KIF13A‐regulated RhoB plasma membrane localization governs membrane blebbing and blebby amoeboid cell migration

Membrane blebbing‐dependent (blebby) amoeboid migration can be employed by lymphoid and cancer cells to invade 3D‐environments. Here, we reveal a mechanism by which the small GTPase RhoB controls membrane blebbing and blebby amoeboid migration. Interestingly, while all three Rho isoforms (RhoA, RhoB and RhoC) regulated amoeboid migration, each controlled motility in a distinct manner. In particular, RhoB depletion blocked membrane blebbing in ALL (acute lymphoblastic leukaemia), melanoma and lung cancer cells as well as ALL cell amoeboid migration in 3D‐collagen, while RhoB overexpression enhanced blebbing and 3D‐collagen migration in a manner dependent on its plasma membrane localization and down‐stream effectors ROCK and Myosin II. RhoB localization was controlled by endosomal trafficking, being internalized via Rab5 vesicles and then trafficked either to late endosomes/lysosomes or to Rab11‐positive recycling endosomes, as regulated by KIF13A. Importantly, KIF13A depletion not only inhibited RhoB plasma membrane localization, but also cell membrane blebbing and 3D‐migration of ALL cells. In conclusion, KIF13A‐mediated endosomal trafficking modulates RhoB plasma membrane localization to control membrane blebbing and blebby amoeboid migration.     

Phosphorylation of RhoB by CK1 impedes actin stress fiber organization and epidermal growth factor receptor stabilization

RhoB is a small GTPase implicated in cytoskeletal organization, EGF receptor trafficking and cell transformation. It is an immediate-early gene, regulated at many levels of its biosynthetic pathway. Herein we show that the serine/threonine protein kinase CK1 phosphorylates RhoB in vitro but not RhoA or RhoC. With the use of specific CK1 inhibitors, IC261 and D4476, we show that the kinase phosphorylates also RhoB in HeLa cells. Mass spectrometry analysis demonstrates that RhoB is monophosphorylated by CK1, in its C-terminal end, on serine 185. The substitution of Ser185 by Ala dramatically inhibited the phosphorylation of RhoB in cultured cells. Lastly we show that the inhibition of CK1 activates RhoB and promotes RhoB dependent actin fiber formation and EGF-R level. Our data provide the first demonstration of RhoB phosphorylation and indicate that this post-translational maturation would be a novel critical mechanism to control the RhoB functions.     

MAP1A light chain-2 interacts with GTP-RhoB to control epidermal growth factor (EGF)-dependent EGF receptor signaling

Rho GTPases have been implicated in the control of several cellular functions, including regulation of the actin cytoskeleton, cell proliferation, and oncogenesis. Unlike RhoA and RhoC, RhoB localizes in part to endosomes and controls endocytic trafficking. Using a yeast two-hybrid screen and a glutathione S-transferase pulldown assay, we identified LC2, the light chain of the microtubule-associated protein MAP1A, as a novel binding partner for RhoB. GTP binding and the 18-amino acid C-terminal hypervariable domain of RhoB are critical for its binding to MAP1A/LC2. Coimmunoprecipitation and immunofluorescence experiments showed that this interaction occurs in U87 cells. Down-regulation of MAP1A/LC2 expression decreased epidermal growth factor (EGF) receptor expression and modified the signaling response to EGF treatment. We concluded that MAP1A/LC2 is critical for RhoB function in EGF-induced EGF receptor regulation. Because MAP1A/LC2 is thought to function as an adaptor between microtubules and other molecules, we postulate that the RhoB and MAP1A/LC2 interactions facilitate endocytic vesicle trafficking and regulate the trafficking of signaling molecules.     

RhoB and the mammalian Diaphanous-related formin mDia2 in endosome trafficking

Rho GTPases and the dynamic assembly and disassembly of actin filaments have been shown to have critical roles in both the internalization and trafficking of growth factor receptors. While all three mammalian Diaphanous-related (mDia1/2/3) formin GTPase effector proteins have been localized on endosomes, a role for their actin nucleation, filament elongation, and/or bundling remains poorly understood in the context of intracellular trafficking. In a study of a functional relationship between RhoB, a GTPase known to associate with both early- and late-endosomes, and the formin mDia2, we show that 1) RhoB and mDia2 interact on endosomes; 2) GTPase activity-the ability to hydrolyze GTP to GDP-is required for the ability of RhoB to govern endosome dynamics; and 3) the actin dynamics controlled by RhoB and mDia2 is necessary for vesicle trafficking. These studies further suggest that Rho GTPases significantly influence the activity of mDia family formins in driving cellular membrane remodeling through the regulation of actin dynamics.     

RhoB protects human keratinocytes from UVB-induced apoptosis through epidermal growth factor receptor signaling

Exposure of the skin to UVB light results in the formation of DNA photolesions that can give rise to cell death, mutations, and the onset of carcinogenic events. Specific proteins are activated by UVB and then trigger signal transduction pathways that lead to cellular responses. An alteration of these signaling molecules is thought to be a fundamental event in tumor promotion by UVB irradiation. RhoB, encoding a small GTPase has been identified as a DNA damage-inducible gene. RhoB is involved in epidermal growth factor (EGF) receptor trafficking, cytoskeletal organization, cell transformation, and survival. We have analyzed the regulation of RhoB and elucidated its role in the cellular response of HaCaT keratinocytes to relevant environmental UVB irradiation. We report here that the activated GTP-bound form of RhoB is increased rapidly within 5 min of exposure to UVB, and then RhoB protein levels increased concomitantly with EGF receptor (EGFR) activation. Inhibition of UVB-induced EGFR activation prevents RhoB protein expression and AKT phosphorylation but not the early activation of RhoB. Blocking UVB-induced RhoB expression with specific small interfering RNAs inhibits AKT and glycogen synthase kinase-3beta phosphorylation through inhibition of EGFR expression. Moreover, down-regulation of RhoB potentiates UVB-induced cell apoptosis. In contrast, RhoB overexpression protects keratinocytes against UVB-induced apoptosis. These results indicated that RhoB is regulated upon UVB exposure by a two-step process consisting of an early EGFR-independent RhoB activation followed by an EGFR-dependent induction of RhoB expression. Moreover, we have demonstrated that RhoB is essential in regulating keratinocyte cell survival after UVB exposure, suggesting its potential role in photocarcinogenesis.     

DLC1 Activation Requires Lipid Interaction through a Polybasic Region Preceding the RhoGAP Domain

Deleted in Liver Cancer 1 (DLC1) is a GTPase-activating protein (GAP) with specificity for RhoA, RhoB, and RhoC that is frequently deleted in various tumor types. By inactivating these small GTPases, DLC1 controls actin cytoskeletal remodeling and biological processes such as cell migration and proliferation. Here we provide evidence that DLC1 binds to phosphatidylinositol-4,5-bisphosphate (PI(4,5)P₂) through a previously unrecognized polybasic region (PBR) adjacent to its RhoGAP domain. Importantly, PI(4,5)P₂-containing membranes are shown to stimulate DLC1 GAP activity in vitro. In living cells, a DLC1 mutant lacking an intact PBR inactivated Rho signaling less efficiently and was severely compromised in suppressing cell spreading, directed migration, and proliferation. We therefore propose that PI(4,5)P₂ is an important cofactor in DLC1 regulation in vivo and that the PBR is essential for the cellular functions of the protein.     

RhoB的翻译后修饰与细胞命运

RhoB作为Rho家族的一员,其生物学活性和蛋白质水平的调控与其他成员有着较大的不同,在肿瘤的发生发展中也起着独特的作用。RhoB作为抑癌蛋白在肿瘤的靶向治疗上受到越来越多的关注,然而在有些类型的肿瘤中RhoB却起着促进肿瘤生长的作用,其中的分子机理还不清楚,亟待研究阐明。可逆的翻译后修饰是快速与精细调控RhoB功能的重要分子机制,对于维持正常细胞的生长、抑制细胞的早期癌变及肿瘤的发生发展至关重要。本文就RhoB翻译后修饰的研究,特别是其泛素化和SUMO化修饰之间的转化在肿瘤细胞命运决定中的作用进行综述,以期为探索RhoB的调控与肿瘤发生发展的机制,以及以RhoB为靶点的癌症治疗提供线索和思路。     

Rho proteins: linking signaling with membrane trafficking

Rho proteins are well known for their effects on the actin cytoskeleton, and are activated in response to a variety of extracellular stimuli. Several Rho family members are localized to vesicular compartments, and increasing evidence suggests that they play important roles in the trafficking of vesicles on both endocytic and exocytic pathways. In particular, RhoA, RhoB, RhoD, Rac and Cdc42 have been shown to affect various steps of membrane trafficking. The underlying molecular basis for these effects of Rho proteins are incompletely understood, but in the case of Cdc42 it appears that it can drive vesicle movement through Arp2/3 complex-mediated actin polymerization at the surface of the vesicle. This is similar to what is believed to happen when Rac and Cdc42 stimulate actin polymerization at the plasma membrane. Rho proteins may also affect membrane trafficking by altering phosphatidylinositide composition of membrane compartments, or through interactions with microtubules.     

Structural Determinants Allowing Endolysosomal Sorting and Degradation of Endosomal GTPases

Rapid control of protein degradation is usually achieved through the ubiquitin‐proteasome pathway. We recently found that the short‐lived GTPase RhoB is degraded in lysosomes. Moreover, the fusion of the RhoB C‐terminal sequence CINCCKVL, containing the isoprenylation and palmitoylation sites, to other proteins directs their sorting into multivesicular bodies (MVBs) and rapid lysosomal degradation. Here, we show that this process is highly specific for RhoB. Alteration of late endosome lipid dynamics produced the accumulation of RhoB, but not of other endosomal GTPases, including Rab5, Rab7, Rab9 or Rab11, into enlarged MVB. Other isoprenylated and bipalmitoylated GTPases, such as H‐Ras, Rap2A, Rap2B and TC10, were not accumulated into MVB and were stable. Remarkably, although TC10, which is highly homologous to RhoB, was stable, a sequence derived from its C‐terminus (CINCCLIT) elicited MVB sorting and degradation of a green fluorescent protein (GFP)‐chimeric protein. This led us to identify a cluster of basic amino acids (KKH) in the TC10 hypervariable region, constituting a secondary signal potentially involved in electrostatic interactions with membrane lipids. Mutation of this cluster allowed TC10 MVB sorting and degradation, whereas inserting it into RhoB hypervariable region rescued this protein from its lysosomal degradation pathway. These findings define a highly specific structural module for entering the MVB pathway and rapid lysosomal degradation.     

Morphological changes and nuclear translocation of DLC1 tumor suppressor protein precede apoptosis in human non-small cell lung carcinoma cells

We have previously shown that reactivation of DLC1, a RhoGAP containing tumor suppressor gene, inhibits tumorigenicity of human non-small cell lung carcinoma cells (NSCLC). After transfection of NSCLC cells with wild type (WT) DLC1, changes in cell morphology were observed. To determine whether such changes have functional implications, we generated several DLC1 mutants and examined their effects on cell morphology, proliferation, migration and apoptosis in a DLC1 deficient NSCLC cell line. We show that WT DLC1 caused actin cytoskeleton-based morphological alterations manifested as cytoplasmic extensions and membrane blebbings in most cells. Subsequently, a fraction of cells exhibiting DLC1 protein nuclear translocation (PNT) underwent caspase 3-dependent apoptosis. We also show that the RhoGAP domain is essential for the occurrence of morphological alterations, PNT and apoptosis, and the inhibition of cell migration. DLC1 PNT is dependent on a bipartite nuclear localizing sequence and most likely is regulated by a serine-rich domain at N-terminal part of the DLC1 protein. Also, we found that DLC1 functions in the cytoplasm as an inhibitor of tumor cell proliferation and migration, but in the nucleus as an inducer of apoptosis. Our analyses provide evidence for a possible link between morphological alterations, PNT and proapoptotic and anti-oncogenic activities of DLC1 in lung cancer.     

RhoB and actin polymerization coordinate Src activation with endosome-mediated delivery to the membrane

We have used a c-Src-GFP fusion protein to address the spatial control of Src activation and the nature of Src-associated intracellular structures during stimulus-induced transit to the membrane. Src is activated during transit, particularly in RhoB-containing cytoplasmic endosomes associated with the perinuclear recycling compartment. Knocking out RhoB or expressing a dominant-interfering Rab11 mutant suppresses both catalytic activation of Src and translocation of active kinase to peripheral membrane structures. In addition, the Src- and RhoB-containing endosomes harbor proteins involved in actin polymerization and filament assembly, for example Scar1, and newly polymerized actin can associate with these endosomes in a Src-dependent manner. This implies that Src may regulate an endosome-associated actin nucleation activity. In keeping with this, Src controls the actin dependence of RhoB endosome movement toward the plasma membrane. This work identifies RhoB as a component of "outside-in" signaling pathways that coordinate Src activation with translocation to transmembrane receptors.     

DLC1 interaction with α-catenin stabilizes adherens junctions and enhances DLC1 antioncogenic activity

The DLC1 (for deleted in liver cancer 1) tumor suppressor gene encodes a RhoGAP protein that inactivates Rho GTPases, which are implicated in regulation of the cytoskeleton and adherens junctions (AJs), a cell-cell adhesion protein complex associated with the actin cytoskeleton. Malignant transformation and tumor progression to metastasis are often associated with changes in cytoskeletal organization and cell-cell adhesion. Here we have established in human cells that the AJ-associated protein α-catenin is a new binding partner of DLC1. Their binding was mediated by the N-terminal amino acids 340 to 435 of DLC1 and the N-terminal amino acids 117 to 161 of α-catenin. These proteins colocalized in the cytosol and in the plasma membrane, where together they associated with E-cadherin and β-catenin, constitutive AJ proteins. Binding of DLC1 to α-catenin led to their accumulation at the plasma membrane and required DLC1 GAP activity. Knocking down α-catenin in DLC1-positive cells diminished DLC1 localization at the membrane. The DLC1-α-catenin complex reduced the Rho GTP level at the plasma membrane, increased E-cadherin's mobility, affected actin organization, and stabilized AJs. This process eventually contributed to a robust oncosuppressive effect of DLC1 in metastatic prostate carcinoma cells. Together, these results unravel a new mechanism through which DLC1 exerts its strong oncosuppressive function by positively influencing AJ stability.     

RhoB in cancer suppression

RhoB is a mainly endosomal small GTPase that regulates actin organization and vesicle trafficking. Expression of RhoB is elevated rapidly by many stimuli, including growth factors, cytokines, and genotoxic stress. In cancer, RhoB can limit cell proliferation, survival, invasion, and metastasis, and during malignant progression its levels are attenuated commonly. In support of its role as a negative modifier of cancer progression, targeted deletion of RhoB in mice can increase tumor formation initiated by Ras mutation. How RhoB acts to suppress different aspects of cancer pathophysiology has emerged as a question of significant interest.     

Farnesylated RhoB inhibits radiation-induced mitotic cell death and controls radiation-induced centrosome overduplication

Our previous results demonstrated that expressing the GTPase ras homolog gene family, member B (RhoB) in radiosensitive NIH3T3 cells increases their survival following 2 Gy irradiation (SF2). We have first demonstrated here that RhoB expression inhibits radiation-induced mitotic cell death. RhoB is present in both a farnesylated and a geranylgeranylated form in vivo. By expressing RhoB mutants encoding for farnesylated (RhoB-F cells), geranylgeranylated or the CAAX deleted form of RhoB, we have then shown that only RhoB-F expression was able to increase the SF2 value by reducing the sensitivity of these cells to radiation-induced mitotic cell death. Moreover, RhoB-F cells showed an increased G2 arrest and an inhibition of centrosome overduplication following irradiation mediated by the Rho-kinase, strongly suggesting that RhoB-F may control centrosome overduplication during the G2 arrest after irradiation. Overall, our results for the first time clearly implicate farnesylated RhoB as a crucial protein in mediating cellular resistance to radiation-induced nonapoptotic cell death.     

The C-Terminal Sequence of RhoB Directs Protein Degradation through an Endo-Lysosomal Pathway

Background

Protein degradation is essential for cell homeostasis. Targeting of proteins for degradation is often achieved by specific protein sequences or posttranslational modifications such as ubiquitination.

Methodology/Principal Findings

By using biochemical and genetic tools we have monitored the localization and degradation of endogenous and chimeric proteins in live primary cells by confocal microscopy and ultra-structural analysis. Here we identify an eight amino acid sequence from the C-terminus of the short-lived GTPase RhoB that directs the rapid degradation of both RhoB and chimeric proteins bearing this sequence through a lysosomal pathway. Elucidation of the RhoB degradation pathway unveils a mechanism dependent on protein isoprenylation and palmitoylation that involves sorting of the protein into multivesicular bodies, mediated by the ESCRT machinery. Moreover, RhoB sorting is regulated by late endosome specific lipid dynamics and is altered in human genetic lipid traffic disease.

Conclusions/Significance

Our findings characterize a short-lived cytosolic protein that is degraded through a lysosomal pathway. In addition, we define a novel motif for protein sorting and rapid degradation, which allows controlling protein levels by means of clinically used drugs.     

Dynein light chain regulates axonal trafficking and synaptic levels of Bassoon

Bassoon and the related protein Piccolo are core components of the presynaptic cytomatrix at the active zone of neurotransmitter release. They are transported on Golgi-derived membranous organelles, called Piccolo-Bassoon transport vesicles (PTVs), from the neuronal soma to distal axonal locations, where they participate in assembling new synapses. Despite their net anterograde transport, PTVs move in both directions within the axon. How PTVs are linked to retrograde motors and the functional significance of their bidirectional transport are unclear. In this study, we report the direct interaction of Bassoon with dynein light chains (DLCs) DLC1 and DLC2, which potentially link PTVs to dynein and myosin V motor complexes. We demonstrate that Bassoon functions as a cargo adapter for retrograde transport and that disruption of the Bassoon–DLC interactions leads to impaired trafficking of Bassoon in neurons and affects the distribution of Bassoon and Piccolo among synapses. These findings reveal a novel function for Bassoon in trafficking and synaptic delivery of active zone material.