A mouse embryonic (days 9.5C10.5) cDNA library was screened for SH3 binding partners using the yeast two-hybrid assay as described (20). Amfenac Sodium Monohydrate activity at the level of the Rho-family GTPases (16, 18, 19). Here we report an interaction between the formin FMNL1 and the RhoGAP family member srGAP2 (Slit-Robo GAP family member 2). This complex forms via binding between the FH1 domain of FMNL1 and the SH3 domain of srGAP2. This binding is temporally regulated by the Rac-mediated activation of FMNL1. Additionally, srGAP2 functions as a selective Rac GAP when compared with Cdc42 or RhoA. Finally, actin filament severing assays show that the srGAP2 SH3 domain also directly inhibits FMNL1 actin severing activity. Together, our data suggest two novel mechanisms for srGAP2-mediated regulation of FMNL1, including GAP domain-mediated regulation of local Rac signaling to FMNL1 and steric/allosteric inhibition of actin severing by FMNL1. EXPERIMENTAL PROCEDURES Yeast Two-hybrid Assay The SH3 domain of srGAP2 was cloned into pLexNA vector, and this vector was transformed into the L40 yeast strain. A mouse embryonic (days 9.5C10.5) cDNA library was screened for SH3 binding partners using the yeast two-hybrid assay as described (20). Positive colonies were cured of the pLexNA-SH3 vector and retransformed with various baits described below to determine the specificity of interaction using 3-aminotriazole and -galactosidase activity. The clones with the strongest and most specific activity for srGAP2 were then sequenced. Cell Culture and Transfections HEK293T cells were cultured in DMEM supplemented with 10% FBS. HeLa cells were cultured in MEM supplemented with 10% FBS, nonessential amino acids, and sodium pyruvate. RAW264.7 cells were cultured in DMEM supplemented with 10% FBS, penicillin, and streptomycin. Transfections were performed using calcium phosphate for HEK293T cells and Lipofectamine 2000 (Invitrogen) as per the manufacturer’s protocol for HeLa cells. Plasmid Constructs A Formin-like 1 ((Uniprot “type”:”entrez-protein”,”attrs”:”text”:”Q9JL26″,”term_id”:”67460975″,”term_text”:”Q9JL26″Q9JL26) FMNL1 WT (amino acids 1C1094)-GFP, FMNL1 L1062D-GFP, and FMNL1 N terminus (amino acids 1C450)-GFP in pAS were generously given by the Michael Rosen laboratory (UT Southwestern). Mutations in srGAP2 (as previously described (21). Full-length srGAP2-V5 for GAP assays was expressed in HEK293T. Cells were lysed with lysis buffer, lysate was precleared, and srGAP2-V5 was purified using anti-V5-conjugated-agarose beads (Sigma) as previously described (22). FMNL1-C (amino acids 449C1094) was purified as previously MMP9 described (14). Briefly, FMNL1-C in pGEX-KT was expressed in BL21-DE3 (BL21) using nickel nitrilotriacetic acid-agarose (Qiagen, Valencia, CA) at 4 C. Purified protein was dialyzed into 1 KMEI Amfenac Sodium Monohydrate (50 mm KCl, 1 mm MgCl2, 1 mm EGTA, 10 mm Amfenac Sodium Monohydrate imidazole, pH 7.0) with 1 mm DTT. Assays were performed as previously described (14). Briefly, actin was polymerized for 1 h in 1 KMEI in G-Mg buffer (2 mm Tris, pH 8.0, 0.5 mm DTT, 0.2 mm ATP, 0.1 mm MgCl2, 0.01% sodium azide). FMNL1-C and srGAP2 SH3 were diluted in 1 KMEI in G-Mg buffer with 0.2 mm nonaethylene glycol monododecyl ether (Thesit) (Sigma). Actin filaments were incubated with FMNL1-C and srGAP2 SH3. The reaction was stopped by adding rhodamine phalloidin (Invitrogen) and diluted into Amfenac Sodium Monohydrate dilution buffer (25 mm imidazole, pH 7.0, 25 mm KCl, 4 mm MgCl2, 1 mm EGTA, 0.5% methylcellulose) supplemented with 250 mm NaCl, 100 mm DTT, 3 mg/ml glucose, 100 mg/ml glucose oxidase, and 18 mg/ml catalase. The solution was placed onto an 18-mm coverslip coated with poly-l-lysine and imaged on a Leica DMAR2 microscope. 10C15 images per coverslip were taken of random fields. Filament lengths were quantified using MetaMorph software (Molecular Devices). Percent severing was calculated for each experiment from fractions of filaments greater than 9 m ((1 ? (? ? = fraction of filaments, GAP assays were performed as previously published (22). Briefly,.