RESUMO
Rho GTPases play a key role in various membrane trafficking processes. RhoU is an atypical small Rho GTPase related to Rac/Cdc42, which possesses unique N- and C-terminal domains that regulate its function and its subcellular localization. RhoU localizes at the plasma membrane, on endosomes and in cell adhesion structures where it governs cell signaling, differentiation and migration. However, despite its endomembrane localization, RhoU function in vesicular trafficking has been unexplored. Here, we identified intersectins (ITSNs) as new binding partners for RhoU and showed that the second PxxP motif at the N terminus of RhoU mediated interactions with the SH3 domains of ITSNs. To evaluate the function of RhoU and ITSNs in vesicular trafficking, we used fluorescent transferrin as a cargo for uptake experiments. We showed that silencing of either RhoU or ITSN2, but not ITSN1, increased transferrin accumulation in early endosomes, resulting from a defect in fast vesicle recycling. Concomitantly, RhoU and ITSN2 colocalized to a subset of Rab4-positive vesicles, suggesting that a RhoU-ITSN2 interaction may occur on fast recycling endosomes to regulate the fate of vesicular cargos.
Assuntos
Endossomos , Proteínas rho de Ligação ao GTP , Proteínas Adaptadoras de Transporte Vesicular , Adesão Celular , Endossomos/metabolismo , Transdução de Sinais , Proteínas rho de Ligação ao GTP/genética , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
WIP (WASP interacting protein) together with N-WASP (neural Wiskott-Aldrich syndrome protein) regulates actin polymerization that is crucial for invadopodia and filopodia formation. Recently, we reported the WIP interaction with ITSN1 which is highly implicated in endo-/exocytosis, apoptosis, mitogenic signaling and cytoskeleton rearrangements. Here we demonstrate that the WIP/ITSN1 complex is involved in the transferrin receptor recycling and partially co-localizes with a marker of the fast recycling endosomes, RAB4. Moreover, ITSN1 recruits WIP to RAB4-positive vesicles upon overexpression. Our data indicate that WIP enhances the interaction of N-WASP with ITSN1 and promotes ITSN1/ß-actin association. Moreover, the WIP/ITSN1-L complex facilitates formation of filopodia-like protrusions in MCF-7 cells. Thus, WIP/ITSN1 complex is involved in the cellular vesicle trafficking and actin-dependent membrane processes.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Vesículas Citoplasmáticas/metabolismo , Pseudópodes/ultraestrutura , Actinas/metabolismo , Transporte Biológico , Proteínas do Citoesqueleto/metabolismo , Endossomos/química , Endossomos/metabolismo , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Células MCF-7 , Transferrina/metabolismo , Proteína Neuronal da Síndrome de Wiskott-Aldrich/metabolismo , Proteínas rab4 de Ligação ao GTP/análiseRESUMO
Actin cytoskeleton rearrangements are required for normal cell functioning, and their deregulation leads to various pathologies. Members of two mammalian protein families - ITSNs (ITSN1 and ITSN2) and verprolins (WIP, CR16 and WIRE) are involved in Cdc42/N-WASP/Arp2/3 signaling pathway-mediated remodeling of the actin cytoskeleton. Recently we demonstrated that ITSNs interact with the actin-regulating protein WIP. Here, we show that other member of verprolin family, CR16, also forms complexes with ITSN1 and ITSN2 in human cell lines. The actin-binding protein CR16 modulates ITSN/ß-actin association. Moreover, overexpressed CR16 promoted co-localization of ITSN1 with F-actin in MCF-7 breast cancer cells. Our data demonstrated that CR16 mRNA is expressed in glioblastoma and breast tumors. These findings provide the basis for further functional investigations of the ITSN/CR16 complex that may play an important role in actin remodeling and cellular invasion.
Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Proteínas do Citoesqueleto/metabolismo , Proteínas dos Microfilamentos/metabolismo , Humanos , Células MCF-7 , Ligação ProteicaRESUMO
Invasive cancer cells form actin-rich membrane protrusions called invadopodia that degrade extracellular matrix and facilitate cell invasion and metastasis. WIP (WASP-interacting protein) together with N-WASP (neural Wiskott-Aldrich syndrome protein) are localized in invadopodia and play a crucial role in their formation. Here we show that WIP interacts with endocytic adaptor proteins of the intersectin (ITSN) family, ITSN1 and ITSN2. The interaction is mediated by the SH3 domains of ITSNs and the middle part of the WIP proline-rich motifs. We have also demonstrated that ITSN1, WIP and N-WASP can form a complex in cells. Endogenous ITSN1 and ITSN2 are located in invasive protrusions of MDA-MB-231 breast cancer cell line. Moreover, data from immunofluorescent analysis revealed co-localization of ITSN1 and WIP at sites of invadopodia formation and in clathrin-coated pits. Together, these findings provide insights into the molecular mechanisms of invadopodia formation and identify ITSNs as scaffold proteins involved in this process.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Proteínas do Citoesqueleto/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Podossomos/metabolismo , Actinas/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/química , Proteínas Adaptadoras de Transporte Vesicular/genética , Animais , Sítios de Ligação , Encéfalo/metabolismo , Linhagem Celular Tumoral , Proteínas do Citoesqueleto/química , Humanos , Imunoprecipitação , Peptídeos e Proteínas de Sinalização Intracelular/química , Camundongos , Microscopia de Fluorescência , Células NIH 3T3 , Ligação Proteica , Proteína Neuronal da Síndrome de Wiskott-Aldrich/metabolismo , Domínios de Homologia de srcRESUMO
Intersectin 1 (ITSN1) is an evolutionarily conserved adaptor protein that functions in clathrin-mediated endocytosis, cell signalling and cytoskeleton rearrangements. The ITSN1 gene encodes two main isoforms: a short form (ITSN1-s), which is ubiquitously expressed and consists of two Eps15 homology (EH) domains and five Src homology 3 (SH3) domains, and a long form (ITSN1-l), which is predominantly expressed in the brain and contains three additional domains, a Dbl homology (DH) domain, a Pleckstrin homology (PH) domain and a C2 domain. Using computational analysis of the EST database and 3' RACE we determined the length of the 3' untranslated region of ITSN1-l and demonstrated that the polyadenylation site is located 11,559 nt downstream of the stop codon of the ITSN1-l mRNA. Recently, additional splicing events affecting ITSN1 transcripts were reported, but full-length transcriptional isoforms with different combinations of alternatively spliced exons remained unknown. Here we report the identification of fifteen novel transcriptional isoforms of the human ITSN1 gene with full-length coding sequences that are the result of different combinations of the alternatively spliced exons 5, 6/6', 20, 23, 25, 26, 26a and 35. The isoforms identified differ in domain organization and expression level in different tissues and more likely contribute to the modulation of many complex protein interactions in which ITSN1 participates.