RESUMO
Numerous observations have suggested a connection between the maintenance of cell polarity and control of cell proliferation; however, the mechanisms underlying these connections remain poorly understood. Here we found that ectopic expression of CRB3, which was previously shown to restore tight junctions and membrane polarity in MCF-10A cells, induced a hyperproliferative phenotype, with significantly enlarged acini in basement membrane culture, similar to structures induced by expression of proliferative oncogenes such as cyclinD1. We found that CRB3-induced proliferation is epidermal growth factor (EGF)-independent and occurs through a mechanism that involves secretion of the EGF-family ligand, amphiregulin (AREG). The increase in AREG secretion is associated with an increase in the number and size of both early and late endosomes. Both the proliferative and endocytic phenotypes associated with CRB3 expression require the FERM-binding domain (FBD) but not the PDZ-binding domain of CRB3, arguing that this proliferative phenotype is independent of the PDZ-dependent polarity signaling by CRB3. We identified the FBD-containing protein, EPB41L4B, as an essential mediator of CRB3-driven proliferation and observed that the CRB3-dependent changes in endocytic trafficking were also dependent on EPB41L4B. Taken together, these data reveal a previously uncharacterized role for CRB3 in regulating proliferation in mammalian cells that is associated with changes in the endocytic trafficking machinery.
Assuntos
Anfirregulina/genética , Polaridade Celular/genética , Proteínas do Citoesqueleto/genética , Glicoproteínas de Membrana/genética , Anfirregulina/biossíntese , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ciclina D1/genética , Células Epiteliais/metabolismo , Domínios FERM/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Glândulas Mamárias Humanas/efeitos dos fármacos , Glândulas Mamárias Humanas/metabolismo , Domínios PDZ/genética , Fenótipo , Ligação Proteica , RNA Interferente Pequeno/genéticaRESUMO
Ciliopathies represent a newly emerging group of human diseases that share a common etiology resulting from dysfunction of the cilium or centrosome. The gene encoding the retinitis pigmentosa 2 protein (RP2) is mutated in X-linked retinitis pigmentosa. RP2 localizes to the ciliary base and this requires the dual acylation of the N-terminus, but the precise mechanism by which RP2 is trafficked to the cilia is unknown. Here we have characterized an interaction between RP2 and Importin ß2 (transportin-1), a member of the Importin-ß family that regulates nuclear-cytoplasmic shuttling. We demonstrate that Importin ß2 is necessary for localization of RP2 to the primary cilium because ablation of Importin ß2 by shRNA blocks entry both of endogenous and exogenous RP2 to the cilium. Furthermore, we identify two distinct binding sites of RP2, which interact independently with Importin ß2. One binding site is a nuclear localization signal (NLS)-like sequence that is located at the N-terminus of RP2 and the other is an M9-like sequence within the tubulin folding cofactor C (TBCC) domain. Mutation of the NLS-like consensus sequence did not abolish localization of RP2 to cilia, suggesting that the sequence is not essential for RP2 ciliary targeting. Interestingly, we found that several missense mutations that cause human disease fall within the M9-like sequence of RP2 and these mutations block entry of RP2 into the cilium, as well as its interaction with Importin ß2. Together, this work further highlights a role of Importin ß2 in regulation of the entry of RP2 and other proteins into the ciliary compartment.