Alternative Splicing as a Mechanism for Regulating 14-3-3 Binding: Interactions between hD53 (TPD52L1) and 14-3-3 Proteins

• Published in: Journal of molecular biology Vol. 332; no. 3; pp. 675 - 687
• Main Authors: Boutros, Rose, Bailey, Angela M, Wilson, Sarah H.D, Byrne, Jennifer A
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 19.09.2003
• Subjects: 14-3-3; 14-3-3 Proteins; Alternative Splicing; Binding Sites; Breast Neoplasms - metabolism; Carcinoma - metabolism; Cytoplasm - metabolism; Exons; Female; gene structure; Humans; Immune Sera; Neoplasm Proteins - genetics; Neoplasm Proteins - immunology; Neoplasm Proteins - metabolism; Precipitin Tests; Protein Binding - physiology; protein–protein interactions; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Solubility; TPD52; Tumor Cells, Cultured; Two-Hybrid System Techniques; Tyrosine 3-Monooxygenase - genetics; Tyrosine 3-Monooxygenase - metabolism; TPD52; 14-3-3; gene structure; protein–protein interactions; PBS; RT; alternative splicing; ins3; TBS; Th; Y2H
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1016/S0022-2836(03)00944-6

D52 (TPD52)-like proteins are coiled-coil motif-bearing proteins first identified through their expression in human breast carcinoma, which have been proposed to represent signalling intermediates and regulators of vesicle trafficking. D52-like gene transcripts are subject to alternative splicing, with sequences encoding a region termed insert 3 being affected in all three D52-like genes. We have now identified a 14-3-3 binding motif within one of two alternatively spliced exons encoding insert 3. As predicted from the distribution of 14-3-3 binding motifs in four hD52-like bait proteins tested, only a hD53 isoform encoding a 14-3-3 binding motif bound both 14-3-3β and 14-3-3ζ preys in the yeast two-hybrid system. Since D53 proteins carrying 14-3-3 binding motifs are predicted to be widely expressed, polyclonal antisera were derived to specifically detect these isoforms. Using soluble protein extracts from breast carcinoma cell lines, pull-down assays replicated interactions between recombinant 14-3-3β and 14-3-3ζ isoforms and exogenously expressed hD53, and co-immunoprecipitation analyses demonstrated interactions between endogenous 14-3-3 and both endogenously and exogenously-expressed hD53 protein. Co-expressed hD53 and 14-3-3 proteins were similarly demonstrated to co-localise within the cytoplasm of breast carcinoma cell lines. These results identify 14-3-3 proteins as partners for hD53, and alternative splicing as a mechanism for regulating 14-3-3 binding.