DBS/MCF2L has been recently identified as a risk locus for osteoarthritis. It encodes a guanine nucleotide exchange factor (Dbs) that has been shown to regulate both normal and tumor cell motility. In the current study, we have determined that endogenous Dbs is predominantly expressed as 2 isoforms, a 130 kDa form (Dbs-130) that is localized to the Golgi complex, and an 80 kDa form (Dbs-80) that is localized to the endoplasmic reticulum (ER). We have previously described an inhibitor that binds to the RhoGEF domain of Dbs and blocks its transforming activity. Here we show that the inhibitor localizes to the Golgi, where it specifically interacts with Dbs-130. Inhibition of endogenous Dbs-130 activity is associated with reduced levels of activated Cdc42, enlarged Golgi, and resistance to Brefeldin A-mediated Golgi dispersal, suggesting a role for Dbs in vesicle transport. Cells treated with the inhibitor exhibit normal protein transport from the ER to the Golgi, but are defective in transport from the Golgi to the plasma membrane. Inhibition of Dbs-130 in MDA-MB-231 human breast tumor cells limits motility in both transwell and wound healing assays, but appears to have no effect on the organization of the microtubule cytoskeleton. The reduced motility is associated with a failure to reorient the Golgi toward the leading edge. This is consistent with the Golgi localization, and suggests that the Dbs-130 regulates aspects of the secretory pathway that are required to support cell polarization during directed migration.
Keywords: BFA, Brefeldin A; COP1, coat protein; DAPI, 4’, 6-diamidino-2-phenylindole; DH, Dbl homology; Dbs; Dbs, Dbl's big sister; ECL, electrochemiluminescence; ER, endoplasmic reticulum; FACS, fluorescence-activated cell sorting; FGD1, faciogenital dysplasia 1 protein; GEF, guanine nucleotide exchange factor; GFP, green fluorescent protein; HA, hemagglutinin; HM, homogenization medium; MCF2L; MTOC, microtubule organizing center; NF-1, neurofibromatosis type 1; PAK3, p21 protein-activated kinase 3; PBS, phosphate buffered saline; PH, pleckstrin homology; Rho; SH3, Src homology 3; VSVG, vesicular stomatitis virus-G; WGA, wheat germ agglutinin; breast cancer; cell motility; golgi complex; guanine nucleotide exchange factor; osteoarthritis; rtPCR, real-time polymerase chain reaction; siRNA, small inhibitory RNA.