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First published online January 21, 2009
doi: 10.1242/10.1242/jcs.027482

Journal of Cell Science 122, 414-424 (2009)
Published by The Company of Biologists 2009
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The SAM domain of the RhoGAP DLC1 binds EF1A1 to regulate cell migration

Dandan Zhong1,*, Jingfeng Zhang2,*, Shuai Yang3,*, Unice J. K. Soh1, Jan Paul Buschdorf1, Yi Ting Zhou1, Daiwen Yang2,4,{ddagger} and Boon Chuan Low1,4,{ddagger}

1 Cell Signaling and Developmental Biology Laboratory, Department of Biological Sciences, 14 Science Drive 4, National University of Singapore, Singapore 117543, Republic of Singapore
2 Department of Biological Sciences, 14 Science Drive 4, National University of Singapore, Singapore 117543, Republic of Singapore
3 Department of Chemistry, 3 Science Drive 3, National University of Singapore, Singapore 117543, Republic of Singapore
4 Structural Biology and Proteomics Research Group, National University of Singapore, Singapore 117543, Republic of Singapore


Figure 1
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  		Fig. 1. Identification of elongation factor 1A1 as a novel partner of DLC1. (A) Schematic diagram showing the composition of protein domains for DLC1: SAM domain, GAP domain and START domain. Residue S329, which corresponds to the phosphorylation site in the rat p122RhoGAP by PKB and the tensin-binding sites are indicated. (B) A unique band around 48 kDa was found to bind to the SAM domain of DLC1 (lane 3, labelled by arrow), when comparing the bound proteins on GST or GST recombinant of DLC1 SAM immobilized on beads after incubation with rat liver lysate (L) or lysis buffer (B). (C) The 48 kDa band was subjected to trypsin digestion followed by MALDI-TOF analysis, and identified as elongation factor 1A1 (EF1A1). Peptide sequences representing 23% coverage of EF1A1 are underlined.

 

Figure 2
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  		Fig. 2. EF1A1 binds to full-length DLC1 and DLC1 SAM. (A) Whole cell lysates (WCL) of untransfected HEK293T cells, or cells transfected with Flag-tagged DLC1 or Flag-tagged EF1A1 were incubated with M2-anti-Flag beads and Flag constructs were immunoprecipitated on the beads. Bound proteins were separated on SDS-PAGE, blotted and probed with indicated antibodies to reveal the bound endogenous EF1A1. (B) GST, GST-DLC1 SAM or GST-DLC2 SAM purified on glutathione-Sepharose beads were incubated with HEK293T cell lysates. The associated proteins were separated on SDS-PAGE, blotted and probed with EF1A1 antibodies (upper panel). The blot was stripped and stained with amido black to reveal loading of GST recombinants (bottom panel). (C) GST or GST-EF1A1 purified on glutathione-Sepharose beads were incubated with or without His-tagged SAM overexpressed and purified from HEK293T cells (see Materials and Methods). GST proteins and the bound His-SAM were separated on SDS-PAGE, blotted and probed with anti-histidine antibodies (upper panel). Blot was stripped and stained with amido black to reveal loading of GST recombinants (bottom panel). (D) Flag-tagged DLC1 was transcribed and translated in vitro and incubated with either GST or GST-EF1A1 beads. GST proteins and the bound Flag-DLC1 were separated on SDS-PAGE, blotted and probed with Flag antibodies (upper panel). Blot was stripped and stained with amido black to reveal loading of GST recombinants (bottom panel). PD, pull down; IP, immunoprecipitation.

 

Figure 3
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  		Fig. 3. Solution structure and van der Waals surface of DLC1 SAM. (A) Ribbon drawing of the ten lowest-energy conformers. The four helices are indicated in red and yellow. (B) Views of the van der Waals surface, illustrating potential protein-protein association interfaces. The side chains are colored: green, hydrophobic; blue, basic; red, acidic; and grey, polar (and main chain). (C) Superimposition of SAM domains of DLC1 (green) and DLC2 (violet). The helices (H) are represented as ribbons. These helices are labeled according to the conserved folds among all known structures of SAM, where helix 3 is absent only in DLC1 and DLC2 SAM. (D) Four hydrophobic residues A16, Y35, F38 and L39 (green) on the EF1A1-binding motif of DLC1 SAM and four corresponding residues of DLC2 SAM (i.e. A60, Y79, S82 and Q83) are shown. The figures were created with MOLMOL (Koradi et al., 1996Go).

 

Figure 4
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  		Fig. 4. Identification of the EF1A1-binding motif in DLC1 SAM. (A,B) HEK293T cells were co-transfected with plasmids encoding (A) HA-EF1A1 and Flag-tagged SAM wild-type (WT), one double mutant K48A/R49A, one triple mutant F38G/L39G/F40G, or two quadruple mutants, K48A/R49A/R64A/R65A, F28G/F53G/L54G/A58G or a Flag-tagged control encoding an unrelated protein (Ctrl); or (B) HA-EF1A1 and Flag-tagged DLC1 SAM wild-type, one triple mutant, F38G/L39G/F40G, three single-point mutants, F38G, L39G, F40G or DLC2 SAM. Whole cell lysates (WCL) were used for immunoprecipitation (IP) with M2-anti-Flag beads. WCL and IP samples were immunoblotted with indicated antibodies. Asterisk indicates the light chain of Flag antibodies. The slower migrating bands for F38G/L39G/F40G, F38G, L39G and F40G mutants were due to their longer polypeptide chains (residues 1-94) compared with the wild type (residues 1-86). This slightly longer version was initially engineered to still encompass the full SAM (residues 11-76) but with eight amino acid residues flanking the C-terminus to help ensure proper folding of the SAM domain. These added residues essentially have no impact on the binding, as shown by F40G mutant and the WT. (C) Homology of DLC1 SAM with other SAM domains of known structures. Multiple sequence alignment was generated using the program CLUSTALW (http://www.ebi.ac.uk/clustalw). Residues totally conserved in all sequences are shaded black, those conserved in most of the sequences are in dark grey, whereas the significant but least conserved residues are in light grey. Species abbreviations: hs, Homo sapiens; mm, Mus musculus; sc, Saccharomyces cerevisiae. GenBank accession numbers: hsDLC1, 33188437; hsDLC2, 28976169; hsDLC3, 31543658; mmEPHA4, 30705030; hsEPHB2, 119615430; hsFLI1, 14603316; scSTE11, 609414; mmBAR, 21313130; scVTS1, 74583753; hsARAP2, 16118245; mmSAMSN1, 10800126. Asterisks indicate the corresponding residues of F38, L39 and F40 in DLC1 SAM.

 

Figure 5
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  		Fig. 5. DLC1 SAM domain facilitates recruitment of EF1A1 to the membrane periphery and membrane ruffles. NIH3T3 cells were transfected with various constructs and made quiescent followed by treatment for 20 minutes with 10 ng/ml fibroblast growth factor (FGF). Cells were then fixed, permeabilized, stained and visualized under confocal immunofluorescent microscopy. (A) SAM domain is not required for DLC1 function in dissolving focal adhesions. Cells were transfected with Flag-DLC1, Flag-DLC1 SAM, Flag-DLC1 {Delta}SAM or HA-EF1A1 and treated with FGF before fixation. Ectopic expression of the proteins was detected by anti-Flag or anti-HA (red), and the focal adhesions are shown by anti-paxillin (green), followed by appropriate fluorophore-conjugated secondary antibodies. Merged signals are presented as overlaid staining (yellow). Arrow indicates untransfected control cell. (B) Cells were cotransfected with HA-EF1A1 and Flag constructs of either DLC1 wild type (DLC1), DLC1 full-length mutant F38G/L39G/F40G (DLC1 FLF), DLC1 full-length mutant R677E (DLC1 R677E), DLC1 full-length with a mutant consisting of a combination of R677E and F38G/L39G/F40G (DLC1 R677E-FLF), DLC1 SAM domain wild type (SAM), SAM domain mutant F38G/L39G/F40G (SAM-FLF), or DLC2 SAM domain. Coexpressed HA-EF1A1 (a; blue), and different Flag-DLC1 constructs (b; green) were detected by appropriate anti-Flag and anti-HA antibodies followed by fluorophore-conjugated secondary antibodies. Cells were labeled with TRITC-phalloidin (c; red) to mark the cell border, the cortical actin on the cell periphery or membrane ruffles. Merged signals are presented in panels labelled d (cyan for panel a overlaid with panel b; purple for a overlaid with c; yellow for b overlaid with c; bright white for a overlaid with both b and c). The boxes highlight regions of tight colocalization whereas dotted boxes indicate regions of low or no colocalization. The P labels punctate structures. The intensity of images was enhanced to capture changes in the cell periphery and cell protrusions. Scale bars: 10 µm.

 

Figure 6
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  		Fig. 6. Effects of DLC1 SAM on cell migration. (A,B,D) The effects of different DLC1 constructs on cell migration were assayed in a Boyden chamber. Data are presented as the means ± s.d. for 3-4 independent experiments. Statistical comparison was made using ANOVA and Newman-Keuls multiple comparisons. NIH3T3 cells were migrated on fibronectin. (A) NIH3T3 cells were transfected with GFP-tagged plasmid as a cell marker together with twice the quantity of Flag-tagged vector (Ctrl), Flag-tagged DLC1 full-length wild-type (DLC1) or FLF full-length mutant (DLC1 FLF). Data denoted by different letters indicate significant difference at P<0.06. (B) NIH3T3 cells were transfected with GFP-tagged plasmid as cell marker together with twice the quantity of Flag-tagged vector (Ctrl), Flag-tagged DLC1 full-length wild-type (DLC1) or R677E full-length mutant (DLC1 R677E). (C) HEK293T cells were transfected with vector control or with Flag-tagged DLC1 or its various deletion or point mutants as indicated. Lysates were then subjected to GST-RBD pull-down assays as described in the Materials and Methods to assess the amount of active Rho being precipitated, hence a measurement for the GAP activity of DLC1. (D) MCF7 cells were transfected with GFP-tagged vector (Ctrl), GFP-tagged DLC1 SAM domain wild type (SAM), FLF mutant (SAM-FLF) or GFP-tagged DLC1 SAM domain. Data denoted by different letters indicate significant difference at P<0.02.

 

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© The Company of Biologists Ltd 2009