Regulation of vesicle transport and cell motility by Golgi-localized Dbs

• Published in: Small GTPases Vol. 5; no. 4; pp. 1 - 12
• Main Authors: Fitzpatrick, Ethan R, Hu, Tinghui, Ciccarelli, Bryan T, Whitehead, Ian P
• Format: Journal Article
• Language: English
• Published: United States Taylor & Francis 02.10.2014
• Subjects: Biological Transport - drug effects; breast cancer; Brefeldin A - pharmacology; Carrier Proteins - chemistry; Carrier Proteins - metabolism; cdc42 GTP-Binding Protein - metabolism; Cell Line, Tumor; cell motility; Cell Movement - drug effects; Cytoskeleton - metabolism; Dbs; Endoplasmic Reticulum - metabolism; Golgi Apparatus - drug effects; Golgi Apparatus - metabolism; golgi complex; guanine nucleotide exchange factor; Guanine Nucleotide Exchange Factors - antagonists & inhibitors; Guanine Nucleotide Exchange Factors - chemistry; Guanine Nucleotide Exchange Factors - metabolism; HeLa Cells; Humans; MCF2L; osteoarthritis; Protein Binding; Protein Isoforms - antagonists & inhibitors; Protein Isoforms - chemistry; Protein Isoforms - metabolism; Protein Structure, Tertiary; Research Paper; Rho; RNA Interference; RNA, Small Interfering - metabolism; Secretory Vesicles - metabolism; WGA, wheat germ agglutinin; PAK3, p21 protein-activated kinase 3; osteoarthritis; DAPI, 4’, 6-diamidino-2-phenylindole; FGD1, faciogenital dysplasia 1 protein; SH3, Src homology 3; siRNA, small inhibitory RNA; golgi complex; Dbs, Dbl's big sister; Dbs; MCF2L; Rho; COP1, coat protein; ECL, electrochemiluminescence; FACS, fluorescence-activated cell sorting; HM, homogenization medium; rtPCR, real-time polymerase chain reaction; GFP, green fluorescent protein; PBS, phosphate buffered saline; MTOC, microtubule organizing center; VSVG, vesicular stomatitis virus-G; breast cancer; NF-1, neurofibromatosis type 1; PH, pleckstrin homology; cell motility; guanine nucleotide exchange factor; GEF, guanine nucleotide exchange factor; HA, hemagglutinin; BFA, Brefeldin A; ER, endoplasmic reticulum; DH, Dbl homology
• ISSN: 2154-1248; 2154-1256
• DOI: 10.4161/sgtp.28570

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.