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Biochem Biophys Res Commun
2009 Oct 09;3881:12-6. doi: 10.1016/j.bbrc.2009.07.114.
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Ca(2+) regulates the subcellular localization of adenomatous polyposis coli tumor suppressor protein.
Togo T
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???displayArticle.abstract??? Microtubule (MT) plus-end tracking proteins (+TIPs) are involved in the regulation of MT plus-end dynamics and stabilization. It was reported previously that an increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) induced by disruption of the plasma membrane stimulates rearrangement of MTs [T. Togo, Disruption of the plasma membrane stimulates rearrangement of microtubules and lipid traffic toward the wound site, J. Cell Sci. 119 (2006) 2780-2786], suggesting that some +TIPs are regulated by Ca(2+). In the present study, the behavior of adenomatous polyposis coli (APC) following an increase in [Ca(2+)](i) was observed using Xenopus A6 epithelial cell expressing GFP-tagged APC. An increase in [Ca(2+)](i) by cell membrane disruption or by ionomycin treatment induced dissociation of APC without depolymerizing MTs. Inhibition of a tyrosine kinase and GSK-3beta suppressed APC dissociation upon an increase in [Ca(2+)](i). Western blotting analysis showed that Ca(2+) transients activated GSK-3beta through a tyrosine kinase. These results suggest that Ca(2+) stimulates redistribution of APC through a tyrosine kinase- and GSK-3beta-dependent pathway.
Fig. 1. Cell membrane disruption induces dissociation of APC and recruitment of EB1 in Xenopus A6 cells. (A,B) An A6 cell expressing both tubulin-RFP and GFP-fAPC was wounded by a glass needle to increase [Ca2+]i. Medium used in this study contained 0.63 mM Ca2+. Asterisk indicates the site of cell membrane disruption. (B) Enlargement of the boxed area in (A). (C) Cells expressing EB1-GFP were wounded by a glass needle. Asterisk indicates the site of cell membrane disruption. A slight increase in the number of EB1-GFP comets was observed throughout the cell. Furthermore, numerous EB1-GFP comets appeared around wound site within 30 s after membrane disruption. This result is consistent with previous study using PtK2 cells [6].
Fig. 2. An increase in [Ca2+]i induces dissociation of APC. (A) A6 cells expressing GFP-fAPC were loaded with Ca2+ indicator Rhod-3 and treated with 1 μM ionomycin. The intensity of GFP-fAPC began to decrease 66.7 ± 11.8 s (n = 18) after the increase in [Ca2+]i. (B) A6 cells expressing EGFP-α-tubulin were treated with 1 μM ionomycin. Ionomycin (1 μM) did not induce depolymerization of MTs.
Fig. 3. An increase in [Ca2+]i results in increased tyrosine phosphorylation of GSK-3β. Parental A6 cells were incubated with or without 50 μM genistein for 15 min, then treated with 1 μM ionomycin for 5 min. Total cell lysates were analyzed by Western blotting either with anti-GSK-3α[pY279]/β[pY216] phospho-specific antibody or anti-GSK-3β antibody. Ionomycin (1 μM) induced phosphorylation on Tyr216 of GSK-3β.
Fig. 4. Inhibition of GSK-3β suppresses APC dissociation. (A) A6 cells expressing GFP-fAPC were incubated with 100 nM BIO for 15 min, then wounded by a glass needle. (B) Fluorescent intensity of GFP at the region indicated by a circle in (A) was measured. Dissociation of APC upon cell membrane disruption was suppressed by BIO. (C) A6 cells expressing GFP-fAPC were incubated with 100 nM BIO for 15 min, then treated with 1 μM ionomycin. Ionomycin did not induce APC dissociation in BIO-treated cells.
