Wnt-3a-dependent Cell Motility Involves RhoA Activation and Is Specifically Regulated by Dishevelled-2

J. Biol. Chem., Vol. 280, Issue 1, 777-786, January 7, 2005

CHO Hamster Chinese ovary, EpithelialCell line

Wound Healing Assay

Migration assay

transwell assay

Phalloidin staining

Wnt-3a induced morphological changes in CHO cells

Wound Healing Assay (24hr) transwell assay (6hr)

Wnt-3a CM-stimulated migration of CHO cells.

b -Catenin pathway was stimulated by Wnt-3a CM but not required for migration in transwell assay.

RhoA was activated by Wnt-3a CM and contributed to Wnt-3a-dependent cell migration.

Wnt-3a induced phosphorylation of Dvl-2 and Dvl-3

Intracellular distribution of Dvl-2 following treatment with Wnt-3a or L CM.

Wnt3a CM L CM

Knock-down of endogenous Dvl-2 by RNAi inhibited Wnt-3a-induced cell motility.

the pull-down assay used to detect active small GTPases

GST-RBD pull-down assay

the N-terminal region of Raf1 protein kinase contains the Ras–binding domain (RBD)the N-terminal regulatory region in p21-activated protein kinase 1 and 2 (Pak1 and 2) contains the p21-binding domain (PBD) for Rac and Cdc42, the N-terminal region of Rhotekin contains the Rho-binding domain (RBD).

The GTPase-binding domains from these downstream effectors are expressed as recombinant glutathione S-transferase (GST) fusion proteins immobilized on glutathione resin and can be used to affinity precipitate (pull-down) the active GTPase from cell lysates. Pulled-down active GTPases are eluted from the resin and detected by immunoblotting with a specific antibody