BMB reports 183 http://bmbreports.org BMB reports *Corresponding author. Shuanglin Xiang, Tel: +86-731-8887-2905; Fax: +86-731-8887-2905; E-mail: xshlin@hunnu.edu.cn; Jian Zhang, T el: +86-731-8887-2792; Fax: +86-731-8887-2792; E-mail: zhang- jian@hunnu.edu.cn # These authors contributed equally to this work. http://dx.doi.org/10.5483/BMBRep.2012.45.3.183 Received 15 September 2011, Revised 6 October 2011, Accepted 6 December 2011 Keywords: Eps8, Interaction, ITSN2, Lysosome pathway, Protein degradation Human Intersectin 2 (ITSN2) binds to Eps8 protein and enhances its degradation Xiaofeng Ding # , Zijian Yang # , Fangliang Zhou, Xiang Hu, Chang Zhou, Chang Luo, Zhicheng He, Qian Liu, Hong Li, Feng Yan, Fangmei Wang, Shuanglin Xiang*& Jian Zhang* Key Laboratory of Protein Chemistry and Development Biology of State Education Ministry of China, College of Life Science, Hunan Normal University, Changsha, China Participates in actin remodeling through Rac and receptor en- docytosis via Rab5. Here, we used yeast two-hybrid system with Eps8 as bait to screen a human brain cDNA library. ITSN2 was identified as the novel binding factor of Eps8. The interaction between ITSN2 and Eps8 was demonstrated by the in vivo co-immunoprecipitation and colocalization assays and the in vitro GST pull-down assays. Furthermore, we mapped the interaction domains to the region between amino acids 260-306 of Eps8 and the coiled-coil domain of ITSN2. In addi- tion, protein stability assays and immunofluorescence analysis showed ITSN2 overexpression induced the degradation of Eps8 proteins, which was markedly alleviated with the lyso- some inhibitor NH4Cl treatment. Taken together, our results suggested ITSN2 interacts with Eps8 and stimulates the degra- dation of Eps8 proteins. [BMB reports 2012; 45(3): 183-188] INTRODUCTION Eps8, originally identified as a substrate for the epidermal growth factor receptor kinase, acts as a multifunctional molecule involved in EGFR signaling (1, 2) and the development of certain malignancies (3-5). The functions of Eps8 have been extensively studied by its domains. The N-terminal phosphotyrosine binding domain (PTB) is a conserved protein-protein interaction domain in a phosphotyrosine-dependent and -independent fashion (6-10). The proline-rich region (PR) is involved in IRSp53/Eps8 complex formation (11). The EGFR binding region (BR) of Eps8 is required for the binding of the juxtamembrane region of EGFR and enhances EGF-dependent mitogenic signals (2). The Src-ho- mology-3 (SH3) domain is a ubiquitous interaction module (12, 13), two interactors, E3b1/Abi-1 and RN-tre, bind to the SH3 do- main of Eps8 and regulate cell growth (14, 15). Further studies showed the Rab5 GTPase-activating protein RN-tre binds with Eps8 to inhibit internalization of the EGFR and control intra- cellular membrane trafficking (16). The Eps8 C-terminal effector region is responsible for the interaction with Sos1 and F-actin (17). Moreover, Abi-1 bridges Eps8 and Sos1 to form a tricom- plex and the complex induces Rac-specific guanine nucleotide exchange factor (GEF) activity, transduces signals from Ras to Rac, enhances Rac-dependent actin remodeling (17-19). Therefore, the Eps8 protein coordinates EGF receptor signaling through Rac and endocytic trafficking through Rab5. Eps8 was implicated in many tumor cell proliferation and invasion. High expression and concomitant tyrosine phosphor- ylation of Eps8 were detected in tumor cell lines (20). Eps8 en- hances EGF-dependent mitogenic signals and leads to malignant transformation (1, 20). Eps8 increases cell growth and motility, by the up-regulation of FOXM1, FAK, MMP-9, ERK, Akt, cyclins D1, D3, and E, the down-regulation of P53 and p21Waf1/Cip1 in squamous cell carcinoma, pancreatic cancer, cervical cancer, colon cancer, pituitary tumors and esophageal carcinomas (4, 5, 21-26). Eps8 is an important signal molecule, we demonstrated here that ITSN2 and Eps8 interact in vivo, and the interaction be- tween both proteins results in the decrease of Eps8 proteins. RESULTS ITSN2 interacts with Eps8 in yeast two-hybrid screening To identify Eps8-interacting proteins, we performed yeast two-hybrid screen. Yeast strain MaV203 was transfected with bait plasmid pDBLeu-Eps8 and then prey plasmid pPC86-cDNA with human brain cDNA library cloned with the GAL4 activa- tion domain of pPC86 vector. After screening on a SD-Leu − , Trp − , uracil − , His − medium supplemented with 25 mM 3-AT, ITSN2 was identified as an interacting partner of Eps8 by X-gal- actosidase filter assays and sequencing analysis (Fig. 1A). Likewise, blue colonies appear in positive control C and positive control D but not in controls expressing pDBLeu-Eps8 and