Abstract
We have previously identified mouse DDA3 as a p53-inducible gene. To explore the functional role of DDA3, we screened a mouse brain cDNA library by the yeast two-hybrid assay, and identified the microtubule plus-end binding protein EB3 as a DDA3-interacting protein. Binding of DDA3 to EB3 was verified by glutathione S-transferase (GST) pull-down assay and subcellular colocalization; co-immunoprecipitation further indicated that interaction of these two proteins within cells required intact microtubules. Domains of DDA3-EB3 interaction were mapped by GST pull-down assay to amino acids 118-241 and 242-329 of DDA3 and the N- and C-termini of EB3. Immunofluorescence analysis revealed colocalization of DDA3 with microtubules in various cell phases, and regions encompassing aa 118-241 and 242-329 contained microtubule-interacting and bundling activities. In vitro microtubule-binding assay showed that DDA3 and EB3 associated directly with microtubules, and cooperated with each other for microtubule binding. In addition, DDA3 bound to the EB3 interacting partner adenomatous polyposis coli 2 (APC2), a homolog of the tumor suppressor APC, which is a component of the beta-catenin destruction complex. Ectopic expression of DDA3 and EB3 enhanced beta-catenin-dependent transactivation and cyclin D1 production, whereas knockdown of endogenous DDA3 or EB3 inhibited beta-catenin-mediated transactivation and the ability of cells to form colonies. Together, our results identify DDA3 as a novel microtubule-associated protein that binds to EB3, and implicate DDA3 and EB3 in the beta-catenin-mediated growth signaling.
Highlights
Mutation of the p53 tumor suppressor is the most common genetic alteration in cancer; about half of the human cancers contain p53 mutation
In response to a variety of cellular stresses, p53 undergoes post-translational modifications and is stabilized and activated, which leads to growth arrest and apoptosis of cells; as a result, damaged cells are prevented from further replication (Vogelstein et al, 2000; Hofseth et al, 2004; Levine et al, 2004). p53 is a sequence-specific transcription factor, the majority of p53 mutations in cancers are point mutations clustered within its DNAbinding domain that render the protein transcriptionally inactive (Hainaut et al, 1998), indicating that transcriptional regulation is a major mechanism by which p53 implements its tumor suppressor function
We further show that DDA3 binds directly to the EB3 interacting partner adenomatous polyposis coli 2 (APC2)
Summary
Mutation of the p53 tumor suppressor is the most common genetic alteration in cancer; about half of the human cancers contain p53 mutation. Activation of Wnt signaling inactivates glycogen synthase kinase 3b (GSK-3b), a member of the multicomponent b-catenin degradation complex that includes adenomatous polyposis coli (APC) and axin/conductin, and leads to inappropriate b-catenin accumulation. APC is a multifunctional protein located at microtubule plus-ends and interacts with many binding partners (Bienz, 2002; Hanson and Miller, 2005; Senda et al, 2005), including the microtubule plusend protein EB1, which binds to the C-terminus of APC (Su et al, 1995; Honnappa et al, 2005) The importance of this interaction in carcinogenesis is shown by the finding that mutation of the APC tumor suppressor that disrupts its interaction with EB1 occurs commonly in the familial and sporadic colon cancers (Su et al, 1995). The biological significance of the APC2EB3 interaction remains to be elucidated
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