Membrane localization of adenomatous polyposis coli protein at cellular protrusions: targeting sequences and regulation by beta-catenin.
Adenomatous polyposis coli protein (APC) translocates to, and stabilizes, the plus-ends of microtubules. In microtubule-dependent cellular protrusions, APC frequently accumulates in peripheral clusters at the basal membrane. APC targeting to membrane clusters is important for cell migration, but the localization mechanism is poorly understood. In this study, we performed deletion mapping and defined a minimal sequence (amino acids 1-2226) that efficiently targets APC to membrane clusters. This sequence lacks DLG-1 and EB1 binding sites, suggesting that these partners are not absolutely required for APC membrane targeting. A series of APC sequences were transiently expressed in cells and compared for their ability to compete endogenous APC at the membrane; potent inhibition of endogenous APC targeting was elicited by the Armadillo- (binds KAP3A, B56alpha, and ASEF) and beta-catenin-binding domains. The Armadillo domain was predicted to inhibit APC membrane localization through sequestration of the kinesin-KAP3A complex. The role of beta-catenin in APC membrane localization was unexpected but affirmed by overexpressing the APC binding sequence of beta-catenin, which similarly reduced APC membrane staining. Furthermore, we used RNA interference to show that loss of beta-catenin reduced APC at membrane clusters in migrating cells. In addition, we report that transiently expressed APC-yellow fluorescent protein co-localized with beta-catenin, KAP3A, EB1, and DLG-1 at membrane clusters, but only beta-catenin stimulated APC anchorage at the membrane. Our findings identify beta-catenin as a regulator of APC targeting to membrane clusters and link these two proteins to cell migration.
Sharma
Manisha
M
Westmead Institute for Cancer Research, University of Sydney, Westmead Millennium Institute at Westmead Hospital, Westmead, New South Wales 2145, Australia.
Leung
Louie
L
Westmead Institute for Cancer Research, University of Sydney, Westmead Millennium Institute at Westmead Hospital, Westmead, New South Wales 2145, Australia.
Brocardo
Mariana
M
Westmead Institute for Cancer Research, University of Sydney, Westmead Millennium Institute at Westmead Hospital, Westmead, New South Wales 2145, Australia.
Henderson
Jasmine
J
Westmead Institute for Cancer Research, University of Sydney, Westmead Millennium Institute at Westmead Hospital, Westmead, New South Wales 2145, Australia.
Flegg
Cameron
C
Westmead Institute for Cancer Research, University of Sydney, Westmead Millennium Institute at Westmead Hospital, Westmead, New South Wales 2145, Australia.
Henderson
Beric R
BR
Westmead Institute for Cancer Research, University of Sydney, Westmead Millennium Institute at Westmead Hospital, Westmead, New South Wales 2145, Australia. Electronic address: beric_henderson@wmi.usyd.edu.au.
D025601:Adenomatous Polyposis Coli Protein / Q000096:biosynthesis* / Q000502:physiology*; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002462:Cell Membrane / Q000378:metabolism*; D004285:Dogs; D005786:Gene Expression Regulation; D051379:Mice; D008870:Microtubules / Q000737:chemistry; D041681:NIH 3T3 Cells; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D014162:Transfection; D051176:beta Catenin / Q000096:biosynthesis* / Q000378:metabolism
D025601:Adenomatous Polyposis Coli Protein; D051176:beta Catenin