A Role for Epsin N-terminal Homology/AP180 N-terminal Homology (ENTH/ANTH) Domains in Tubulin Binding

The epsin N-terminal homology (ENTH) domain is a protein module of ∼150 amino acids found at the N terminus of a variety of proteins identified in yeast, plants, nematode, frog, and mammals. ENTH domains comprise multiple α-helices folded upon each other to form a compact globular structure that has been implicated in interactions with lipids and proteins. In characterizing this evolutionarily conserved domain, we isolated and identified tubulin as an ENTH domain-binding partner. The interaction, which is direct and has a dissociation constant of ∼1 μm, was observed with ENTH domains of proteins present in various species. Tubulin is co-immunoprecipitated from rat brain extracts with the ENTH domain-containing proteins, epsins 1 and 2, and punctate epsin staining is observed along the microtubule cytoskeleton of dissociated cortical neurons. Consistent with a role in microtubule processes, the over-expression of epsin ENTH domain in PC12 cells stimulates neurite outgrowth. These data demonstrate an evolutionarily conserved property of ENTH domains to interact with tubulin and microtubules. The epsin N-terminal homology (ENTH) domain is a protein module of ∼150 amino acids found at the N terminus of a variety of proteins identified in yeast, plants, nematode, frog, and mammals. ENTH domains comprise multiple α-helices folded upon each other to form a compact globular structure that has been implicated in interactions with lipids and proteins. In characterizing this evolutionarily conserved domain, we isolated and identified tubulin as an ENTH domain-binding partner. The interaction, which is direct and has a dissociation constant of ∼1 μm, was observed with ENTH domains of proteins present in various species. Tubulin is co-immunoprecipitated from rat brain extracts with the ENTH domain-containing proteins, epsins 1 and 2, and punctate epsin staining is observed along the microtubule cytoskeleton of dissociated cortical neurons. Consistent with a role in microtubule processes, the over-expression of epsin ENTH domain in PC12 cells stimulates neurite outgrowth. These data demonstrate an evolutionarily conserved property of ENTH domains to interact with tubulin and microtubules. The epsin N-terminal homology (ENTH) 1The abbreviations used are: ENTH, epsin N-terminal homology; ANTH, AP180 N-terminal homology; E/ANTH, epsin/AP180 N-terminal homology; DH, Dbl homology; GFP, green fluorescent protein; GST, glutathione S-transferase; HIP, Huntingtin-interacting protein; MAP, microtubule-associated protein; MES, 4-morpholineethanesulfonic acid; MP90, mitotic phosphoprotein of 90 kDa; PH, pleckstrin homology; PIPES, 1,4-piperazinediethanesulfonic acid; PtdIns(4,5)P2, phosphatidylinositol 4,5-bisphosphate; SH3, Src homology 3; TRITC, tetramethylrhodamine isothiocyanate. domain is an evolutionarily conserved globular module of ∼150 amino acids that occurs at the amino terminus of a variety of proteins (1Kay B.K. 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