The RhoGEF TEM4 Contains an F-Actin Binding Domain Figure 2. N-terminus of TEM4 is essential for localization of TEM4 to cytoskeleton. A, Endogenous TEM4 associates with F-actin. TEM4 was immunoprecipitated from HUVECs pretreated with LatA and b-actin was detected by western blotting. B, Schematic of GFP-TEM4 fusion constructs. DH, Dbl homology; PH, pleckstrin homology. C, The N-terminus of TEM4 possesses the actin association signal. HUVECs were electroporated with GFP or GFP-tagged TEM4 constructs as indicated and imaged live. Scale bar, 20 mm. doi:10.1371/journal.pone.0041876.g002 blot analysis with antibodies raised against epitope shared between TEM4 and p164-RhoGEF proteins. Consistent, with the current curated NCBI annotation, expression of only the full-length, p222 kDa TEM4 protein was detected in multiple human and rodent cell lines (Fig. 1B), confirming that the sequence of TEM4 used in this report constitutes the authentic full-length TEM4. To determine the specificity profile of TEM4 towards Rho GTPases, a bacterially expressed DH-PH fragment of TEM4 (residues 1050–1478) was used in fluorescent-based GEF assays in vitro [15] using bacterially expressed recombinant Rho GTPases (Fig. 1C–G). In this assay, TEM4 accelerated the intrinsic guanine nucleotide exchange rate of RhoA, as previously shown [20,21], and the closely related RhoB and RhoC isoforms (Fig. 1C–E), but not Rac1 or Cdc42 (Fig. 1F, G). The Rac1 and Cdc42 proteins were functional as shown by their ability to be stimulated by the isolated DH-PH domains of P-Rex1 [22]. These data demonstrate that the catalytic activity of TEM4 is specific for RhoA/B/C in vitro. Finally, the presence of a PH domain C-terminal to the DH domain of TEM4 was put into question by a previous study [20]. Indeed, conventional domain prediction and annotation based solely on primary sequence alignment, such as is performed by the Simple Modular Architecture Research Tool (SMART) [23] does not identify a PH domain in TEM4 with statistical significance. However, three-dimensional structure prediction algorithms, which more reliably identify remote homologous sequences, readily detect a PH domain within TEM4 residues 1286–1476 (Fig. 1A, H). For example, the Phyre2 web server predicts a TEM4 PH domain with 99.4% confidence [24]. The Phyre2 generated PH domain homology model reveals that an ,70 residue a-helical domain (aa 1366–1433) is inserted in the b5/b6 turn of the TEM4 PH domain. Furthermore, residues Arg 1306, Lys 1321 and Lys 1348 within the PH domain are likely to participate in forming a functional phospholipid binding site. To assess the ability of the PH domain of TEM4 to bind phosphoinositides [25], we performed lipid dot blot analyses using bacterially expressed GST-DH-PH fusion protein. We found that the TEM4 DH-PH domains bound primarily PI(3)P, PI(4)P and PI(5)P (Fig. 1I). Since DH domains do not characteristically bind phosphoinositides PLoS ONE | www.plosone.org 4 July 2012 | Volume 7 | Issue 7 | e41876