Abstract
Adenomatous Polyposis Coli (APC) is a tumor suppressor gene product involved in colon cancer. APC is a large multidomain molecule of 2843 amino acid residues and connects cell-cell adhesion, the F-actin/microtubule cytoskeleton and the nucleus. Here we show that Cdc42 interacts directly with the first three armadillo repeats of APC by yeast two-hybrid screens. We confirm the Cdc42-APC interaction using pulldown assays in vitro and FRET assays in vivo. Interestingly, Cdc42 interacts with APC at leading edge sites where F-actin is enriched. In contrast, Cdc42 interacts with the truncated mutant APC1–1638 in cellular puncta associated with the golgi-lysozome pathway in transfected CHO cells. In HCT116 and SW480 cells, Cdc42 induces the relocalization of endogenous APC and the mutant APC1–1338 to the plasma membrane and cellular puncta, respectively. Taken together, these data indicate that the Cdc42-APC interaction induces localization of both APC and mutant APC and may thus play a direct role in the functions of these proteins.
Highlights
Adenomatous Polyposis Coli (APC) is a tumor suppressor gene product involved in cellular processes including Wnt signaling, cell growth, cell fate determination and cell migration
GFP-Cdc42V12, HA-Cdc42 and GFPCdc42N17 were generated by subcloning the respective inserts into the pXJ40 vector. mRFP-Cdc42V12 and mRFP-Cdc42N17 were created by subcloning the respective gene into the pXJ40mRFP vector in between the BamHI and BglII restriction sites. mRFP-APC222–653 was made by inserting the amino acid sequences of APC222–653 into the pXJ40-mRFP vector in between the BamHI and NotI restriction sites
APC222–452 and APC453–653 were made by inserting the corresponding amino acid sequences of APC into the pXJ40-mRFP vector in between the BamHI and NotI restriction sites. mRFP-GFP tandem fusion was made by inserting GFP between the BamHI and NotI sites in the pXJ40-mRFP vector. mRFPSBP-Cdc42V12 and mRFP-SBP-GFP were made by inserting streptavidin binding protein (SBP) into the BamHI site
Summary
Adenomatous Polyposis Coli (APC) is a tumor suppressor gene product involved in cellular processes including Wnt signaling, cell growth, cell fate determination and cell migration. APC is a multidomain scaffold protein of 2843 amino acid (aa) residues linking the nucleus to cell surface events through associations with the cytoskeleton [1]. The APC N-terminal is a region that contains seven armadillo repeats [2] through which it binds KAP3 (kinesinassociated protein 3), Asef (Cdc42/Rac-specific guanine nucleotide exchange factor) and IQGAP1 (IQ motif containing GTPase activating protein 1) [3,4,5]. The C-terminal region of APC consists of positively charged amino acid residues responsible for binding microtubules and EB1 (end-binding protein 1). The binding site for a PDZ domain [8] is the last domain of the C-terminus
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