Glycogen Synthase Kinase 3 and h-prune Regulate Cell Migration by Modulating Focal Adhesions

Abstract

h-prune, which has been suggested to be involved in cell migration, was identified as a glycogen synthase kinase 3 (GSK-3)-binding protein. Treatment of cultured cells with GSK-3 inhibitors or small interfering RNA (siRNA) for GSK-3 and h-prune inhibited their motility. The kinase activity of GSK-3 was required for the interaction of GSK-3 with h-prune. h-prune was localized to focal adhesions, and the siRNA for GSK-3 or h-prune delayed the disassembly of paxillin. The tyrosine phosphorylation of focal adhesion kinase (FAK) and the activation of Rac were suppressed in GSK-3 or h-prune knocked-down cells. GSK-3 inhibitors suppressed the disassembly of paxillin and the activation of FAK and Rac. Furthermore, h-prune was highly expressed in colorectal and pancreatic cancers, and the positivity of the h-prune expression was correlated with tumor invasion. These results suggest that GSK-3 and h-prune cooperatively regulate the disassembly of focal adhesions to promote cell migration and that h-prune is useful as a marker for tumor aggressiveness.

Phosphorylation of nm23-H1 by CKI induces its complex formation with h-prune and promotes cell motility

“…At this time, only the data reported by Kobayashi et al (2006) and Garzia et al (2006) link h-prune to GSK3-b, a protein involved in the Wnt activation pathway, and to cell migration. Our findings show that IC261 impairs cell motility, thus raising the hypothesis that h-prune, nm23 and CKIe, the last of which has already been found to be involved in the modulation of the signaling of dishevelled (Cong et al, 2004), have roles in the Wnt pathway.…”

Wnt5a signaling is involved in the aggressiveness of prostate cancer and expression of metalloproteinase

“…To measure the cell migration activity, we performed transwell assays using a modified Boyden chamber (tissue culture treated, 6.5-mm-diameter, 10-mm-thickness, 8-mm-pores; Transwell, Costar, Cambridge, MA, USA) as described previously (Kobayashi et al, 2006). The lower surface of the filters was coated with 10 mg/ml fibronectin for LNCap cells and 10 mg/ml type I collagen for DU145 cells.…”

“…The monolayer of DU145 cells was then scratched manually with a plastic pipette tip, and after being washed with PBS, the wounded monolayers of Wnt5a and aggressiveness of prostate cancer H Yamamoto et al cells were allowed to heal for 18-24 h in RPMI-1640 medium containing 10% fetal bovine serum. The length of the wounds was measured and expressed as a percentage of the initial length at zero time (Kobayashi et al, 2006). When necessary, the anti-Wnt5a antibody (10 mg/ml) or CM containing sFRP2 was added to the medium.…”

AIP regulates stability of Aurora-A at early mitotic phase coordinately with GSK-3β

“…Immunoprecipitation, western blotting and immunofluorescence studies were performed as described (Yamamoto et al, 2003;Kobayashi et al, 2006), and the results shown are representative of three independent experiments. The data of western blotting were quantified with Image J software.…”

“…Although the mechanisms by which GSK-3b regulates the functions of many substrates specifically and selectively are not fully understood, one possibility is that GSK-3b determines the substrate specificity by binding to it directly or indirectly. So far we have identified several GSK-3b-binding proteins including Axin, Axil, AKAP220 and h-prune Yamamoto et al, 1998;Tanji et al, 2002;Kobayashi et al, 2006). The complexes including GSK-3b and the binding protein have distinct localization and specific functions.…”