Effects of Structure of Rho GTPase-activating Protein DLC-1 on Cell Morphology and Migration

Tai Young Kim,Kevin D Healy,Channing J Der,Noah Sciaky,Yung-Jue Bang,Rudy L Juliano

doi:10.1074/jbc.m800617200

Tai Young Kim, Kevin D Healy + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m800617200

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Abstract

DLC-1 encodes a Rho GTPase-activating protein (RhoGAP) and negative regulator of specific Rho family proteins (RhoA-C and Cdc42). DLC-1 is a multi-domain protein, with the RhoGAP catalytic domain flanked by an amino-terminal sterile α motif (SAM) and a carboxyl-terminal START domain. The roles of these domains in the regulation of DLC-1 function remain to be determined. We undertook a structure-function analysis involving truncation and missense mutants of DLC-1. We determined that the amino-terminal SAM domain functions as an autoinhibitory domain of intrinsic RhoGAP activity. Additionally, we determined that the SAM and START domains are dispensable for DLC-1 association with focal adhesions. We then characterized several mutants for their ability to regulate cell migration and identified constitutively activated and dominant negative mutants of DLC-1. We report that DLC-1 activation profoundly alters cell morphology, enhances protrusive activity, and can increase the velocity but reduce directionality of cell migration. Conversely, the expression of the amino-terminal domain of DLC-1 acts as a dominant negative and profoundly inhibits cell migration by displacing endogenous DLC-1 from focal adhesions.

Highlights

Members of the Rho family of small GTPases are intimately involved in many aspects of cell function including cell cycle progression, intracellular trafficking, and control of cell division [1]
To further evaluate the contribution of amino- and carboxyl-terminal sequences in regulation of DLC-1, we generated expression vectors that encoded more precise deletion and truncation mutants that were coupled at the carboxyl terminus to enhanced green fluorescent protein (EGFP) to allow us to monitor DLC-1 expression and localization in live cells (Fig. 1)
In agreement with previous studies using amino-terminally tagged DLC-1 fusion proteins (20 –22), we found that fulllength DLC-1 with a carboxyl-terminally fused GFP partially localized at the tips of actin filaments in MDA-MB-468 cells (Fig. 2A) as well as showing some diffuse cytosolic distribution

Summary

EXPERIMENTAL PROCEDURES

Cell Culture—HEK293 cells were maintained in Dulbecco’s modification of Eagle’s medium (DMEM) supplemented with. Plasmids—For mammalian cell expression of green fluorescent protein tagged DLC-1 proteins, the full-length (1–1091 amino acids) and truncated sequences of the DLC-1 cDNA (GenBankTM accession number NM_006094) were generated by PCR and subcloned into the BamHI site of pEGFP-N1 (BD Biosciences). After 12–18 h of transfection, the cells were trypsinized, replated sparsely on fibronetin-coated (10 ␮g/ml) glass-bottomed dishes in DMEM supplemented with 10% fetal bovine serum and placed in a temperature- and CO2-controlled chamber of a microscope equipped with 40ϫ objective lenses. To explore the effects of Rho kinase (ROCK) inhibition on cell migration, HEK293 cells on fibronectin-coated (10 ␮g/ml) glass-bottomed dishes in DMEM supplemented with 10% fetal bovine serum were treated for 3 h with 50 ␮M of the ROCK inhibitor Y27632 (Calbiochem Inc.) before starting time lapse recording. The velocity was calculated as [total length of migration paths (␮M)/time (min)] and the persistence of migration was calculated as [net displacement (␮M)/total length of migration paths (␮M)]

RESULTS

The SAM Domain Functions as an Autoinhibitory Regulator of

DISCUSSION