Interaction between Tumor Suppressor Adenomatous Polyposis Coli and Topoisomerase IIα: Implication for the G2/M Transition

Abstract

The tumor suppressor adenomatous polyposis coli (APC) is implicated in regulating multiple stages of the cell cycle. APC participation in G1/S is attributed to its recognized role in Wnt signaling. APC function in the G2/M transition is less well established. To identify novel protein partners of APC that regulate the G2/M transition, APC was immunoprecipitated from colon cell lysates and associated proteins were analyzed by matrix-assisted laser desorption ionization/time of flight (MALDI-TOF). Topoisomerase II␣ (topo II␣) was identified as a potential binding partner of APC. Topo II␣ is a critical regulator of G2/M transition. Evidence supporting an interaction between endogenous APC and topo II␣ was obtained by coimmunoprecipitation, colocalization, and Fö rster resonance energy transfer (FRET). The 15-amino acid repeat region of APC (M2-APC) interacted with topo II␣ when expressed as a green fluorescent protein (GFP)-fusion protein in vivo.Although lacking defined nuclear localization signals (NLS) M2-APC predominantly localized to the nucleus. Furthermore, cells expressing M2-APC displayed condensed or fragmented nuclei, and they were arrested in the G2 phase of the cell cycle. Although M2-APC contains a ␤-catenin binding domain, biochemical studies failed to implicate ␤-catenin in the observed phenotype. Finally, purified recombinant M2-APC enhanced topo II␣ activity in vitro. Together, these data support a novel role for APC in the G2/M transition, potentially through association with topo II␣. INTRODUCTIONThe tumor suppressor protein adenomatous polyposis coli (APC) is inactivated in Ͼ80% of all colorectal cancers (Kinzler and Vogelstein, 1996). The detection of mutant APC in the earliest stages of polyp development supports the idea that mutation of APC is an initiating event in colon carcinogenesis. The most common form of APC mutation results in elimination of the carboxy-terminal half of the APC protein.Because APC is a large, multidomain protein, APC truncation is predicted to impact several cellular mechanisms, the extent of which we are only beginning to understand.There is accumulating evidence supporting a role for APC in the regulation of cell cycle. Overexpression of APC in NIH3T3 fibroblasts and colon cancer cell lines leads to G1 cell cycle arrest (Ishidate et al., 2000;Heinen et al., 2002), presumably by repressing transcription of Wnt targets such as cyclin D1. APC may also participate directly in mitosis because it is transiently hyperphosphorylated in the M phase of the cell cycle (Bhattacharjee et al., 1996), accumulates at the microtubule-organizing center (Olmeda et al., 2003), and associates with the kinetochore in dividing cells (Fodde et al., 2001;Kaplan et al., 2001). A role for APC in mitosis might be critical for regulation of genomic stability and proper chromosome segregation. APC stabilized by zinc treatment induces G2/M cell cycle arrest in colon cancer cells (Jaiswal and Narayan, 2004). However, to date, little is known about the underlying mechanism by which APC participat...

Nup358 interacts with APC and plays a role in cell polarization

“…Moreover, a recent study suggests that the mitotic role of Nup358 includes SUMOylation of topoisomerase II and regulation of its activity at the kinetochores (Dawlaty et al, 2008). In a different report, APC has been shown to interact with topoisomerase II and regulate its activity during G2-M transition (Wang et al, 2008). An interesting possibility is that the mitotic roles of APC, topoisomerase II and Nup358 could involve functioning of these players at the kinetochores in a coordinated manner.…”

Interactions and functions of the adenomatous polyposis coli (APC) protein at a glance

“…Many of the known interaction partners of APC, such as bcatenin, are accounted for. However, not all of the previously published protein-protein interactions of APC, including those with the Ras GTPase-activating-like protein IQGAP1, topoisomerase 2, and the APC membrane recruitment proteins AMER1 and AMER2, were included in the BioGRID database, suggesting that it is not complete (Pfister et al, 2012a;Pfister et al, 2012b;Wang et al, 2008;Wang et al, 2010;Watanabe et al, 2004). The entire set of the protein interactions of APC reported by BioGRID, plus the omissions we detected on the basis of published data, is shown on the accompanying poster grouped into colour-coded clusters showing the functions of the interactors (Breitkreutz et al, 2003).…”

New insights from animal models of colon cancer: inflammation control as a new facet on the tumor suppressor APC gem

“…36 APC interacts with Topoisomerase IIα, an enzyme essential in DNA replication and cell cycle progression. 39,40 APC also interacts with PCNA, FEN-1, and polymerase-β, components of long patch-base excision repair (LP-BER), [41][42][43][44][45] and affects CREB-C/EBP-mediated transcription. 46 Although the significance is not completely understood, APC appears to directly interact with A/T-rich DNA sequences.…”