Alternative splicing as a mechanism for regulating 14-3-3 binding: interactions between hD53 (TPD52L1) and 14-3-3 proteins.
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 14-3-3beta and 14-3-3zeta 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 14-3-3beta and 14-3-3zeta 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.
Boutros
Rose
R
Molecular Oncology Laboratory, Oncology Research Unit, The Children's Hospital at Westmead, Locked Bag 4001, Westmead, NSW, 2145, Australia.
Bailey
Angela M
AM
Wilson
Sarah H D
SH
Byrne
Jennifer A
JA
D048948:14-3-3 Proteins; D017398:Alternative Splicing; D001665:Binding Sites; D001943:Breast Neoplasms / Q000378:metabolism; D002277:Carcinoma / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D005091:Exons; D005260:Female; D006801:Humans; D007106:Immune Sera; D009363:Neoplasm Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D011233:Precipitin Tests; D011485:Protein Binding / Q000502:physiology; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012995:Solubility; D014407:Tumor Cells, Cultured; D020798:Two-Hybrid System Techniques; D014446:Tyrosine 3-Monooxygenase / Q000235:genetics* / Q000378:metabolism*
D048948:14-3-3 Proteins; D007106:Immune Sera; D009363:Neoplasm Proteins; D011994:Recombinant Proteins; C114751:TPD52 protein, human; C486416:TPD52L1 protein, human; C482982:YWHAB protein, human; D014446:Tyrosine 3-Monooxygenase