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

Natasha K Hussain,Montarop Yamabhai,Asha L Bhakar,Martina Metzler,Stephen S.G Ferguson,Michael R Hayden,Peter S Mcpherson,Brian K Kay

doi:10.1074/jbc.m300995200

Abstract

The epsin N-terminal homology (ENTH) domain is a protein module of approximately 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 alpha-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 approximately 1 microm, 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.

Highlights

The epsin N-terminal homology (ENTH) domain is a protein module of ϳ150 amino acids found at the N terminus of a variety of proteins identified in yeast, plants, nematode, frog, and mammals
Adaptor protein 180 (AP180), clathrin assembly lymphoid myeloid leukemia protein (CALM), Huntingtin-interacting protein-1 (HIP1) and HIP12, and the yeast proteins yAP180 and Sla2p contain a module that is so similar in structure to the epsin ENTH domain that they were initially denoted ENTHbearing proteins [11,12,13]
Recent studies have demonstrated that the E/ANTH domains of epsin and AP180 can mediate lipid binding, to phosphatidylinositol 4,5-bisphosphate (PtdIns[4,5]P2), and that this interaction is required for efficient clathrin-mediated endocytosis in COS-7 cells [12, 13]

Summary

ENTH Domains Bind Tubulin

Lipid-ligand pocket is coordinated by residues in ␣1, the ␣1–2 loop, ␣3, and ␣4 and is dependent on the formation and binding of ␣0, a helix that is not present or generated in the ANTH domain [12,13,14]. Aguilar et al [26] have identified an ENTH domain mutant that fails to rescue Ent1⌬Ent2⌬ cells despite the fact that this mutant continues to bind PtdIns[4,5]P2 Based on these findings, the authors suggest that at least in yeast, another critical protein-binding partner(s) must exist to assist ENTH domain functions [26]. Previous studies into the function of this module have revealed that ENTH domains can mediate interactions with proteins; the ENTH domain of mammalian epsin 1 binds weakly to the vesicular coat protein coatomer and more robustly to human promyelocytic leukemia Zn2ϩ finger protein (PLZF), a transcriptional repressor [11]. We propose that interaction with tubulin is an evolutionarily conserved property of E/ANTH domains

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION