Abstract
We observed previously that combined small interfering RNAs (siRNAs) targeting CrkII and CrkL, known activators of guanine nucleotide exchange factor DOCK1, strongly inhibit Caco-2 intestinal epithelial cell spreading and migration on collagen IV. DOCK1 siRNA reduced its expression >95% in Caco-2 cells but inhibited spreading much less than combined CrkII/CrkL siRNAs, suggesting that CrkII/CrkL interact with additional DOCK proteins. siRNA targeting DOCK5, a closely related DOCK1 family member, inhibited Caco-2 spreading similarly to DOCK1 siRNA, and the combined siRNAs synergistically inhibited spreading. Similar results were observed in human umbilical vein endothelial cells, and reverse transcriptase PCR demonstrated DOCK5 siRNA reduction of DOCK5 expression in both cell types. Combined DOCK1/DOCK5 siRNAs also inhibited Caco-2 migration and lamellipodial extension. Expression of DOCK5 cDNA, with silent mutations in the siRNA target region allowing expression simultaneously with DOCK5 siRNA, required CrkII/CrkL to restore cell spreading and DOCK5 coimmunoprecipitated with CrkII and CrkL. DOCK5 association with CrkII and CrkL was greatly reduced by mutations in their NH2-terminal SH3 domains. Expression of the DOCK5 COOH-terminal region (Met1738-Gln1870), containing potential Src homology 3 domain-binding proline-rich sites but lacking other functional regions, inhibited Caco-2 spreading and coimmunoprecipitated with CrkL. Coimmunoprecipitation of full-length DOCK5 with CrkL was strongly reduced by deletion of DOCK5 COOH-terminal amino acids 1832-1870. Green fluorescent protein-tagged DOCK5 localized to the membrane of Caco-2 cells spreading on collagen IV. In these studies, we describe human DOCK5 cloning and expression, our results indicating that, along with DOCK1, DOCK5 is an important mediator of CrkII/CrkL regulation of Caco-2 spreading and migration on collagen IV.
Highlights
The Rac guanine-nucleotide exchange factor (GEF)2 DOCK1 [1] belongs to a family of GEFs that
We examine the role of DOCK1 and DOCK5 in regulation of Caco-2 intestinal epithelial cell spreading and migration on collagen IV using siRNAs that target these proteins and confirm these results in human umbilical vein endothelial cells (HUVECs)
DOCK1 siRNA Only Partially Reduces Caco-2 Cell Spreading— We have previously observed that combined siRNA inhibition of the adaptor proteins CrkII and CrkL strongly inhibits Caco-2 cell spreading and migration on collagen IV by more than 40% and strongly inhibits lamellipodial formation at the leading edge of the migrating front [26]. siRNA targeting the GEF DOCK1, which is a downstream effector of CrkII and CrkL in numerous cell systems [1, 30, 31], only reduced cell spreading by 15% despite reducing DOCK1 protein levels by more than 95% (Fig. 1A)
Summary
Materials—Dulbecco’s modified Eagle’s medium, Oligofectamine, Lipofectamine, and Plus Reagent and the -galactosidase detection kit were obtained from Invitrogen. CrkII monoclonal and polyclonal antibodies were obtained from Transduction Laboratories (Lexington, KY) and Santa Cruz Biotechnology, Inc. CrkL polyclonal antibody was obtained from Santa Cruz Biotechnology. Glutathione S-transferase (GST) polyclonal antibody was obtained from Cell Signaling Technology (Danvers, MA). Human DOCK5 Cloning and Expression—RNA from Caco-2 cells and HUVECs was obtained using TRIzol reagent (Invitrogen), and cDNA libraries were made using Superscript reverse transcriptase (Invitrogen). Eukaryotic expression expression vectors containing CrkII and CrkII W169L were obtained from Dr Michiyuki Matsuda (Kyoto University, Kyoto, Japan), and PCR was used for cloning of cDNAs into pGEX4T-1 GST fusion protein expression vector (GE Healthcare). GST-CrkL expression vector was obtained from Dr Ayako Arai and Dr Osamu Miura (Tokyo Medical and Dental University), and PCR was used to introduce W160L mutation into the amino-terminal SH3 domain of this protein. Expression of Glutathione S-Transferase Fusion Proteins— BL21(DE3)pLysS Escherichia coli (Invitrogen) were transformed with control pGEX vector or pGEX vectors expressing CrkII, CrkL, or the corresponding mutant proteins
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