Protease inhibitor cocktail tablets (Complete Mini, catalog number 11-836-153-001, and Complete Mini EDTA free, catalog number 11-836-170-001) were purchased from Roche Diagnostics. An ECL Plus Western blotting detection kit (catalog number RPN2132) was purchased from GE Healthcare. Phosphatase inhibitor cocktail 1 (catalog number P2850) was purchased from Sigma. Control small interfering RNA (siRNA) (catalog number sc-37007), profilin-1-specific siRNAs (catalog number sc-36316), and profilin-1/profilin-2a-specific siRNAs (catalog number sc-44045) were purchased from Santa Cruz Biotechnology. Profilin-1-specific siRNAs contain three different sequences: GUGUCCUGGUUGGCAAAGA, CACGGUGGUUUGAUCAACA, and CCCCAUACCCCUUAUUGCU. Profilin-1/profilin 2a siRNAs contain four different sequences: two targeting profilin-1 (GCAAAGACCGGUCAAGUUU and CACGGUGGUUUGAUCAACA) and the other two targeting profilin-2a (GUAGAGCAUUGGUUAUAGU and CCAGGGACAUUCCAUCAUU). Lysophosphatidic acid (LPA) was purchased from Sigma.
cDNAs encoding ARN127(Q65) or Htt exon 1 fused to cyan fluorescent protein (CFP) or yellow fluorescent protein (YFP) (36
) were subcloned from p6R (36
) into the backbone of pEYFP.N1 (Clontech) to drive expression under the cytomegalovirus promoter. For bacterial expression, glutathione S
-transferase (GST)-ARN127(Q25) YFP was cloned into the pGEX-4T-1 vector (Amersham Biosciences). pGEX-Htt exon 1 (Q20 or Q53) constructs were provided by Paul J. Muchowski (31
). Human His6
-profilin-1 (wild type [wt], S137A, and S137D) was PCR amplified and cloned into the bacterial expression vector pRK172. For expression in HEK293 cells, human profilin-1 was PCR amplified and cloned into pcDNA3.1. Mutations at Ser-137 were introduced into the PCR primers, and others were introduced by QuikChange mutagenesis (Stratagene). Human profilin-2a was PCR amplified and cloned into the gWIZ blank vector. For expression in primary neurons, profilin-1 (wt versus mutants) and Htt exon 1(Q72) YFP were cloned into vector pCAGGS (provided by Robert Edwards) to drive expression under the chicken β-actin promoter. pCAG-ROCK1(wt) or pCAG-ROCK1(KDIA) was provided by Shuh Narumiya (20
). pXJ40-ROCK2 was provided by Thomas Leung (24
Cell culture and transfection.
HEK293 cells were cultured in Dulbecco's modified Eagle's medium-5% fetal bovine serum (FBS) and transfected as described previously (10
). For LPA treatment and phospho-profilin-1 detection, primary basal ganglion neurons were dissected from embryonic day 14 rat embryos and maintained in Dulbecco's modified Eagle's medium-10% FBS for 4 days prior to use. For effects of profilin on aggregation, primary cortical neurons were dissected from embryonic day 19 rat embryos and electroporated with Htt exon 1(Q72) YFP and profilin-1 (wt versus mutants) using a rat neuron Nucleofector kit (catalog number DPG-1003; Amaxa). They were cultured in Neurobasal medium (Gibco) supplemented with 1% FBS, 2% B27, and 2 mM Glutamax for 4 days prior to use.
FRET measurements and calculations.
FRET measurements were made as described previously (10
). The relative FRET/donor ratio was calculated as follows: relative FRET/donor ratio = [(FRET/donor)a − (FRET/donor)b]/(FRET/donor)b, where a represents cells cotransfected or treated with aggregation modulators and b represents control cells untreated or cotransfected with empty vector or control siRNAs.
Protein expression and affinity purification.
pGEX constructs were transformed into Rosetta(DE3) (Novagen) or Sure competent cells (Stratagene). Cells were grown at 37°C until an optical density at 600 nm of 0.6 to 0.9 was reached and induced with 0.5 mM IPTG (isopropyl-β-d-thiogalactopyranoside) at 30°C for 5 h. Cells were lysed by sonication in phosphate saline buffer, and lysate was mixed with glutathione-Sepharose beads (Amersham Biosciences) at 4°C for 2 h. Beads were washed and eluted with 50 mM Tris-HCl (pH 8.0) containing 10 mM reduced glutathione. pRK172/His-profilin (wt, S137A, and S137D) was transformed into Rosetta(DE3) cells and induced with 0.5 mM IPTG at 30°C for 5 h. The lysate was sonicated and bound to Ni-nitrilotriacetic acid agarose (Qiagen) at 4°C for 2 h. Beads were washed and eluted with 250 mM imidazole. Proteins were dialyzed against a solution containing 50 mM Tris-HCl (pH 7.4), 100 mM NaCl, 0.2 mM ATP, 5% sucrose, and 0.5 mM dithiothreitol (DTT) for in vitro kinase assays or against a solution containing 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, and 1 mM DTT for pull-down assays.
Immunoprecipitation and pull-down assays.
For coimmunoprecipitation of endogenous actin with Myc-profilin, HEK293 cells were lysed in a solution containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Triton X-100, and protease inhibitor cocktail (Roche). Cleared lysates were mixed with anti-mouse immunoglobulin G (IgG) beads (Sigma) bound with mouse anti-Myc antibody. After 2 h of mixing at 4°C, beads were washed with lysis buffer and analyzed by Western blotting using anti-Myc and antiactin antibodies. For interactions between GST-Htt exon 1 and profilin, Htt exon 1 was immobilized on glutathione-Sepharose and incubated with bacterial lysate or HEK293 cell lysate. Beads were washed with phosphate-buffered saline-1 mM EDTA-0.1% Tween 20 and analyzed by Coomassie blue staining and Western blotting against profilin. For interactions between His-profilin and Htt exon 1, His6-profilin was immobilized on Ni-nitrilotriacetic acid agarose and mixed with bacterial lysate containing GST-HA-Htt exon 1 (Q25). Beads were washed with phosphate-buffered saline and analyzed by Coomassie blue staining and Western blotting against Htt. For immunoprecipitation of phospho-profilin, HEK293 cells were lysed with a solution containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 50 mM NaF, 1% Triton X-100, and protease inhibitor cocktail. The cleared lysate was incubated with anti-rabbit IgG beads (Sigma) bound with rabbit antiphosphoserine antibody. After 2 h of mixing at 4°C, beads were washed with a solution containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 50 mM NaF, and 1% Triton X-100 and analyzed by Western blotting against the Myc tag.
In vitro kinase assay.
Immunoprecipitated Myc-ROCK1(wt) or Myc-ROCK1(KDIA) was mixed with 50 ng/μl His6-profilin (wt or S137A) in 25 μl kinase reaction buffer (25 mM Tris-HCl [pH 7.4], 4 mM MgCl2, 3.6 mM EDTA, 1 mM DTT, 0.1 μM calyculin A [Calbiochem], phosphatase inhibitor cocktail 1 [Sigma], 50 μM arachidonic acid [Sigma], 80 μM cold ATP, 20 μM [γ-32P]ATP [5 μCi; Perkin-Elmer]) for 60 min at 30°C. Reactions were stopped with sodium dodecyl sulfate (SDS) sample buffer and analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) and autoradiography.
Commercial primary antibodies purchased were as follows: mouse anti-Myc tag (catalog number sc-40; Santa Cruz), mouse anti-Htt (MAB5374, clone mEM48; Chemicon), rabbit anti-actin (catalog number sc-1616-R; Santa Cruz), rabbit anti-phosphoserine (AB1603; Chemicon), and mouse anti-tetra-His (catalog number 34670; Qiagen). Secondary antibodies used for Western blotting include alkaline phosphatase-conjugated secondary antibodies (catalog number A3562; Sigma) and horseradish peroxidase-conjugated secondary antibodies (NA9340V for anti-rabbit and NA931V for anti-mouse antibodies; Amersham Biosciences). Mouse true blot secondary antibody was purchased from eBioscience. Secondary antibody-conjugated agarose beads used for immunoprecipitation are anti-mouse IgG-agarose (catalog number A6531; Sigma) and anti-rabbit IgG immunoprecipitation beads (catalog number 00-8800-25; eBioscience). A rabbit polyclonal antibody (P3490) selective for phospho-Ser-137 of profilin was raised using a synthetic phosphopeptide [Ac-CMASHLRR(pS)QY-OH] derived from the C terminus of human profilin-1 and affinity purified by New England Peptide, Inc. Polyclonal antibodies against profilin-1 and -2a were generated by Open Biosystems (Huntsville, AL) by injecting the linear peptides KCYEMASHLRRSQY and KAYSMAKYLRDSGF, respectively, conjugated to keyhole limpet hemocyanin into rabbits. Serum was purified with an affinity column containing the respective profilin peptide. A polyclonal antibody against the N-terminal portion of profilin-1 was purchased from Cell Signaling.