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Abstract
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.