Exp Mol Med.  2009 Aug;41(8):555-568. 10.3858/emm.2009.41.8.061.

Ubiquitylation of Fe65 adaptor protein by neuronal precursor cell expressed developmentally down regulated 4-2 (Nedd4-2) via the WW domain interaction with Fe65

• Lee EJ
• Hyun S
• Chun J
• Shin SH
• Kang SS

• KMID: 1119321
• DOI: http://doi.org/10.3858/emm.2009.41.8.061

Abstract

Fe65 has been characterized as an adaptor protein, originally identified as an expressed sequence tag (EST) corresponding to an mRNA expressed at high levels in the rat brain. It contains one WW domain and two phosphotyrosine interaction/phosphotyrosine binding domains (PID1/PID2). As the neuronal precursor cell expressed developmentally down regulated 4-2 (Nedd4-2) has a putative WW domain binding motif (72PPLP75) in the N-terminal domain, we hypothesized that Fe65 associates with Nedd4-2 through a WW domain interaction, which has the characteristics of E3 ubiquitin-protein ligase. In this paper, we present evidence for the interaction between Fe65 WW domain and Nedd4-2 through its specific motif, using a pull down approach and co-immunoprecipitation. Additionally, the co-localization of Fe65 and Nedd4-2 were observed via confocal microscopy. Co-localization of Fe65 and Nedd4-2 was disrupted by either the mutation of Fe65 WW domain or its putative binding motif of Nedd4-2. When the ubiquitin assay was performed, the interaction of Nedd4-2 (wt) with Fe65 is required for the cell apoptosis and the ubiquitylation of Fe65. We also observed that the ubiquitylation of Fe65 (wt) was augmented depending on Nedd4-2 expression levels, whereas the Fe65 WW domain mutant (W243KP245K) or the Nedd4-2 AL mutant (72PPLP75 was changed to 72APLA75) was under-ubiquitinated significantly. Thus, our observations implicated that the protein-protein interaction between the WW domain of Fe65 and the putative binding motif of Nedd4-2 down-regulates Fe65 protein stability and subcellular localization through its ubiquitylation, to contribute cell apoptosis.

Keyword

APBB2 protein, human; Nedd4 ubiquitin protein ligases; protein interaction domains and motifs; protein interaction mapping; ubiquitin

MeSH Terms

Adaptor Proteins, Signal Transducing/chemistry/genetics/*metabolism
Cell Line
*Down-Regulation
Endosomal Sorting Complexes Required for Transport/genetics/*metabolism
*Gene Expression Regulation, Developmental
Humans
Immunoprecipitation
Microscopy, Confocal
Mutation
Protein Interaction Mapping
Protein Structure, Tertiary/*physiology
Transfection
Ubiquitin-Protein Ligases/genetics/*metabolism
Ubiquitination