Published Online: 27 December, 1999 | Supp Info: http://doi.org/10.1083/jcb.147.7.1379 Downloaded from jcb.rupress.org on July 31, 2018 Brief Report The Eps15 Homology (EH) Domain-based Interaction between Eps15 and Hrb Connects the Molecular Machinery of Endocytosis to That of Nucleocytosolic Transport Margherita Doria,* Anna Elisabetta Salcini,* Emanuela Colombo,* Tristram G. Parslow,‡ Pier Giuseppe Pelicci,*§ and Pier Paolo Di Fiore*i *Department of Experimental Oncology, European Institute of Oncology, 20141 Milan, Italy; ‡Department of Pathology, University of California, San Francisco California 94193; §Istituto di Patologia Speciale Medica, University of Parma, 43100 Italy; and iIstituto di Microbiologia, University of Bari, 70123 Italy Abstract. The Eps15 homology (EH) module is a pro- to enhance the function of Rev in the export pathway. tein–protein interaction domain that establishes a net- In addition, the EH-mediated association between work of connections involved in various aspects of en- Eps15 and Hrb is required for the synergistic effect. docytosis and sorting. The finding that EH-containing The interaction between Eps15 and Hrb occurs in the proteins bind to Hrb (a cellular cofactor of the Rev pro- cytoplasm, thus pointing to an unexpected site of action tein) and to the related protein Hrbl raised the possi- of Hrb, and to a possible role of the Eps15–Hrb com- bility that the EH network might also influence the plex in regulating the stability of Rev. so-called Rev export pathway, which mediates nucleo- cytoplasmic transfer of proteins and RNAs. In this Key words: EH • Eps15 • Hrb • endocytosis • nucleo- study, we demonstrate that Eps15 and Eps15R, two cytoplasmic transport EH-containing proteins, synergize with Hrb and Hrbl HE Eps15 homology (EH)1 domain is a protein–pro- Eps15, Eps15R, and intersectin, interact with Hrb (also tein interaction module originally identified in the called hRip or RAB, Human Gene Nomenclature Com- Ttyrosine kinase substrates Eps15 (Fazioli et al., mittee approved symbols are used in this paper), a cellular 1993) and Eps15R (Wong et al., 1995; Coda et al., 1998). cofactor for the HIV-1 Rev protein (Bogerd et al., 1995; Several EH-binding proteins have been subsequently Fritz et al., 1995), and with the related protein Hrbl (Sal- identified (Salcini et al., 1997). Biochemical and functional cini et al., 1997; Yamabhai et al., 1998). studies implicated proteins of the EH network in endocy- By shuttling between the nucleus and cytoplasm of an tosis and actin cytoskeleton organization (reviewed in San- infected cell, Rev induces nucleocytoplasmic export of un- tolini et al., 1999). Other evidence points to additional spliced and partially spliced viral mRNAs that contain a functions. In particular, the EH network might be in- unique Rev binding sequence (Rev response element, volved in the control of nucleocytoplasmic export, as sug- [RRE]; Cullen, 1998). Rev functions through a cellular gested by the finding that three EH-containing proteins, pathway, operationally defined as the Rev export path- way, which normally exports endogenous RNAs (Fischer et al., 1995), and proteins that, like Rev, contain nuclear Address correspondence to Pier Paolo Di Fiore, Istituto Europeo di On- cologia, Via Ripamonti 435, 20141 Milan, Italy. Tel.: 39-02-57489855. Fax: export signals (NESs; Fridell et al., 1996; Fritz and Green, 39-02-57489851. E-mail: [email protected] 1996; Roth et al., 1998). Efforts to identify cellular cofac- Dr. Doria’s present address is Ospedale Pediatrico Bambino Gesù, tors mediating Rev export led to the isolation of the dis- D0133 Rome, Italy. tantly related human Hrb (Bogerd et al., 1995; Fritz et al., 1. Abbreviations used in this paper: aa, amino acids; EH, Eps15 homology; 1995) and yeast Rip1p (Stutz et al., 1995) proteins, which FG, phenylalanine-glycine; NESs, nuclear export signals; RRE, Rev re- were shown to enhance Rev function in cells. Both pro- sponse element. teins also are related to nucleoporins, in that they display The Rockefeller University Press, 0021-9525/99/12/1379/6 $5.00 The Journal of Cell Biology, Volume 147, Number 7, December 27, 1999 1379–1384 http://www.jcb.org 1379 phenylalanine-glycine (FG) repeats, a common feature of used were: anti-Eps15 (Fazioli et al., 1993) and anti-Eps15R (Coda et al., this class of proteins. These observations led to the hy- 1998) sera; polyclonal anti-Hrb IgGs directed against the last 19 amino ac- ids of Hrb (Santa Cruz Biotechnology); a polyclonal anti-Hrbl serum rec- pothesis that Rev recruits RRE-containing RNAs to the ognizing amino acids 280–481 of the protein. Routinely, immunoblots nuclear pore, through direct interaction with Hrb or other were stripped and reprobed with an anti-tubulin antibody, to ensure equal nucleoporins. protein loading in the various lanes (not shown). Recent observations suggest a more complex picture. The interaction between Hrb (or Rip1p) and Rev, ob- served in the yeast two-hybrid system, could not be dem- Results onstrated using purified proteins in vitro, suggesting that it might be indirect (Stutz et al., 1996; Henderson and Perci- Eps15 and Eps15R Influence the Rev Export Pathway palle, 1997; Neville et al., 1997). Indeed, Crm1, a shuttling protein that belongs to the transport receptor family, To gain insight into the contribution of Eps15 and Eps15R serves as a bridging factor between Hrb and Rev (re- to the nucleocytoplasmic export pathway used by Rev, we viewed in Ullman et al., 1997). Crm1 binds to NESs and used a previously described assay based on cotransfection mediates the export of NES-containing proteins (For- of Rev with the reporter plasmid pDM128 (Huang et al., nerod et al., 1997; Stade et al., 1997). In addition, Crm1 in- 1991). Transcripts from this latter construct contain a CAT teracts with the FG repeats of Hrb and of several FG- coding sequence within an intron from the env region of nucleoporins (Neville et al., 1997). Thus, Rev might be re- HIV-1, which includes an RRE. Upon cotransfection, cruited to nuclear pores indirectly through sequential in- Rev binds to the RRE, thus allowing cytoplasmic translo- teractions with Crm1 and Hrb. There are, however, addi- cation and expression of the unspliced transcripts (Huang tional difficulties with this model. First, the FG-containing et al., 1991). In preliminary experiments (not shown), we region of Rip1p was shown to make only limited contribu- transiently cotransfected CV-1 cells with pDM128 and in- tions to Rev-mediated export (Stutz et al., 1997). Second, creasing amounts of a Rev expression vector pDM121. it is not yet certain whether Hrb is an authentic constituent Transactivation of CAT by Rev was linear in a range from of the nuclear pore complex (Bogerd et al., 1995; Fritz et al., 4–100-fold activation. For all subsequent experiments we 1995). Thus, while the finding that overexpression of Hrb used an amount of pDM121 yielding z20% of the maxi- enhances Rev activity, albeit modestly, suggests a role for mal transactivation. Hrb in the Rev export pathway, the mechanisms remain to When expression vectors for Eps15 or Eps15R were be determined. Based on the observation of a physical in- cotransfected with Rev, an increase in CAT activity, teraction between Eps15 (and Eps15R) with Hrb (and z50% greater than the value obtained with Rev alone, Hrbl; Salcini et al., 1997), this study was undertaken to test was reproducibly detected (Fig. 1 A). Expression vectors whether EH-mediated interactions are involved in the for Hrb, a known cofactor of Rev, and for Hrbl yielded a control of the Rev export pathway. comparable 50% increase in Rev activity (Fig. 1 A). All responses were Rev dependent, since they could be abol- ished by replacing pDM128 with pDM138, a variant con- Materials and Methods struct lacking the RRE sequence (Fig. 1 A), or by omitting the pDM121 construct (not shown). The effects were also Vectors not due to influence of Eps15, Eps15R, Hrb, or Hrbl on transcription, since none of the corresponding vectors af- pDM128, pDM138, pDM121 (Huang et al., 1991), pCEVEps15, and fected expression of an exonic CAT gene under the con- pCEVEps15R (Wong et al., 1995; Coda et al., 1998) have been previously described. pMTHrb and pMTHrbl were generated by subcloning the open trol of an SV-40 or RSV promoter in the absence of the reading frame of the human cDNAs into the pMT2 vector. pCEVaEps15 RRE (not shown). harbors the Eps15 cDNA in the anti-sense orientation in the LTR-based To prove the physiological relevance of Eps15 to the pCEV vector. pMTHA-HrbNPV was generated by changing the se- Rev export pathways, we used an antisense Eps15 con- quences coding for the four NPF motifs to sequences coding for NPV in the pMTHA-Hrb vector (Salcini et al., 1997). pCEVEps15DEH, encoding struct (pCEVaEps15). Transfection of pCEVaEps15 into a Eps15 protein devoid of its EH domains (and encompassing amino acids CV-1 cells significantly reduced the steady state levels of [aa] 342–897), was engineered by PCR and subcloned into pCEV. All con- Eps15, as revealed by both immunoblotting and immuno- structs were sequenced in the regions that underwent genetic manipula- fluorescence analyses (Fig. 1 B). When pCEVaEps15 was tions. cotransfected with pDM128 and the Rev expression vec- CAT Assays tor, a dose-dependent reduction in the activity of Rev was observed (Fig. 1 C). CV-1 cells were transfected by the calcium phosphate method, with the CAT (chloramphenycol acetyltransferase) reporter plasmid pDM128 (100 Eps15 and Eps15R Synergize with Hrb and Hrbl ng) together with the Rev expression vector pDM121 (10 ng) and the pCMVbgal reporter plasmid (Clontech, 100 ng). Various combinations of The finding that Eps15 and Eps15R can function as cofac- pMT-Hrb (0.25 mg), pMT-Hrbl (0.25 mg), pCEVEps15 (1 mg), and tors for the Rev export pathway prompted us to inves- pCEVEps15R (1 mg) were also transfected.