' interaction with E3 ligase Trim31 and hence inhibits MAVS ubiquitination, aggregation, and activation. Rac1 also facilitates the recruitment of caspase-8 and cFLIP to the MAVS signalosome and the subsequent cleavage of Ripk1 that terminates MAVS signaling. Consistently, mice with myeloid deficiency of protein geranylgeranylation showed improved survival upon influenza A virus infection. Our work revealed a critical role of protein geranylgeranylation in regulating antiviral innate immune response.
Reactive oxygen species (ROS) have an important pathogenic role in the development of many diseases, including kidney disease. Major ROS generators in the glomerulus of the kidney are the p47(phox)-containing NAPDH oxidases NOX1 and NOX2. The cytosolic p47(phox) subunit is a key regulator of the assembly and function of NOX1 and NOX2 and its expression and phosphorylation are upregulated in the course of renal injury, and have been shown to exacerbate diabetic nephropathy. However, its role in nondiabetic-mediated glomerular injury is unclear. To address this, we subjected p47(phox)-null mice to either adriamycin-mediated or partial renal ablation-mediated glomerular injury. Deletion of p47(phox) protected the mice from albuminuria and glomerulosclerosis in both injury models. Integrin α1-null mice develop more severe glomerulosclerosis compared with wild-type mice in response to glomerular injury mainly due to increased production of ROS. Interestingly, the protective effects of p47(phox) knockout were more profound in p47(phox)/integrin α1 double knockout mice. In vitro analysis of primary mesangial cells showed that deletion of p47(phox) led to reduced basal levels of superoxide and collagen IV production. Thus, p47(phox)-dependent NADPH oxidases are a major glomerular source of ROS, contribute to kidney injury, and are potential targets for antioxidant therapy in fibrotic disease.
CD148 is a transmembrane tyrosine phosphatase that is expressed at cell junctions. Recent studies have shown that CD148 associates with the cadherin/catenin complex and p120 catenin (p120) may serve as a substrate. However, the role of CD148 in cadherin cell-cell adhesion remains unknown. Therefore, here we addressed this issue using a series of stable cells and cell-based assays. Wild-type (WT) and catalytically inactive (CS) CD148 were introduced to A431D (lacking classical cadherins), A431D/E-cadherin WT (expressing wild-type E-cadherin), and A431D/E-cadherin 764AAA (expressing p120-uncoupled E-cadherin mutant) cells. The effects of CD148 in cadherin adhesion were assessed by Ca2+ switch and cell aggregation assays. Phosphorylation of E-cadherin/catenin complex and Rho family GTPase activities were also examined. Although CD148 introduction did not alter the expression levels and complex formation of E-cadherin, p120, and β-catenin, CD148 WT, but not CS, promoted cadherin contacts and strengthened cell-cell adhesion in A431D/E-cadherin WT cells. This effect was accompanied by an increase in Rac1, but not RhoA and Cdc42, activity and largely diminished by Rac1 inhibition. Further, we demonstrate that CD148 reduces the tyrosine phosphorylation of p120 and β-catenin; causes the dephosphorylation of Y529 suppressive tyrosine residue in Src, a well-known CD148 site, increasing Src activity and enhancing the phosphorylation of Y228 (a Src kinase site) in p120, in E-cadherin contacts. Consistent with these findings, CD148 dephosphorylated both p120 and β-catenin in vitro. The shRNA-mediated CD148 knockdown in A431 cells showed opposite effects. CD148 showed no effects in A431D and A431D/E-cadherin 764AAA cells. In aggregate, these findings provide the first evidence that CD148 promotes E-cadherin adhesion by regulating Rac1 activity concomitant with modulation of p120, β-catenin, and Src tyrosine phosphorylation. This effect requires E-cadherin and p120 association.
Phosphatidylinositol 3-kinase (PI3K) promotes cancer cell survival, migration, growth and proliferation by generating phosphatidylinositol 3,4,5-trisphosphate (PIP3) in the inner leaflet of the plasma membrane. PIP3 recruits pleckstrin homology domain-containing proteins to the membrane to activate oncogenic signaling cascades. Anticancer therapeutics targeting the PI3K/AKT/mTOR (mammalian target of rapamycin) pathway are in clinical development. In a mass spectrometric screen to identify PIP3-regulated proteins in breast cancer cells, levels of the Rac activator PIP3-dependent Rac exchange factor-1 (P-REX1) increased in response to PI3K inhibition, and decreased upon loss of the PI3K antagonist phosphatase and tensin homolog (PTEN). P-REX1 mRNA and protein levels were positively correlated with ER expression, and inversely correlated with PI3K pathway activation in breast tumors as assessed by gene expression and phosphoproteomic analyses. P-REX1 increased activation of Rac1, PI3K/AKT and MEK/ERK signaling in a PTEN-independent manner, and promoted cell and tumor viability. Loss of P-REX1 or inhibition of Rac suppressed PI3K/AKT and MEK/ERK, and decreased viability. P-REX1 also promoted insulin-like growth factor-1 receptor activation, suggesting that P-REX1 provides positive feedback to activators upstream of PI3K. In support of a model where PIP3-driven P-REX1 promotes both PI3K/AKT and MEK/ERK signaling, high levels of P-REX1 mRNA (but not phospho-AKT or a transcriptomic signature of PI3K activation) were predictive of sensitivity to PI3K inhibitors among breast cancer cell lines. P-REX1 expression was highest in estrogen receptor-positive breast tumors compared with many other cancer subtypes, suggesting that neutralizing the P-REX1/Rac axis may provide a novel therapeutic approach to selectively abrogate oncogenic signaling in breast cancer cells.
Non-muscle myosin II (MyoII) contractility is central to the regulation of numerous cellular processes, including migration. Rho is a well-characterized modulator of actomyosin contractility, but the function of other GTPases, such as Rac, in regulating contractility is currently not well understood. Here, we show that activation of Rac by the guanine nucleotide exchange factor Asef2 (also known as SPATA13) impairs migration on type I collagen through a MyoII-dependent mechanism that enhances contractility. Knockdown of endogenous Rac or treatment of cells with a Rac-specific inhibitor decreases the amount of active MyoII, as determined by serine 19 (S19) phosphorylation, and negates the Asef2-promoted increase in contractility. Moreover, treatment of cells with blebbistatin, which inhibits MyoII activity, abolishes the Asef2-mediated effect on migration. In addition, Asef2 slows the turnover of adhesions in protrusive regions of cells by promoting large mature adhesions, which has been linked to actomyosin contractility, with increased amounts of active β1 integrin. Hence, our data reveal a new role for Rac activation, promoted by Asef2, in modulating actomyosin contractility, which is important for regulating cell migration and adhesion dynamics.
The spatial distribution of molecular signals within cells is crucial for cellular functions. Here, as a model to study the polarized spatial distribution of molecular activities, we used cells on micropatterned strips of fibronectin with one end free and the other end contacting a neighbouring cell. Phosphoinositide 3-kinase and the small GTPase Rac display greater activity at the free end, whereas myosin II light chain and actin filaments are enriched near the intercellular junction. Phosphoinositide 3-kinase and Rac polarization depend specifically on the N-cadherin-p120 catenin complex, whereas myosin II light chain and actin filament polarization depend on the N-cadherin-β-catenin complex. Integrins promote high phosphoinositide 3-kinase/Rac activities at the free end, and the N-cadherin-p120 catenin complex excludes integrin α5 at the junctions to suppress local phosphoinositide 3-kinase and Rac activity. We hence conclude that N-cadherin couples with distinct effectors to polarize phosphoinositide 3-kinase/Rac and myosin II light chain/actin filaments in migrating cells.
Migrating cells possess intracellular gradients of active Rho GTPases, which serve as central hubs in transducing signals from extracellular receptors to cytoskeletal and adhesive machinery. However, it is unknown whether shallow exogenously induced intracellular gradients of Rho GTPases are sufficient to drive cell polarity and motility. Here, we use microfluidic control to generate gradients of a small molecule and thereby directly induce linear gradients of active, endogenous Rac without activation of chemotactic receptors. Gradients as low as 15% were sufficient not only to trigger cell migration up the chemical gradient but to induce both cell polarization and repolarization. Cellular response times were inversely proportional to the steepness of Rac inducer gradient in agreement with a mathematical model, suggesting a function for chemoattractant gradient amplification upstream of Rac. Increases in activated Rac levels beyond a well-defined threshold augmented polarization and decreased sensitivity to the imposed gradient. The threshold was governed by initial cell polarity and PI3K activity, supporting a role for both in defining responsiveness to Rac activation. Our results reveal that Rac can serve as a starting point in defining cell polarity. Furthermore, our methodology may serve as a template to investigate processes regulated by intracellular signaling gradients.
The small GTPase Rac is known to be an important regulator of cell polarization, cytoskeletal reorganization, and motility of mammalian cells. In recent microfluidic experiments, HeLa cells endowed with appropriate constructs were subjected to gradients of the small molecule rapamycin leading to synthetic membrane recruitment of a Rac activator and direct graded activation of membrane-associated Rac. Rac activation could thus be triggered independent of upstream signaling mechanisms otherwise responsible for transducing activating gradient signals. The response of the cells to such stimulation depended on exceeding a threshold of activated Rac. Here we develop a minimal reaction-diffusion model for the GTPase network alone and for GTPase-phosphoinositide crosstalk that is consistent with experimental observations for the polarization of the cells. The modeling suggests that mutual inhibition is a more likely mode of cell polarization than positive feedback of Rac onto its own activation. We use a new analytical tool, Local Perturbation Analysis, to approximate the partial differential equations by ordinary differential equations for local and global variables. This method helps to analyze the parameter space and behaviour of the proposed models. The models and experiments suggest that (1) spatially uniform stimulation serves to sensitize a cell to applied gradients. (2) Feedback between phosphoinositides and Rho GTPases sensitizes a cell. (3) Cell lengthening/flattening accompanying polarization can increase the sensitivity of a cell and stabilize an otherwise unstable polarization.
The heritable form of pulmonary arterial hypertension (PAH) is typically caused by a mutation in bone morphogenic protein receptor type 2 (BMPR2), and mice expressing Bmpr2 mutations develop PAH with features similar to human disease. BMPR2 is known to interact with the cytoskeleton, and human array studies in PAH patients confirm alterations in cytoskeletal pathways. The goal of this study was to evaluate cytoskeletal defects in BMPR2-associated PAH. Expression arrays on our Bmpr2 mutant mouse lungs revealed cytoskeletal defects as a prominent molecular consequence of universal expression of a Bmpr2 mutation (Rosa26-Bmpr2(R899X)). Pulmonary microvascular endothelial cells cultured from these mice have histological and functional cytoskeletal defects. Stable transfection of different BMPR2 mutations into pulmonary microvascular endothelial cells revealed that cytoskeletal defects are common to multiple BMPR2 mutations and are associated with activation of the Rho GTPase, Rac1. Rac1 defects are corrected in cell culture and in vivo through administration of exogenous recombinant human angiotensin-converting enzyme 2 (rhACE2). rhACE2 reverses 77% of gene expression changes in Rosa26-Bmpr2(R899X) transgenic mice, in particular, correcting defects in cytoskeletal function. Administration of rhACE2 to Rosa26-Bmpr2(R899X) mice with established PAH normalizes pulmonary pressures. Together, these findings suggest that cytoskeletal function is central to the development of BMPR2-associated PAH and that intervention against cytoskeletal defects may reverse established disease.
Brain-derived neurotrophic factor (BDNF) was shown to play a role in Schwann cell myelination by recruiting Par3 to the axon-glial interface, but the underlying mechanism has remained unclear. Here we report that Par3 regulates Rac1 activation by BDNF but not by NRG1-Type III in Schwann cells, although both ligands activate Rac1 in vivo. During development, active Rac1 signaling is localized to the axon-glial interface in Schwann cells by a Par3-dependent polarization mechanism. Knockdown of p75 and Par3 individually inhibits Rac1 activation, whereas constitutive activation of Rac1 disturbs the polarized activation of Rac1 in vivo. Polarized Rac1 activation is necessary for myelination as Par3 knockdown attenuates myelination in mouse sciatic nerves as well as in zebrafish. Specifically, Par3 knockdown in zebrafish disrupts proper alignment between the axon and Schwann cells without perturbing Schwann cell migration, suggesting that localized Rac1 activation at the axon-glial interface helps identify the initial wrapping sites. We therefore conclude that polarization of Rac1 activation is critical for myelination.