An effector region in Eps8 is responsible for the activation of the Rac-specific GEF activity of Sos-1 and for the proper localization of the Rac-based actin–polymerizing machine

Scita, Giorgio; Tenca, Pierluigi; Areces, Liliana B.; Tocchetti, Arianna; Frittoli, Emanuela; Giardina, Giuseppina; Ponzanelli, Isabella; Sini, Patrizia; Innocenti, Metello; Fiore, Pier Paolo Di

doi:10.1083/jcb.200103146

Cited by 124 publications

(147 citation statements)

References 37 publications

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“…Remarkably, inhibition of N-WASP by wiskostatin in the testis in vivo did not lead to germ cell detachment or BTB damage, unlike Eps8, which is an actin-regulating protein with multiple functions, whose knockdown by RNAi would lead to germ cell loss and BTB damage in vivo (17). Although the Arp2/3 complex can serve as a downstream effector of Eps8 through the activation of Rac and WASP family verprolin-homologous protein (WAVE) (5,18), Eps8 has more diverse effects on the actin cytoskeleton that are not mediated through Arp2/3 complex, such as actin capping and bundling (19,20). Thus, the activation of Arp2/3 complex by N-WASP can be limited to actin cytoskeletal restructuring via actin branching, instead of maintaining junction integrity in the testis.…”

Section: Discussionmentioning

confidence: 99%

Restricted Arp3 expression in the testis prevents blood–testis barrier disruption during junction restructuring at spermatogenesis

Lie

Chan²,

Mruk³

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

132

181

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In epithelia, a primary damage of tight junctions (TJ) always leads to a secondary disruption of adherens junction (AJ), and vice versa. This response, if occurring in the testis, would disrupt spermatogenesis because the blood-testis barrier (BTB) must remain intact during the transit of spermatids in the seminiferous epithelium, which is associated with extensive apical ectoplasmic specialization (apical ES, a testis-specific AJ type) restructuring. As such, apical ES restructuring accompanied with the transit of developing spermatids during spermiogenesis must be segregated from the BTB to avoid an immunological barrier breakdown in all stages of the seminiferous epithelial cycle, except at stage VIII when spermiation and BTB restructuring take place concurrently. We report herein a mechanism involving restricted spatial and temporal expression of Arp2/3 complex and N-WASP, whose actin branching activity associated with apical ES and BTB restructuring in the seminiferous epithelium. High expression of Arp3 at the apical ES was shown to correlate with spermatid movement and proper spermatid orientation. Likewise, high Arp3 level at the BTB associated with its restructuring to accommodate the transit of preleptotene spermatocytes at stage VIII of the epithelial cycle. These findings were validated by in vitro and in vivo studies using wiskostatin, an inhibitor that blocks N-WASP from activating Arp2/3 complex to elicit actin branching. Inhibition of actin branching caused a failure of spermatid transit plus a loss of proper orientation in the epithelium, and a "tightened" Sertoli cell TJ permeability barrier, supporting the role of Arp2/3 complex in segregating the events of AJ and BTB restructuring.actin dynamics | ectoplasmic specialization | seminiferous epithelial cycle | spermiogenesis

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Section: Discussionmentioning

confidence: 99%

Restricted Arp3 expression in the testis prevents blood–testis barrier disruption during junction restructuring at spermatogenesis

Lie

Chan²,

Mruk³

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

132

181

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“…This is similar to the mechanism of stimulation of Sos exchange activity. The Sos DH domain is active for Rac1 exchange only when Sos is in a ternary complex with Eps8 and E3b1 (Scita et al, 1999(Scita et al, , 2001Innocenti et al, 2002). We investigated whether binding of interacting proteins to the SH3 domains can derepress the exchange activity in vitro.…”

Section: Discussionmentioning

confidence: 99%

“…As in the case for the Vav GEF, intersectin 1L may need to be phosphorylated in order to derepress the DH domain (Crespo et al, 1997;Han et al, 1997;Schuebel et al, 1998;Aghazadeh et al, 2000). Similarly, binding of an additional protein in vivo may be necessary, as in the case of activation of Sos Rac1 exchange activity by formation of a ternary complex (Scita et al, 1999(Scita et al, , 2001Innocenti et al, 2002). Because our studies have utilized recombinant proteins that are missing the amino-terminal domains, a third possibility is that a missing amino-terminal component of intersectin 1L is involved in the regulation of exchange activity.…”

Section: Discussionmentioning

confidence: 99%

Intersectin 1L Guanine Nucleotide Exchange Activity Is Regulated by Adjacent src Homology 3 Domains That Are Also Involved in Endocytosis

Zamanian

Kelly

2003

MBoC

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Intersectin 1L is a scaffolding protein involved in endocytosis that also has guanine nucleotide exchange activity for Cdc42. In the context of the full-length protein, the catalytic exchange activity of the DH domain is repressed. Here we use biochemical methods to dissect the mechanism for this inhibition. We demonstrate that the intersectin 1L SH3 domains, which bind endocytic proteins, directly inhibit the activity of the DH domain in assays for both binding and exchange of Cdc42. This inhibitory mechanism seems to act through steric hindrance of Cdc42 binding by an intramolecular interaction between the intersectin 1L SH3 domain region and the adjacent DH domain. Surprisingly, the mode of SH3 domain binding is other than through the proline peptide binding pocket. The dual role of the SH3 domains in endocytosis and repression of exchange activity suggests that the intersectin 1L exchange activity is regulated by endocytosis. We show that the endocytic protein, dynamin, competes for binding to the SH3 domains with the neural Wiskott-Aldrich Syndrome protein, an actin filament nucleation protein that is a substrate for activated Cdc42. Swapping of SH3 domain binding partners might act as a switch controlling the actin nucleation activity of intersectin 1L. INTRODUCTIONRho family guanine nucleotide exchange factors (GEFs) are critical regulatory proteins of cellular pathways that require regulation of actin cytoskeleton rearrangements (for review see Zheng, 2001;Hoffman and Cerione, 2002). Their conserved catalytic domain, the Dbl homology (DH) domain (Hart et al., 1991(Hart et al., , 1994 catalyzes the release of GDP from Rho GTPases and thus subsequent activation by GTP binding. DH domains are invariably found upstream and adjacent to pleckstrin homology (PH) domains, which are thought to influence their activity (Liu et al., 1998;Das et al., 2000) and membrane localization (Whitehead et al., 1999). The noncatalytic parts of the structurally complex GEFs link the exchange activity to cellular processes and inhibit the DH domain exchange activity (Zheng, 2001;Hoffman and Cerione, 2002). Cellular inputs, such as protein (Hart et al., 1998;Scita et al., 1999;Innocenti et al., 2002) and phospholipid binding to (Han et al., 1998;Nimnual et al., 1998;Crompton et al., 2000;Das et al., 2000;Russo et al., 2001), and phosphorylation of (Crespo et al., 1997;Han et al., 1997;Schuebel et al., 1998;Aghazadeh et al., 2000) these regulatory regions derepress exchange activity. Integration of cellular signals by Rho GEFs can focus GTPase activity to allow temporal and spatially localized activation of actin cytoskeletal rearrangements.Intersectin 1L, a neuronal splice variant of the endocytic scaffolding protein intersectin 1, is a Rho family GEF that is composed of two N-terminal EH domains (EH1, EH2), a large region of putative coiled-coils, five SH3 domains (SH3A, B, C, D, and E; Roos and Kelly, 1998;Yamabhai et al., 1998;Okamoto et al., 1999;Sengar et al., 1999), followed by the DH and PH domains and a carboxyl-terminal ...

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“…36,37 Several studies have shown that internalized EGFR are enzymatically active, are still phosphorylated and maintain association with many adaptor proteins. [43][44][45] In addition, it has been recently shown that interaction with some adaptor proteins, such as eps8, a protein involved in cytoskeletal reorganization and actin remodeling, 46 occurs only at endosomal level. 46 Furthermore, blocking of EGFR endocytosis by using a dynamin mutant results in downregulation of ERK and PI3K activation in response to EGF 47 and insulin.…”

Section: Egfr Internalization Is Altered In Pc3-ar Cellsmentioning

confidence: 99%

“…[43][44][45] In addition, it has been recently shown that interaction with some adaptor proteins, such as eps8, a protein involved in cytoskeletal reorganization and actin remodeling, 46 occurs only at endosomal level. 46 Furthermore, blocking of EGFR endocytosis by using a dynamin mutant results in downregulation of ERK and PI3K activation in response to EGF 47 and insulin. 48 Whether EGFR-AR interaction may be responsible for decreased EGFR signaling and internalization remains to be addressed; however, it is possible that such interaction disrupts the ability of the receptor to autophosphorylate by attenuating its intrinsic tyrosine kinase activity, determining as a consequence a reduction of internalization and PI3K activation.…”

Section: Egfr Internalization Is Altered In Pc3-ar Cellsmentioning

confidence: 99%

EGF receptor (EGFR) signaling promoting invasion is disrupted in androgen‐sensitive prostate cancer cells by an interaction between EGFR and androgen receptor (AR)

Bonaccorsi

Carloni

Muratori

et al. 2004

Intl Journal of Cancer

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We previously demonstrated that expression of androgen receptor (AR) by transfection of the androgen-independent prostate cancer cell line PC3 decreases invasion and adhesion of these cells (PC3-AR) through modulation of ␣6␤4 integrin expression. The treatment with androgens further reduced invasion of the cells without modifying ␣6␤4 expression, suggesting an interference with the invasion process by androgens. Here, we investigated EGF-mediated signal transduction processes that lead to invasion in PC3-AR cells. We show that EGF-induced EGFR autotransphosphorylation is reduced in PC3-AR cells compared to PC3 cells transfected only with the vector (PC3-Neo). EGF-stimulated PI3K activity, a key signaling pathway for invasion of these cells, and EGF-PI3K interaction are also decreased in PC3 Key words: prostate cancer; epidermal growth factor receptor; androgen receptor; PI3K; invasionProstate Cancer (PC) is one of the most common cancer and the second leading cause of death in American men. 1 Since prostate cancer cell growth is enhanced by androgens, in the advanced stages of the disease, androgen ablation therapy represents a valuable tool for the treatment of these patients. However the development in most patients after few years of treatment of androgenindependent clones, characterized by higher invasiveness and metastatic properties, has focused attention on the molecular mechanisms that lead to loss of androgen-dependence as well as on the pathways that are regulated by androgens in these cells besides proliferation. Indeed, although androgens are the major stimulus for proliferation of prostate cancer cells, maintenance of androgensensitivity appears to keep a more differentiated and less malignant phenotype of these cells. The ability to produce tumors in nude mice, for instance, is higher in androgen-insensitive cell lines (such as PC3 and DU145) with respect to androgen sensitive (LNCaP). 2 In this light, the role of androgens in the regulation of the pathways involved in invasion and metastasis represents a major task in studies on prostate cancer biology. 3 As a result, some androgen-regulated genes involved in signaling pathways that lead to invasion have been recently identified 4 -6 and their role in decreasing invasion ability of androgen-sensitive prostate cancer cells indicated. Migration and invasion of cancer cells is regulated by multiple pathways that employ various growth factor and their receptors, integrins and cytoskeletal elements. A key role is played by the EGF receptor (EGFR), which, following interaction with the integrin ␣6␤4, promotes cell migration through activation of PI3K and other downstream pathways. 7,8 In a previous study, we demonstrated that the expression of androgen receptor in PC3 cells by transfection with a full-length human androgen receptor expression vector (PC3-AR) determined a decrease in the expression of the integrin ␣6␤4 and in the ability of these cells to invade Matrigel in response to EGF. 6 The treatment with the synthetic androgen R1881 determined a ...

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An effector region in Eps8 is responsible for the activation of the Rac-specific GEF activity of Sos-1 and for the proper localization of the Rac-based actin–polymerizing machine

Cited by 124 publications

References 37 publications

Restricted Arp3 expression in the testis prevents blood–testis barrier disruption during junction restructuring at spermatogenesis

Restricted Arp3 expression in the testis prevents blood–testis barrier disruption during junction restructuring at spermatogenesis

Intersectin 1L Guanine Nucleotide Exchange Activity Is Regulated by Adjacent src Homology 3 Domains That Are Also Involved in Endocytosis

EGF receptor (EGFR) signaling promoting invasion is disrupted in androgen‐sensitive prostate cancer cells by an interaction between EGFR and androgen receptor (AR)

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