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The loss of E-cadherin causes dysfunction of the cell-cell junction machinery, which is an initial step in epithelial-to-mesenchymal transition (EMT), facilitating cancer cell invasion and the formation of metastases. A set of transcriptional repressors of E-cadherin (CDH1) gene expression, including Snail1, Snail2 and Zeb2 mediate E-cadherin downregulation in breast cancer. However, the molecular mechanisms underlying the control of E-cadherin expression in breast cancer progression remain largely unknown. Here, by using global gene expression approaches, we uncover a novel function for Cdc42 GTPase-activating protein (CdGAP) in the regulation of expression of genes involved in EMT. We found that CdGAP used its proline-rich domain to form a functional complex with Zeb2 to mediate the repression of E-cadherin expression in ErbB2-transformed breast cancer cells. Conversely, knockdown of CdGAP expression led to a decrease of the transcriptional repressors Snail1 and Zeb2, and this correlated with an increase in E-cadherin levels, restoration of cell-cell junctions, and epithelial-like morphological changes. In vivo, loss of CdGAP in ErbB2-transformed breast cancer cells impaired tumor growth and suppressed metastasis to lungs. Finally, CdGAP was highly expressed in basal-type breast cancer cells, and its strong expression correlated with poor prognosis in breast cancer patients. Together, these data support a previously unknown nuclear function for CdGAP where it cooperates in a GAP-independent manner with transcriptional repressors to function as a critical modulator of breast cancer through repression of E-cadherin transcription. Targeting Zeb2-CdGAP interactions may represent novel therapeutic opportunities for breast cancer treatment.

Kinases are known to have kinase activity independent functions. To gain further insights into potential kinase-independent functions of SLK/STK2, we have developed a kinase-dead allele, SLK K63R using in vivo CRISPR/Cas technology. Our studies show that blastocysts homozygote for SLK K63R do not develop into viable mice. However, heterozygotes are viable and fertile with no overt phenotypes. Analyses of mouse embryonic fibroblasts show that expression of SLK K63R results in a 50% decrease in kinase activity in heterozygotes. In contrast to previous studies, our data show that SLK does not form homodimers and that the kinase defective allele does not act in a dominant negative fashion. Expression of SLK K63R leads to altered Rac1 and RhoA activity, increased stress fiber formation and delayed focal adhesion turnover. Our data support a previously observed role for SLK in cell migration and suggest that at least 50% kinase activity is sufficient for embryonic development., Competing Interests: Declaration of competing interest None., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Epithelial-to-mesenchymal transition (EMT) plays a crucial role in metastasis, which is the leading cause of death in breast cancer patients. Here, we show that Cdc42 GTPase-activating protein (CdGAP) promotes tumor formation and metastasis to lungs in the HER2-positive (HER2 + ) murine breast cancer model. CdGAP facilitates intravasation, extravasation, and growth at metastatic sites. CdGAP depletion in HER2 + murine primary tumors mediates crosstalk with a Dlc1-RhoA pathway and is associated with a transforming growth factor β (TGF-β)-induced EMT transcriptional signature. CdGAP is positively regulated by TGF-β signaling during EMT and interacts with the adaptor talin to modulate focal adhesion dynamics and integrin activation. Moreover, HER2 + breast cancer patients with high CdGAP mRNA expression combined with a high TGF-β-EMT signature are more likely to present lymph node invasion. Our results suggest CdGAP as a candidate therapeutic target for HER2 + metastatic breast cancer by inhibiting TGF-β and integrin/talin signaling pathways., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

A central factor in the maintenance of tissue integrity is the response of stem cells to variations in the levels of niche signals. In the gut, intestinal stem cells (ISCs) depend on Wnt ligands for self-renewal and proliferation. Transient increases in Wnt signaling promote regeneration after injury or in inflammatory bowel diseases, whereas constitutive activation of this pathway leads to colorectal cancer. Here, we report that Discs large 1 (Dlg1), although dispensable for polarity and cellular turnover during intestinal homeostasis, is required for ISC survival in the context of increased Wnt signaling. RNA sequencing (RNA-seq) and genetic mouse models demonstrated that DLG1 regulates the cellular response to increased canonical Wnt ligands. This occurs via the transcriptional regulation of Arhgap31, a GTPase-activating protein that deactivates CDC42, an effector of the non-canonical Wnt pathway. These findings reveal a DLG1-ARHGAP31-CDC42 axis that is essential for the ISC response to increased niche Wnt signaling., Competing Interests: Declaration of interests D.C.-A. is an employee of Genentech, Inc. and a shareholder of Roche., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Purpose: The study aimed to identify novel genes for idiopathic hypogonadotropic hypogonadism (IHH)., Methods: A cohort of 1387 probands with IHH underwent exome sequencing and de novo, familial, and cohort-wide investigations. Functional studies were performed on 2 p190 Rho GTPase-activating proteins (p190 RhoGAP), ARHGAP35 and ARHGAP5, which involved in vivo modeling in larval zebrafish and an in vitro p190A-GAP activity assay., Results: Rare protein-truncating variants (PTVs; n = 5) and missense variants in the RhoGAP domain (n = 7) in ARHGAP35 were identified in IHH cases (rare variant enrichment: PTV [unadjusted P = 3.1E-06] and missense [adjusted P = 4.9E-03] vs controls). Zebrafish modeling using gnrh3:egfp phenotype assessment showed that mutant larvae with deficient arhgap35a, the predominant ARHGAP35 paralog in the zebrafish brain, display decreased GnRH3-GFP+ neuronal area, a readout for IHH. In vitro GAP activity studies showed that 1 rare missense variant [ARHGAP35 p.(Arg1284Trp)] had decreased GAP activity. Rare PTVs (n = 2) also were discovered in ARHGAP5, a paralog of ARHGAP35; however, arhgap5 zebrafish mutants did not display significant GnRH3-GFP+ abnormalities., Conclusion: This study identified ARHGAP35 as a new autosomal dominant genetic driver for IHH and ARHGAP5 as a candidate gene for IHH. These observations suggest a novel role for the p190 RhoGAP proteins in GnRH neuronal development and integrity., Competing Interests: Conflict of Interest The authors declare no conflict of interest., (Copyright © 2022 American College of Medical Genetics and Genomics. All rights reserved.)

Rho GTPases are regulators of the actin cytoskeleton and their activity is modulated by GTPase-activating proteins (GAPs) and guanine nucleotide exchanging factors (GEFs). Glomerular podocytes have numerous actin-based projections called foot processes and their alteration is characteristic of proteinuric kidney diseases. We reported previously that Rac1 hyperactivation in podocytes causes proteinuria and glomerulosclerosis in mice. However, which GAP and GEF modulate Rac1 activity in podocytes remains unknown. Here, using a proximity-based ligation assay, we identified CdGAP (ARHGAP31) and β-PIX (ARHGEF7) as the major regulatory proteins interacting with Rac1 in human podocytes. CdGAP interacted with β-PIX through its basic region, and upon EGF stimulation, they both translocated to the plasma membrane in podocytes. CdGAP-depleted podocytes had altered cell motility and increased basal Rac1 and Cdc42 activities. When stimulated with EGF, CdGAP-depleted podocytes showed impaired β-PIX membrane-translocation and tyrosine phosphorylation, and reduced activities of Src kinase, focal adhesion kinase, and paxillin. Systemic and podocyte-specific CdGAP-knockout mice developed mild but significant proteinuria, which was exacerbated by Adriamycin. Collectively, these findings show that CdGAP contributes to maintain podocyte function and protect them from injury., (© 2022. The Author(s).)

Microexons are small sized (≤51 bp) exons which undergo extensive alternative splicing in neurons, microglia, embryonic stem cells, and cancer cells, giving rise to cell type specific protein isoforms. Due to their small sizes, microexons provide a unique challenge for the splicing machinery. They frequently lack exon splicer enhancers/repressors and require specialized neighboring trans-regulatory and cis-regulatory elements bound by RNA binding proteins (RBPs) for their inclusion. The functional consequences of including microexons within mRNAs have been extensively documented in the central nervous system (CNS) and aberrations in their inclusion have been observed to lead to abnormal processes. Despite the increasing evidence for microexons impacting cellular physiology within CNS, mechanistic details illustrating their functional importance in diseases of the CNS is still limited. In this review, we discuss the unique characteristics of microexons, and how RBPs participate in regulating their inclusion and exclusion during splicing. We consider recent findings of microexon alternative splicing and their implication for regulating the function of small GTPases in the context of the microglia, and we extrapolate these findings to what is known in neurons. We further discuss the emerging evidence for dysregulation of the Rho GTPase pathway in CNS diseases and the consequences contributed by the mis-splicing of microexons. This article is categorized under: RNA Processing > Splicing Mechanisms RNA Processing > Splicing Regulation/Alternative Splicing RNA in Disease and Development > RNA in Disease., (© 2021 Wiley Periodicals LLC.)

High mortality of prostate cancer patients is primarily due to metastasis. Understanding the mechanisms controlling metastatic processes remains essential to develop novel therapies designed to prevent the progression from localized disease to metastasis. CdGAP plays important roles in the control of cell adhesion, migration, and proliferation, which are central to cancer progression. Here we show that elevated CdGAP expression is associated with early biochemical recurrence and bone metastasis in prostate cancer patients. Knockdown of CdGAP in metastatic castration-resistant prostate cancer (CRPC) PC-3 and 22Rv1 cells reduces cell motility, invasion, and proliferation while inducing apoptosis in CdGAP-depleted PC-3 cells. Conversely, overexpression of CdGAP in DU-145, 22Rv1, and LNCaP cells increases cell migration and invasion. Using global gene expression approaches, we found that CdGAP regulates the expression of genes involved in epithelial-to-mesenchymal transition, apoptosis and cell cycle progression. Subcutaneous injection of CdGAP-depleted PC-3 cells into mice shows a delayed tumor initiation and attenuated tumor growth. Orthotopic injection of CdGAP-depleted PC-3 cells reduces distant metastasic burden. Collectively, these findings support a pro-oncogenic role of CdGAP in prostate tumorigenesis and unveil CdGAP as a potential biomarker and target for prostate cancer treatments., (© 2021. The Author(s).)

Internalization and intracellular trafficking of G protein-coupled receptors (GPCRs) play pivotal roles in cell responsiveness. Dysregulation in receptor trafficking can lead to aberrant signaling and cell behavior. Here, using an endosomal BRET-based assay in a high-throughput screen with the prototypical GPCR angiotensin II type 1 receptor (AT1R), we sought to identify receptor trafficking inhibitors from a library of ~115,000 small molecules. We identified a novel dual Ras and ARF6 inhibitor, which we named Rasarfin, that blocks agonist-mediated internalization of AT1R and other GPCRs. Rasarfin also potently inhibits agonist-induced ERK1/2 signaling by GPCRs, and MAPK and Akt signaling by EGFR, as well as prevents cancer cell proliferation. In silico modeling and in vitro studies reveal a unique binding modality of Rasarfin within the SOS-binding domain of Ras. Our findings unveil a class of dual small G protein inhibitors for receptor trafficking and signaling, useful for the inhibition of oncogenic cellular responses., (© 2021. The Author(s).)

The role of RNA binding proteins in regulating the phagocytic and cytokine-releasing functions of microglia is unknown. Here, we show that microglia deficient for the QUAKING (QKI) RNA binding protein have increased proinflammatory cytokine release and defects in processing phagocytosed cargo. Splicing analysis reveals a role for QKI in regulating microexon networks of the Rho GTPase pathway. We show an increase in RhoA activation and proinflammatory cytokines in QKI-deficient microglia that are repressed by treating with a Rock kinase inhibitor. During the cuprizone diet, mice with QKI-deficient microglia are inefficient at supporting central nervous system (CNS) remyelination and cause the recruited oligodendrocyte precursor cells to undergo apoptosis. Furthermore, the expression of QKI in microglia is downregulated in preactive, chronic active, and remyelinating white matter lesions of multiple sclerosis (MS) patients. Overall, our findings identify QKI as an alternative splicing regulator governing a network of Rho GTPase microexons with implications for CNS remyelination and MS patients., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Liver metastases remain a major cause of death from gastrointestinal tract cancers and other malignancies, such as breast and lung carcinomas. Understanding the underlying biology is essential for the design of effective therapies. We previously identified the chemokine CCL7 and its receptor CCR3 as critical mediators of invasion and metastasis in lung and colon carcinoma cells. Here we show that the CCL7/CCR3 axis regulates a late stage in invadopodia genesis namely, the targeting of MMP-9 to the invadopodia complex, thereby promoting invadopodia maturation and collagen degradation. We show that this process could be blocked by overexpression of a dominant negative RhoA in highly invasive cells, while a constitutively active RhoA upregulated invadopodia maturation in CCL7-silenced and poorly invasive and metastatic cells and also enhanced their metastatic potential in vivo, collectively, implicating RhoA activation in signaling downstream of CCL7. Blockade of the ERK or PI3K pathways by chemical inhibitors also inhibited invadopodia formation, but affected the initiation stage of invadopodia genesis. Our data implicate CCL7/CCR3 signaling in invadopodia maturation and suggest that chemokine signaling acts in concert with extracellular matrix-initiated signals to promote invasion and liver metastasis., Competing Interests: Declaration of competing interest The authors of this manuscript have no conflict of interest to declare., (Copyright © 2020 Elsevier B.V. All rights reserved.)

During development, neurons extend axons toward their appropriate synaptic targets to establish functional neuronal connections. The growth cone, a highly motile structure at the tip of the axon, is capable of recognizing extracellular guidance cues and translating them into directed axon outgrowth through modulation of the actin cytoskeleton. Netrin-1 mediates its attractive function through the receptor deleted in colorectal cancer (DCC) to promote axon outgrowth and guidance. The calcium-activated protease calpain is involved in the cleavage of cytoskeletal proteins, which plays an important role during adhesion turnover and cell migration. However, its function during neuronal development is less understood. Here we demonstrate that netrin-1 activated calpain in embryonic rat cortical neurons in an extracellular-regulated kinase 1/2-dependent manner. In addition, we found that netrin-1 stimulation led to an increase in calpain-1 localization in the axon, whereas its endogenous inhibitor calpastatin was decreased in the growth cones of cortical neurons by indirect immunofluorescence. Interestingly, calpain-1 was able to cleave DCC in vitro. Furthermore, netrin-1 induced the cleavage of the cytoskeletal proteins spectrin and focal adhesion kinase concomitantly with the intracellular domain of DCC in a calpain-dependent manner in embryonic rat cortical neurons. Cortical neurons over-expressing calpastatin or calpain-depleted neurons displayed increased basal axon length and were unresponsive to netrin-1 stimulation. Altogether, we propose a novel model whereby netrin-1/DCC-mediated axon outgrowth is modulated by calpain-mediated proteolysis of DCC and cytoskeletal targets in embryonic cortical neurons. Open Science: This manuscript was awarded with the Open Materials Badge For more information see: https://cos.io/our-services/open-science-badges/., (© 2019 International Society for Neurochemistry.)

One of the fundamental steps during development of the nervous system is the formation of proper connections between neurons and their target cells-a process called neural wiring, failure of which causes neurological disorders ranging from autism to Down's syndrome. Axons navigate through the complex environment of a developing embryo toward their targets, which can be far away from their cell bodies. Successful implementation of neuronal wiring, which is crucial for fulfillment of all behavioral functions, is achieved through an intimate interplay between axon guidance and neural activity. In this review, our focus will be on axon pathfinding and the implication of some of its downstream molecular components in neurological disorders. More precisely, we will talk about axon guidance and the molecules implicated in this process. After, we will briefly review the Rho family of small GTPases, their regulators, and their involvement in downstream signaling pathways of the axon guidance cues/receptor complexes. We will then proceed to the final and main part of this review, where we will thoroughly comment on the implication of the regulators for Rho GTPases-GEFs (Guanine nucleotide Exchange Factors) and GAPs (GTPase-activating Proteins)-in neurological diseases and disorders.

Liver metastases remain a major cause of death from gastrointestinal tract cancers as well as from other malignancies such as breast and lung carcinomas and melanoma. Understanding the underlying biology is essential for the design of effective targeted therapies. We previously reported that collagen IV α1/α2 overexpression in non-metastatic lung carcinoma (M27 colIV ) cells increased their metastatic ability, specifically to the liver and documented high collagen IV levels in surgical resections of liver metastases from diverse tumor types. Here, we aimed to elucidate the functional relevance of collagen IV to metastatic outgrowth in the liver. Gene expression profiling revealed in M27 colIV cells significant increases in the expression of chemokines CCL5 (5.7-fold) and CCL7 (2.6-fold) relative to wild-type cells, and this was validated by qPCR and western blotting. Similarly, in human colon carcinoma KM12C and KM12SM cells with divergent liver-colonizing potentials, CCL7 and CCL5 production correlated with type IV collagen expression and the metastatic phenotype. CCL7 silencing by short hairpin RNA (shRNA) reduced experimental liver metastasis in both cell types, whereas CCL5 silencing reduced metastasis of M27 colIV cells, implicating these cytokines in metastatic expansion in the liver. Subsequent functional analyses implicated both MEK/ERK and PI3K signaling upstream of CCL7 upregulation and identified CCL7 (but not CCL5) as a critical migration/invasion factor, acting via the chemokine receptor CCR3. Chemokine CCL5 was identified as a regulator of the T-cell immune response in the liver. Loss of CCL7 in KM12SM cells was also associated with altered E-cadherin and reduced vimentin and Snail expression, implicating it in epithelial-to-mesenchymal transition in these cells. Moreover, in clinical specimens of colon cancer liver metastases analyzed by immunohistochemistry, CCL5 and CCL7 levels paralleled those of collagen IV. The results identify the chemokines CCL5 and CCL7 as type IV collagen-regulated genes that promote liver metastasis by distinct and complementary mechanisms.

Cdc42 GTPase-activating protein (CdGAP, also named ARHGAP31) is a negative regulator of the GTPases Rac1 and Cdc42. Associated with the rare developmental disorder Adams-Oliver Syndrome (AOS), CdGAP is critical for embryonic vascular development and VEGF-mediated angiogenesis. Moreover, CdGAP is an essential component in the synergistic interaction between TGFβ and ErbB-2 signaling pathways during breast cancer cell migration and invasion, and is a novel E-cadherin transcriptional co-repressor with Zeb2 in breast cancer. CdGAP is highly phosphorylated on serine and threonine residues in response to growth factors and is a substrate of ERK1/2 and GSK-3. Here, we identified Ser1093 and Ser1163 in the C-terminal region of CdGAP, which are phosphorylated by RSK in response to phorbol ester. These phospho-residues create docking sites for binding to 14-3-3 adaptor proteins. The interaction between CdGAP and 14-3-3 proteins inhibits the GAP activity of CdGAP and sequesters CdGAP into the cytoplasm. Consequently, the nucleocytoplasmic shuttling of CdGAP is inhibited and CdGAP-induced cell rounding is abolished. In addition, 14-3-3β inhibits the ability of CdGAP to repress the E-cadherin promoter and to induce cell migration. Finally, we show that 14-3-3β is unable to regulate the activity and subcellular localization of the AOS-related mutant proteins lacking these phospho-residues. Altogether, we provide a novel mechanism of regulation of CdGAP activity and localization, which impacts directly on a better understanding of the role of CdGAP as a promoter of breast cancer and in the molecular causes of AOS., Competing Interests: CONFLICTS OF INTEREST The authors have no conflicts of interest to disclose.

Gangliosides (sialylated glycolipids) play an essential role in the CNS by regulating recognition and signaling in neurons. Metabolic blocks in processing and catabolism of gangliosides result in the development of severe neurologic disorders, including gangliosidoses manifesting with neurodegeneration and neuroinflammation. We demonstrate that 2 mammalian enzymes, neuraminidases 3 and 4, play important roles in catabolic processing of brain gangliosides by cleaving terminal sialic acid residues in their glycan chains. In neuraminidase 3 and 4 double-knockout mice, G M3 ganglioside is stored in microglia, vascular pericytes, and neurons, causing micro- and astrogliosis, neuroinflammation, accumulation of lipofuscin bodies, and memory loss, whereas their cortical and hippocampal neurons have lower rate of neuritogenesis in vitro Double-knockout mice also have reduced levels of G M1 ganglioside and myelin in neuronal axons. Furthermore, neuraminidase 3 deficiency drastically increased storage of G M2 in the brain tissues of an asymptomatic mouse model of Tay-Sachs disease, a severe human gangliosidosis, indicating that this enzyme is responsible for the metabolic bypass of β-hexosaminidase A deficiency. Together, our results provide the first in vivo evidence that neuraminidases 3 and 4 have important roles in CNS function by catabolizing gangliosides and preventing their storage in lipofuscin bodies.-Pan, X., De Britto Pará De Aragão, C., Velasco-Martin, J. P., Priestman, D. A., Wu, H. Y., Takahashi, K., Yamaguchi, K., Sturiale, L., Garozzo, D., Platt, F. M., Lamarche-Vane, N., Morales, C. R., Miyagi, T., Pshezhetsky, A. V. Neuraminidases 3 and 4 regulate neuronal function by catabolizing brain gangliosides., (© FASEB.)

Haploinsufficiency of the SYNGAP1 gene, which codes for a Ras GTPase-activating protein, impairs cognition both in humans and in mice. Decrease of Syngap1 in mice has been previously shown to cause cognitive deficits at least in part by inducing alterations in glutamatergic neurotransmission and premature maturation of excitatory connections. Whether Syngap1 plays a role in the development of cortical GABAergic connectivity and function remains unclear. Here, we show that Syngap1 haploinsufficiency significantly reduces the formation of perisomatic innervations by parvalbumin-positive basket cells, a major population of GABAergic neurons, in a cell-autonomous manner. We further show that Syngap1 haploinsufficiency in GABAergic cells derived from the medial ganglionic eminence impairs their connectivity, reduces inhibitory synaptic activity and cortical gamma oscillation power, and causes cognitive deficits. Our results indicate that Syngap1 plays a critical role in GABAergic circuit function and further suggest that Syngap1 haploinsufficiency in GABAergic circuits may contribute to cognitive deficits.

Mutations in the CdGAP/ARHGAP31 gene, which encodes a GTPase-activating protein for Rac1 and Cdc42, have been reported causative in the Adams-Oliver developmental syndrome often associated with vascular defects. However, despite its abundant expression in endothelial cells, CdGAP function in the vasculature remains unknown. Here, we show that vascular development is impaired in CdGAP-deficient mouse embryos at E15.5. This is associated with superficial vessel defects and subcutaneous edema, resulting in 44% embryonic/perinatal lethality. VEGF-driven angiogenesis is defective in CdGAP(-/-) mice, showing reduced capillary sprouting from aortic ring explants. Similarly, VEGF-dependent endothelial cell migration and capillary formation are inhibited upon CdGAP knockdown. Mechanistically, CdGAP associates with VEGF receptor-2 and controls VEGF-dependent signaling. Consequently, CdGAP depletion results in impaired VEGF-mediated Rac1 activation and reduced phosphorylation of critical intracellular mediators including Gab1, Akt, PLCγ and SHP2. These findings are the first to demonstrate the importance of CdGAP in embryonic vascular development and VEGF-induced signaling, and highlight CdGAP as a potential therapeutic target to treat pathological angiogenesis and vascular dysfunction.

Background Information: Rho GTPases play an essential role during the development of the nervous system. They induce cytoskeletal rearrangements that are critical for the regulation of axon outgrowth and guidance. It is generally accepted that Rac1 and Cdc42 are positive regulators of axon outgrowth and guidance, whereas RhoA is a negative regulator. However, spatiotemporal control of their activity can modify the function of Rho GTPases during axonal morphogenesis. Signalling downstream of the axon guidance cue netrin-1 and its receptor deleted in colorectal cancer (DCC) triggers the activation of Rac1 and the inhibition of RhoA to promote axon outgrowth. However, our previous work also suggests that netrin-1/DCC signalling can activate RhoA in a time- and region-specific manner., Results: Here, we visualised RhoA activation in response to netrin-1 in live embryonic cortical neurons using fluorescence resonance energy transfer. RhoA activity oscillated in unstimulated neurons and netrin-1 increased the amplitude of the oscillations in growth cones after 5 min of stimulation. Within this period of time, netrin-1 transiently increased RhoA activity and modulated the pattern of RhoA oscillations. We found that the timing of netrin-1-induced RhoA activation was different in whole neurons, cell bodies and growth cones., Conclusions: We conclude that netrin-1 modulates the spatiotemporal activation of RhoA in embryonic cortical neurons., Significance: This study demonstrates for the first time the short-term localised activation of RhoA in neuronal growth cones by the axon guidance cue netrin-1., (© 2016 Société Française des Microscopies and Société de Biologie Cellulaire de France. Published by John Wiley & Sons Ltd.)

The receptor deleted in colorectal cancer (DCC) mediates the attraction of growing axons to netrin-1 during brain development. In response to netrin-1 stimulation, DCC becomes a signaling platform to recruit proteins that promote axon outgrowth and guidance. The Ras GTPase-activating protein (GAP) p120RasGAP inhibits Ras activity and mediates neurite retraction and growth cone collapse in response to repulsive guidance cues. Here we show an interaction between p120RasGAP and DCC that positively regulates netrin-1-mediated axon outgrowth and guidance in embryonic cortical neurons. In response to netrin-1, p120RasGAP is recruited to DCC in growth cones and forms a multiprotein complex with focal adhesion kinase and ERK. We found that Ras/ERK activities are elevated aberrantly in p120RasGAP-deficient neurons. Moreover, the expression of p120RasGAP Src homology 2 (SH2)-SH3-SH2 domains, which interact with the C-terminal tail of DCC, is sufficient to restore netrin-1-dependent axon outgrowth in p120RasGAP-deficient neurons. We provide a novel mechanism that exploits the scaffolding properties of the N terminus of p120RasGAP to tightly regulate netrin-1/DCC-dependent axon outgrowth and guidance., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Rho family GTPases act as molecular switches regulating actin cytoskeleton dynamics. Attenuation of their signaling capacity is provided by GTPase-activating proteins (GAPs), including p190A, that promote the intrinsic GTPase activity of Rho proteins. In the current study we have performed a small-scale ENU mutagenesis screen and identified a novel loss of function allele of the p190A gene Arhgap35, which introduces a Leu1396 to Gln substitution in the GAP domain. This results in decreased GAP activity for the prototypical Rho-family members, RhoA and Rac1, likely due to disrupted ordering of the Rho binding surface. Consequently, Arhgap35-deficient animals exhibit hypoplastic and glomerulocystic kidneys. Investigation into the cystic phenotype shows that p190A is required for appropriate primary cilium formation in renal nephrons. P190A specifically localizes to the base of the cilia to permit axoneme elongation, which requires a functional GAP domain. Pharmacological manipulations further reveal that inhibition of either Rho kinase (ROCK) or F-actin polymerization is able to rescue the ciliogenesis defects observed upon loss of p190A activity. We propose a model in which p190A acts as a modulator of Rho GTPases in a localized area around the cilia to permit the dynamic actin rearrangement required for cilia elongation. Together, our results establish an unexpected link between Rho GTPase regulation, ciliogenesis and glomerulocystic kidney disease.

CUX1 and CUX2 proteins are characterized by the presence of three highly similar regions called Cut repeats 1, 2, and 3. Although CUX1 is ubiquitously expressed, CUX2 plays an important role in the specification of neuronal cells and continues to be expressed in postmitotic neurons. Cut repeats from the CUX1 protein were recently shown to stimulate 8-oxoguanine DNA glycosylase 1 (OGG1), an enzyme that removes oxidized purines from DNA and introduces a single strand break through its apurinic/apyrimidinic lyase activity to initiate base excision repair. Here, we investigated whether CUX2 plays a similar role in the repair of oxidative DNA damage. Cux2 knockdown in embryonic cortical neurons increased levels of oxidative DNA damage. In vitro, Cut repeats from CUX2 increased the binding of OGG1 to 7,8-dihydro-8-oxoguanine-containing DNA and stimulated both the glycosylase and apurinic/apyrimidinic lyase activities of OGG1. Genetic inactivation in mouse embryo fibroblasts or CUX2 knockdown in HCC38 cells delayed DNA repair and increased DNA damage. Conversely, ectopic expression of Cut repeats from CUX2 accelerated DNA repair and reduced levels of oxidative DNA damage. These results demonstrate that CUX2 functions as an accessory factor that stimulates the repair of oxidative DNA damage. Neurons produce a high level of reactive oxygen species because of their dependence on aerobic oxidation of glucose as their source of energy. Our results suggest that the persistent expression of CUX2 in postmitotic neurons contributes to the maintenance of genome integrity through its stimulation of oxidative DNA damage repair., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

During development, netrin-1 is both an attractive and repulsive axon guidance cue and mediates its attractive function through the receptor Deleted in Colorectal Cancer (DCC). The activation of Rho guanosine triphosphatases within the extending growth cone facilitates the dynamic reorganization of the cytoskeleton required to drive axon extension. The Rac1 guanine nucleotide exchange factor (GEF) Trio is essential for netrin-1-induced axon outgrowth and guidance. Here, we identify the molecular chaperone heat shock cognate protein 70 (Hsc70) as a novel Trio regulator. Hsc70 dynamically associated with the N-terminal region and Rac1 GEF domain of Trio. Whereas Hsc70 expression supported Trio-dependent Rac1 activation, adenosine triphosphatase-deficient Hsc70 (D10N) abrogated Trio Rac1 GEF activity and netrin-1-induced Rac1 activation. Hsc70 was required for netrin-1-mediated axon growth and attraction in vitro, whereas Hsc70 activity supported callosal projections and radial neuronal migration in the embryonic neocortex. These findings demonstrate that Hsc70 chaperone activity is required for Rac1 activation by Trio and this function underlies netrin-1/DCC-dependent axon outgrowth and guidance., (© 2015 DeGeer et al.)

In the last decade, several mouse models for RhoA, Rac1, and Cdc42 have emerged and have contributed a great deal to understanding the precise functions of Rho GTPases at early stages of development. This review summarizes our current knowledge of various mouse models of tissue-specific ablation of Cdc42, Rac1, and RhoA with emphasis on early embryogenesis, epithelial and skin morphogenesis, tubulogenesis, development of the central nervous system, and limb development.

Rho GTPases are molecular switches that modulate multiple intracellular signaling processes by means of various effector proteins. As a result, Rho GTPase activities are tightly spatiotemporally regulated in order to ensure homeostasis within the cell. Though the roles of Rho GTPases during neural development have been well documented, their participation during neurodegeneration has been far less characterized. Herein we discuss our current knowledge of the role and function of Rho GTPases and regulators during neurodegeneration, and highlight their potential as targets for therapeutic intervention in common neurodegenerative disorders., (© 2013 Elsevier Inc. All rights reserved.)

The chemotropic guidance cue netrin-1 mediates attraction of migrating axons during central nervous system development through the receptor Deleted in Colorectal Cancer (DCC). Downstream of netrin-1, activated Rho GTPases Rac1 and Cdc42 induce cytoskeletal rearrangements within the growth cone. The Rho guanine nucleotide exchange factor (GEF) Trio is essential for Rac1 activation downstream of netrin-1/DCC, but the molecular mechanisms governing Trio activity remain elusive. Here, we demonstrate that Trio is phosphorylated by Src family kinases in the embryonic rat cortex in response to netrin-1. In vitro, Trio was predominantly phosphorylated at Tyr(2622) by the Src kinase Fyn. Though the phospho-null mutant Trio(Y2622F) retained GEF activity toward Rac1, its expression impaired netrin-1-induced Rac1 activation and DCC-mediated neurite outgrowth in N1E-115 neuroblastoma cells. Trio(Y2622F) impaired netrin-1-induced axonal extension in cultured cortical neurons and was unable to colocalize with DCC in growth cones, in contrast to wild-type Trio. Furthermore, depletion of Trio in cortical neurons reduced the level of cell surface DCC in growth cones, which could be restored by expression of wild-type Trio but not Trio(Y2622F). Together, these findings demonstrate that Trio(Y2622) phosphorylation is essential for the regulation of the DCC/Trio signaling complex in cortical neurons during netrin-1-mediated axon outgrowth.

The Rho family of small GTPases are membrane-associated molecular switches involved in the control of a wide range of cellular activities, including cell migration, adhesion, and proliferation. Cdc42 GTPase-activating protein (CdGAP) is a phosphoprotein showing GAP activity toward Rac1 and Cdc42. CdGAP activity is regulated in an adhesion-dependent manner and more recently, we have identified CdGAP as a novel molecular target in signaling and an essential component in the synergistic interaction between TGFβ and Neu/ErbB-2 signaling pathways in breast cancer cells. In this study, we identified a small polybasic region (PBR) preceding the RhoGAP domain that mediates specific binding to negatively charged phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3). In vitro reconstitution of membrane vesicles loaded with prenylated Rac1 demonstrates that the PBR is required for full activation of CdGAP in the presence of PI(3,4,5)P3. In fibroblast cells, the expression of CdGAP protein mutants lacking an intact PBR shows a significant reduced ability of the protein mutants to induce cell rounding or to mediate negative effects on cell spreading. Furthermore, an intact PBR is required for CdGAP to inactivate Rac1 signaling into cells, whereas it is not essential in an in vitro context. Altogether, these studies reveal that specific interaction between negatively charged phospholipid PI(3,4,5)P3 and the stretch of polybasic residues preceding the RhoGAP domain regulates CdGAP activity in vivo and is required for its cellular functions.

The receptor Deleted in Colorectal Cancer (DCC) mediates the attractive response of axons to the guidance cue netrin-1 during development. On netrin-1 stimulation, DCC is phosphorylated and induces the assembly of signaling complexes within the growth cone, leading to activation of cytoskeleton regulators, namely the GTPases Rac1 and Cdc42. The molecular mechanisms that link netrin-1/DCC to the actin machinery remain unclear. In this study we seek to demonstrate that the actin-binding proteins ezrin-radixin-moesin (ERM) are effectors of netrin-1/DCC signaling in embryonic cortical neurons. We show that ezrin associates with DCC in a netrin-1-dependent manner. We demonstrate that netrin-1/DCC induces ERM phosphorylation and activation and that the phosphorylation of DCC is required in that context. Moreover, Src kinases and RhoA/Rho kinase activities mediate netrin-1-induced ERM phosphorylation in neurons. We also observed that phosphorylated ERM proteins accumulate in growth cone filopodia, where they colocalize with DCC upon netrin-1 stimulation. Finally, we show that loss of ezrin expression in cortical neurons significantly decreases axon outgrowth induced by netrin-1. Together, our findings demonstrate that netrin-1 induces the formation of an activated ERM/DCC complex in growth cone filopodia, which is required for netrin-1-dependent cortical axon outgrowth.

The establishment of neural connectivity implicates tight regulation of the intracellular signaling pathways mediated by axon guidance molecules. The Rho family of small GTPases, in particular Rho, Rac, and Cdc42, are important regulators of the cytoskeleton in neuronal cells acting, downstream of most, if not all, guidance cue receptors. Furthermore, recent studies using in vivo knockout mouse models provide new evidence of the primary role played by Rho GTPase signaling during the development of the nervous system. Here, we review our recent understanding of Rho GTPase signaling in response to classical axon guidance cues in mammalian cells. We also describe how in vivo knockout mouse models have been useful to implicate Rho GTPase signaling during the formation of the nervous system. Finally, we present several lines of evidence showing the involvement of Rho GTPase signaling in the development and progression of neurodegenerative diseases.

Regulation of cell proliferation and motility is essential for normal development. The Rho family of GTPases plays a critical role in the control of cell polarity and migration by effecting the cytoskeleton, membrane trafficking, and cell adhesion. We investigated a recognized developmental disorder, Adams-Oliver syndrome (AOS), characterized by the combination of aplasia cutis congenita (ACC) and terminal transverse limb defects (TTLD). Through a genome-wide linkage analysis, we detected a locus for autosomal-dominant ACC-TTLD on 3q generating a maximum LOD score of 4.93 at marker rs1464311. Candidate-gene- and exome-based sequencing led to the identification of independent premature truncating mutations in the terminal exon of the Rho GTPase-activating protein 31 gene, ARHGAP31, which encodes a Cdc42/Rac1 regulatory protein. Mutant transcripts are stable and increase ARHGAP31 activity in vitro through a gain-of-function mechanism. Constitutively active ARHGAP31 mutations result in a loss of available active Cdc42 and consequently disrupt actin cytoskeletal structures. Arhgap31 expression in the mouse is substantially restricted to the terminal limb buds and craniofacial processes during early development; these locations closely mirror the sites of impaired organogenesis that characterize this syndrome. These data identify the requirement for regulated Cdc42 and/or Rac1 signaling processes during early human development., (Copyright © 2011 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

The small GTPases Rac1 and Cdc42 are key regulators of the cytoskeleton. We have previously identified the endocytic protein Intersectin as a binding partner and regulator of Cdc42 GTPase-activating protein (CdGAP) with activity towards Rac1 and Cdc42. This interaction is mediated through the SH3D domain of Intersectin and the central domain of CdGAP, which does not contain any typical proline-rich domain or known SH3-binding motif. Here, we have characterized the Intersectin-SH3D/CdGAP interaction. We show that Intersectin-SH3D interacts directly with a small region of CdGAP highly enriched in basic residues and comprising a novel conserved xKx(K/R)K motif., (Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Netrin-1 attracts or repels growing axons during development. The UNC5 receptors mediate the repulsive response, either alone or in complex with DCC receptors. The signaling mechanisms activated by UNC5 are poorly understood. Here, we examined the role of Rho GTPases in UNC5a signaling. We found that UNC5a induced neurite outgrowth in N1E-115 neuroblastoma cells in a netrin-1- and Rac1-dependent manner. UNC5a lacking its cytoplasmic tail also mediated this effect. In fibroblasts, UNC5a was able to activate RhoA and to a lower extent Rac1 and Cdc42 in response to netrin-1. Using Fluorescence Resonance Energy Transfer (FRET) intermolecular probes, we visualized the spatial and temporal activation of Rac1, Cdc42 and RhoA in live N1E-115 cells expressing UNC5a during neurite outgrowth. We found that Rac1 but not Cdc42 was transiently activated at the leading edge of the cell during neurite initiation. However, at later times when well-developed neurites were formed, active RhoA was found in the cell body and at the base of the neuronal leading process in UNC5a-expressing cells. Together, these findings demonstrate that the netrin-1 receptor UNC5a is able to induce neurite outgrowth and to differentially activate RhoA and Rac1 during neurite extension in a spatial and temporal manner.

Recent studies have identified Rab35 in the endocytic pathway and as a regulator of cytokinesis; however its molecular mechanisms are currently unknown. Here, we find that Rab35 colocalizes with actin filaments and with Cdc42, Rac1 and RhoA, and that Rab35 can activate Cdc42 both in vivo and in vitro. We find activated Rab35 stimulates neurite outgrowth in PC12 and N1E-115 cells via a Cdc42-dependent pathway and that siRNA knockdown of Rab35 activity abolishes neurite outgrowth in these cell lines. We conclude that one function of Rab35 is to regulate Rho-family GTPases and that this role has consequences for neurite outgrowth.

The Src-associated substrate during mitosis with a molecular mass of 68 kDa (Sam68) is predominantly nuclear and is known to associate with proteins containing the Src homology 3 (SH3) and SH2 domains. Although Sam68 is a Src substrate, little is known about the signaling pathway that link them. Src is known to be activated transiently after cell spreading, where it modulates the activity of small Rho GTPases. Herein we report that Sam68-deficient cells exhibit loss of cell polarity and cell migration. Interestingly, Sam68-deficient cells exhibited sustained Src activity after cell attachment, resulting in the constitutive tyrosine phosphorylation and activation of p190RhoGAP and its association with p120rasGAP. Consistently, we observed that Sam68-deficient cells exhibited deregulated RhoA and Rac1 activity. By using total internal reflection fluorescence microscopy, we observed Sam68 near the plasma membrane after cell attachment coinciding with phosphorylation of its C-terminal tyrosines and association with Csk. These findings show that Sam68 localizes near the plasma membrane during cell attachment and serves as an adaptor protein to modulate Src activity for proper signaling to small Rho GTPases.

Background Information: Netrin-1 is a bi-functional cue that attracts or repels different classes of neurons during development. The netrin-1 receptor DCC (deleted in colorectal cancer) acts as a tyrosine kinase-associated receptor to mediate the attractive response towards netrin-1. The lipid raft-localized Src family kinase Fyn is required for DCC-mediated axon guidance. DCC functions are also dependent on lipid rafts, membrane microdomains corresponding to a low-density, detergent-resistant membrane fraction. However, it remains unclear how the association of DCC with lipid rafts controls netrin-1 signalling., Results: DCC targeted to lipid rafts represented a minor proportion of total DCC inside the cell, but predominated on the cell surface of both IMR-32 human neuroblastoma cells and embryonic cortical neurons. Netrin-1 accumulated in lipid rafts, but had no effect on the targeting of DCC to that compartment, with DCC remaining on the cell surface in lipid rafts through 60 min post-treatment. However, DCC was able to interact with Fyn, both in the lipid rafts and soluble compartments isolated from embryonic E19 rat brains, whereas early downstream signalling components such as Nck-1, and total and active focal adhesion kinase were mainly localized to the non-lipid raft compartment., Conclusions: Together, these results suggest that DCC can be found in raft and non-raft portions of the plasma membrane, with early signalling events propagated by non-raft associated DCC.

The chemotropic guidance cue netrin-1 promotes neurite outgrowth through its receptor Deleted in Colorectal Cancer (DCC) via activation of Rac1. The guanine nucleotide exchange factor (GEF) linking netrin-1/DCC to Rac1 activation has not yet been identified. Here, we show that the RhoGEF Trio mediates Rac1 activation in netrin-1 signaling. We found that Trio interacts with the netrin-1 receptor DCC in mouse embryonic brains and that netrin-1-induced Rac1 activation in brain is impaired in the absence of Trio. Trio(-/-) cortical neurons fail to extend neurites in response to netrin-1, while they are able to respond to glutamate. Accordingly, netrin-1-induced commissural axon outgrowth is reduced in Trio(-/-) spinal cord explants, and the guidance of commissural axons toward the floor plate is affected by the absence of Trio. The anterior commissure is absent in Trio-null embryos, and netrin-1/DCC-dependent axonal projections that form the internal capsule and the corpus callosum are defective in the mutants. Taken together, these findings establish Trio as a GEF that mediates netrin-1 signaling in axon outgrowth and guidance through its ability to activate Rac1.

RhoA, Rac1, and Cdc42, the founding members of the Rho subfamily of small GTPases, have been the focus of many research studies since the first discovery of their primary roles in the reorganisation of the actin cytoskeleton. Since then, it is clear that they are involved in a great deal of cellular functions, including cell migration and adhesion, cell growth control, and membrane trafficking. The complete sequencing of the human genome has now highlighted a total of 20 genes encoding Rho-like proteins. Little is known about their distinct cellular functions, however, numerous studies are now beginning to unravel that each of the Rho GTPase must play a specific role in the cell in a timely and spatially regulated fashion. Here, we are presenting a brief overview of the distinct functional roles and similarities known to date for each of the Rho members.

Visceral glomerular epithelial cells (GEC) are essential for maintenance of normal glomerular permselectivity. The actin cytoskeleton is a key determinant of GEC morphology and function. In the rat passive Heymann nephritis (PHN) model of membranous nephropathy, complement C5b-9 induces nonlytic GEC injury associated with morphological changes of GEC and proteinuria. The current study addresses the role of Rho family of small GTPases in complement-mediated GEC injury. When cultured rat GEC were stimulated with complement C5b-9 for 18 h, RhoA activity increased, whereas Rac1/Cdc42 activities decreased, compared with control cells. Similar changes in Rho-GTPase activities were observed in glomeruli from rats with PHN. The amount of active p190RhoGAP, a negative upstream regulator of RhoA, was decreased in complement-stimulated GEC, potentially contributing to increased RhoA activity. To address the functional effects of Rho-GTPases, GEC were transfected with constitutively active (CA) or dominant negative (DN) Rho-GTPase mutants. GEC transfected with CA-RhoA showed a smaller and round contour and prominent cortical F-actin. In contrast, GEC transfected with CA-Rac1 demonstrated morphological changes that resembled process formation. In addition, expression of CA-RhoA attenuated complement-mediated cytotoxicity, whereas cytotoxicity was augmented by DN-RhoA. Thus exposure of GEC to complement alters the balance of RhoA, Rac1, and Cdc42 activities. The activity of Rac1 may contribute to process formation, while activation of RhoA (e.g., in the setting of complement attack), with or without blunting of Rac1 activity, may have an opposite effect, i.e., contribute to foot process effacement. Activation of RhoA increases the resistance of GEC to complement-mediated injury.

Rho GTPases regulate a multitude of cellular processes from cytoskeletal reorganization to gene transcription and are negatively regulated by GTPase-activating proteins (GAPs). Cdc42 GTPase-activating protein (CdGAP) is a ubiquitously expressed GAP for Rac1 and Cdc42. In this study, we set out to identify CdGAP-binding partners and, using a yeast two-hybrid approach, glycogen synthase kinase 3alpha (GSK-3alpha) was identified as a partner for CdGAP. GSK-3 exists in two isoforms, alpha and beta, and is involved in regulating many cellular functions from insulin response to tumorigenesis. We show that GSK-3alpha and -beta interact with CdGAP in mammalian cells. We also demonstrate that GSK-3 phosphorylates CdGAP both in vitro and in vivo on Thr-776, which we have previously shown to be an ERK 1/2 phosphorylation site involved in CdGAP regulation. We report that the mRNA and protein levels of CdGAP are increased upon serum stimulation and that GSK-3 activity is necessary for the up-regulation of the protein levels of CdGAP but not for the increase in mRNA. We conclude that GSK-3 is an important regulator of CdGAP and that regulation of CdGAP protein levels by serum presents a novel mechanism for cells to control Cdc42/Rac1 GTPase signaling pathways.

The Rho GTPases are implicated in almost every fundamental cellular process. They act as molecular switches that cycle between an active GTP-bound and an inactive GDP-bound state. Their slow intrinsic GTPase activity is greatly enhanced by RhoGAPs (Rho GTPase-activating proteins), thus causing their inactivation. To date, more than 70 RhoGAPs have been identified in eukaryotes, ranging from yeast to human, and based on sequence homology of their RhoGAP domain, we have grouped them into subfamilies. In the present Review, we discuss their regulation, biological functions and implication in human diseases.

Background Information: Rho GTPases regulate a wide range of cellular functions affecting both cell proliferation and cytoskeletal dynamics. They cycle between inactive GDP- and active GTP-bound states. This cycle is tightly regulated by GEFs (guanine nucleotide-exchange factors) and GAPs (GTPase-activating proteins). Mouse CdGAP (mCdc42 GTPase-activating protein) has been previously identified and characterized as a specific GAP for Rac1 and Cdc42, but not for RhoA. It consists of an N-terminal RhoGAP domain and a C-terminal proline-rich region. In addition, CdGAP-related genes are present in both vertebrates and invertebrates. We have recently reported that two predominant isoforms of CdGAP (250 and 90 kDa) exist in specific mouse tissues., Results: In the present study, we have identified and characterized human CdGAP (KIAA1204) which shares 76% sequence identity to the long isoform of mCdGAP (mCdGAP-l). Similar to mCdGAP, it is active in vitro and in vivo on both Cdc42 and Rac1, but not RhoA, and is phosphorylated in vivo on serine and threonine residues. In contrast with mCdGAP-l, human CdGAP interacts with ERK1/2 (extracellular-signal-regulated kinase 1/2) through a region that does not involve a DEF (docking site for ERK Phe-Xaa-Phe-Pro) domain. Also, the tissue distribution of CdGAP proteins appears to be different between human and mouse species. Interestingly, we found that CdGAP proteins cause membrane blebbing in COS-7 cells., Conclusions: Our results suggest that CdGAP properties are well conserved between human and mouse species, and that CdGAP may play an unexpected role in apoptosis.

Background: Integrin signaling, stimulated by cell adhesion to the extracellular matrix, plays a critical role in coordinating changes in cell morphology and migration. The requisite remodeling of the cytoskeleton is controlled by the Rho family of small GTPases, which are, in turn, regulated via activation by guanine-nucleotide exchange factors (GEFs) and inactivation by GTPase-activating proteins (GAPs). However, the mechanisms contributing to the precise spatial and temporal regulation of these Rho GTPase modulators remain poorly understood., Results: The Cdc42/Rac GAP CdGAP has previously been implicated as an inhibitor of growth-factor-induced lamellipodia formation. Herein, CdGAP is shown to localize to focal adhesions, potentially through its direct association with the amino terminus of actopaxin, a paxillin and actin binding protein. CdGAP activity is regulated in an adhesion-dependent manner and, through the overexpression of wild-type CdGAP and a GAP-deficient mutant, as well as RNA interference, is shown to be required for normal cell spreading, polarized lamellipodia formation, and cell migration. Introduction of an actopaxin mutant defective for CdGAP binding, or reduction of actopaxin by using RNAi, significantly attenuated these effects., Conclusions: We have established that CdGAP is an important regulator of integrin-induced Rho family signaling to the cytoskeleton and that its interaction with the focal-adhesion protein actopaxin is critical for the correct spatial and/or temporal regulation of CdGAP function. A complete understanding of the coordination of signaling events downstream of integrin engagement with the extracellular matrix will provide valuable insight into the regulation of cell migration during processes such as wound repair, development, and tumor cell metastasis.

The adenovirus early region 4 ORF4 protein (E4orf4) triggers a novel death program that bypasses classical apoptotic pathways in human cancer cells. Deregulation of the cell cytoskeleton is a hallmark of E4orf4 killing that relies on Src family kinases and E4orf4 phosphorylation. However, the cytoskeletal targets of E4orf4 and their role in the death process are unknown. Here, we show that E4orf4 translocates to cytoplasmic sites and triggers the assembly of a peculiar juxtanuclear actin-myosin network that drives polarized blebbing and nuclear shrinkage. We found that E4orf4 activates the myosin II motor and triggers de novo actin polymerization in the perinuclear region, promoting endosomes recruitment to the sites of actin assembly. E4orf4-induced actin dynamics requires interaction with Src family kinases and involves a spatial regulation of the Rho GTPases pathways Cdc42/N-Wasp, RhoA/Rho kinase, and Rac1, which make distinct contributions. Remarkably, activation of the Rho GTPases is required for induction of apoptotic-like cell death. Furthermore, inhibition of actin dynamics per se dramatically impairs E4orf4 killing. This work provides strong support for a causal role for endosome-associated actin dynamics in E4orf4 killing and in the regulation of cancer cell fate.

Cyclic AMP (cAMP) is a pleiotropic second messenger that regulates numerous cellular processes. In vascular smooth muscle cells (VSMCs), these include cell proliferation, migration, and contractility. Here we show that cAMP-elevating agents induce dramatic morphological changes in VSMCs, characterized by cell rounding and formation of long branching processes. The stellate morphology is associated with disassembly of actin stress fibers and lamellipodia, loss of focal adhesions, and the formation of small F-actin rings. Because of the importance of Rho family GTPases in regulating actin dynamics, we analyzed their individual roles in the cAMP phenotype. We found that pharmacological or genetic inhibition of Rac mimics cAMP effect in inducing a stellate morphology of VSMCs. Expression of activated Rac1 prevents forskolin-induced cAMP stellation, suggesting that cAMP affects cell morphology by inhibiting Rac function. Consistent with this, treatment with forskolin inhibits agonist-stimulated Rac activation in VSMCs. We further show that activated Rac1 containing the F37A effector loop substitution fails to rescue the cAMP phenotype. Our results suggest that cAMP modulates the morphology of VSMCs by inhibiting a Rac-dependent signaling pathway., ((c) 2005 Wiley-Liss, Inc.)

Rho GTPases regulate multiple cellular processes affecting both cell proliferation and cytoskeletal dynamics. Their cycling between inactive GDP- and active GTP-bound states is tightly regulated by guanine nucleotide exchange factors and GTPase-activating proteins (GAPs). We have previously identified CdGAP (for Cdc42 GTPase-activating protein) as a specific GAP for Rac1 and Cdc42. CdGAP consists of an N-terminal RhoGAP domain and a C-terminal proline-rich region. In addition, CdGAP is a member of the impressively large number of mammalian RhoGAP proteins that is well conserved among both vertebrates and invertebrates. In mice, we find two predominant isoforms of CdGAP differentially expressed in specific tissues. We report here that CdGAP is highly phosphorylated in vivo on serine and threonine residues. We find that CdGAP is phosphorylated downstream of the MEK-extracellular signal-regulated kinase (ERK) pathway in response to serum or platelet-derived growth factor stimulation. Furthermore, CdGAP interacts with and is phosphorylated by ERK-1 and RSK-1 in vitro. A putative DEF (docking for ERK FXFP) domain located in the proline-rich region of CdGAP is required for efficient binding and phosphorylation by ERK1/2. We identify Thr776 as an in vivo target site of ERK1/2 and as an important regulatory site of CdGAP activity. Together, these data suggest that CdGAP is a novel substrate of ERK1/2 and mediates cross talk between the Ras/mitogen-activated protein kinase pathway and regulation of Rac1 activity.

The GTPase-activating proteins (GAPs) accelerate the hydrolysis of GTP to GDP by small GTPases. The GTPases play diverse roles in many cellular processes, including proliferation, cell motility, endocytosis, nuclear import/export, and nuclear membrane formation. Little is known about GAP-domain proteins in spermatogenesis. We isolated a novel RhoGAP domain-containing tGAP1 protein from male germ cells that exhibits unusual properties. The tGAP1 is expressed at low levels in early spermatogonia. Robust transcription initiates in midpachytene spermatocytes and continues after meiosis. The 175-kDa tGAP1 protein localizes to the cytoplasm of spermatocytes and to the cytoplasm and nucleus in spermatids. The protein contains four GAP domain-related sequences, in contrast to all other GAP proteins that harbor one such domain. No activity toward RhoA, Rac1, or Cdc42 could be detected. Results of transfection studies in various somatic cells indicated that low-level tGAP1 expression significantly slows down the cell cycle. Expression of higher levels of tGAP1 by infection of somatic cells with recombinant adenoviruses demonstrated that tGAP1 efficiently induces apoptosis, which to our knowledge is the first such demonstration for a RhoGAP protein. Based on its subcellular location in spermatids and its activity, tGAP1 may play a role in nuclear import/export.