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Regulation and function of the interaction between the APC tumour suppressor protein and EB1

Oncogene volume 19, pages 1950–1958 (2000)Cite this article

Abstract

The interaction between the adenomatous polyposis coli (APC) tumour suppressor and the microtubule-associated protein EB1 was examined. Immunoprecipitation suggested that APC and EB1 were not associated in cultures of HCT116 cells arrested in mitosis. The C-terminal 170 amino acids of APC, purified as a bacterial fusion protein, precipitated EB1 from cell extracts, significantly refining the location of the EB1 interaction domain in APC. In vitro phosphorylation of this fusion protein by either protein kinase A or p34cdc2 reduced its ability to bind to EB1. Expression of GFP fusions to C-terminal APC sequences lacking or including the APC basic domain but encompassing the EB1 binding region in SW480 cells revealed a microtubule tip association which co-localized with that of EB1. Expression of the basic domain alone revealed a non-specific microtubule localization. In vitro interaction studies confirmed that the APC basic domain did not contribute to EB1 binding. These findings strongly suggest that the interaction between APC and EB1 targets APC to microtubule tips, and that the interaction between the two proteins is down-regulated during mitosis by the previously described mitotic phosphorylation of APC.

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Abbreviations

APC:

adenomatous polyposis coli

GST:

glutathione-S-transferase

DTT:

dithiothreitol

PBS:

phosphate buffered saline

PCR:

polymerase chain reaction

TX100:

Triton X-100

AS:

asynchronous

M:

mitosis

PKA:

protein kinase A

PKC:

protein kinase C

CKII:

casein kinase II

eGFP:

enhanced green fluorescent protein

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Acknowledgements

This work was supported by grants from the Medical Research Council (UK), Yorkshire Cancer Research and the West Riding Medical Research Trust.

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  1. Molecular Medicine Unit, University of Leeds, Clinical Sciences Building, St. James's University Hospital, Leeds, LS9 7TF, UK

    J M Askham, P Moncur, A F Markham & E E Morrison

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  1. J M Askham
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Askham, J., Moncur, P., Markham, A. et al. Regulation and function of the interaction between the APC tumour suppressor protein and EB1. Oncogene 19, 1950–1958 (2000). https://doi.org/10.1038/sj.onc.1203498

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