Profilin inhibits AR and Htt aggregation in cultured HEK293 cells. (A) HEK293 cells were cotransfected with constitutively active cofilin(S3A) or inactive cofilin(S3E) with Htt exon 1(Q72) CFP/YFP or ARN127(Q65) CFP/YFP. Aggregation was measured by FRET, and the effects were expressed as relative values as described in Materials and Methods. Cofilin(S3A) increased the aggregation of both Htt exon 1(Q72) and ARN127(Q65), whereas cofilin(S3E) had little effect on Htt exon 1(Q72) aggregation (**, P < 0.005 by paired t test) and decreased ARN127(Q65) aggregation. Error bars represent the standard errors of the means (SEM). (B and C) HEK293 cells were cotransfected with Htt exon 1(Q72) CFP/YFP or ARN127(65) CFP/YFP and increasing amounts of profilin-1 (which will subsequently be referred to as profilin, unless otherwise stated) (B) or profilin-2a (C). Relative aggregation was measured by FRET. Both profilin isoforms dose-dependently reduced aggregation. Error bars represent the SEM. (D) HEK293 cells were cotransfected with Htt exon 1(Q72) YFP or ARN127(Q65) YFP and profilin-1. Cells were cultured for 2 days, fixed, and analyzed using fluorescence microscopy. Profilin-1 reduced the number of inclusions formed by both Htt exon 1(Q72) YFP and ARN127(Q65) YFP. Profilin-2a had the same effect (data not shown). Representative images (magnification, ×10) are shown.

Actin binding by profilin is required to inhibit polyglutamine aggregation. (A) Myc-tagged profilin(wt) or profilin(Y59A) was immunoprecipitated from HEK293 cells, and coprecipitated actin was detected by Western blotting. Untagged profilin was used as the negative control (first lane). Profilin(Y59A) failed to bind actin. IP, immunoprecipitation. (B) Sepharose-bound GST or GST-Htt exon 1(Q25) was incubated with HEK293 cell lysate containing profilin(wt) or profilin(Y59A). Both profilin proteins bound Htt exon 1 comparably. (C) HEK293 cells were cotransfected with profilin(wt) or profilin(Y59A) with Htt exon 1(Q72) CFP/YFP or ARN127(Q65) CFP/YFP. Relative aggregation was measured by FRET. Profilin(wt) reduced the aggregation of Htt exon 1(Q72) and ARN127(Q65), whereas profilin(Y59A) partially reduced Htt exon 1(Q72) aggregation (***, P < 0.0005 by unpaired t test) and increased ARN127(Q65) aggregation. Error bars represent the SEM.

Rho/ROCK signaling regulates endogenous profilin phosphorylation at Ser-137. (A to C) Characterization of phosphospecific profilin antibody selective for P-Ser-137 (P3490). (A) HEK293 cells were transfected with Myc-tagged profilin(wt) or profilin(S137A). The cell lysate was analyzed by SDS-PAGE and Western blotting using P3490 or the anti-Myc antibody. While equal amounts of Myc-profilin (wt versus S137A) were present, P3490 preferentially recognized Myc-profilin(wt) versus Myc-profilin(S137A). (B) HEK293 cell lysate was incubated with a fixed amount of calf intestinal phosphatase and increasing concentrations of urea at 37°C for 6 h. Urea was used to partially denature profilin to make Ser-137 accessible to calf intestinal phosphatase. Equal amounts of lysate were analyzed by SDS-PAGE and Western blotting using P3490 or the anti-profilin antibody. Alkaline phosphatase treatment decreased the reactivity of P3490 with endogenous profilin in HEK293 cell lysate. (C) Equal amounts of bacterially expressed His-tagged profilin (wt, S137A, or S137D) were analyzed by SDS-PAGE and Western blotting using P3490 or the anti-His tag antibody. P3490 preferentially recognizes profilin(S137D). (D) HEK293 cells were transfected with pcDNA3 or ROCK1, followed by Y-27632 treatment for 24 h. Lysates were probed for phospho-profilin at Ser-137 (P3490) or total profilin. ROCK1 increased P3490 reactivity by over twofold without changing the total profilin level and was inhibited by Y-27632. (E) HEK293 cells or rat primary striatal neurons were treated with or without 50 μM Y-27632 for 24 h, followed by 10 μM LPA treatment for 45 min. Lysates were analyzed for phospho-profilin or total profilin as above (D). LPA increased P3490 reactivity in both cell types (58% for HEK293 and 60% for neurons) without changing the total profilin level. This was inhibited by Y-27632.

Phosphorylation at Ser-137 inhibits profilin as an aggregation suppressor in primary neurons. Rat embryonic cortical neurons were cotransfected with Htt exon 1(Q72) YFP with either an empty vector or different profilin constructs (wt, S137A, or S137D). Cells were cultured for 4 days and fixed prior to counting inclusions in 10 to 20 fields per coverslip. (A) Representative images of transfected cells showing inclusions formed by Htt exon 1(Q72) YFP (magnification, ×10). Profilin(wt) and profilin(S137A) each reduced the number of inclusions, while profilin(S137D) did not. (B) Averages of three independent experiments showing the effect of profilin expression on inclusion formation. The y axis represents relative inclusion numbers normalized against those in the cells cotransfected with the empty vector. Profilin(wt) and profilin(S137A) each reduced inclusions, whereas profilin(S137D) did not (*, P < 0.05 by paired t test).

Rho/ROCK1/profilin signaling regulates AR and Htt aggregation via overlapping but distinct mechanisms. (A) Rho activation of ROCK1 leads to its phosphorylation of profilin at Ser-137. This inhibits profilin's interaction with G actin and inactivates profilin as an inhibitor of AR aggregation. (B) In addition to G actin binding, phosphorylation at Ser-137 also inhibits the polyproline binding of profilin and disrupts its direct interaction with Htt. In combination, this inactivates profilin as an inhibitor of Htt aggregation. In both cases, Y-27632 blocks the phosphorylation of profilin by ROCK1, which restores profilin's ability to bind G actin and Htt and its antiaggregation activity. For both AR and Htt, phosphorylation at Ser-137 could also affect other unknown functions of profilin (broken lines) that are important for its antiaggregation activity.

Inhibition of Htt aggregation by profilin requires direct interaction. (A) Sepharose-bound GST, GST-ARN127(Q25) YFP, or GST-Htt exon 1(Q25) was mixed with bacterial lysate containing His-profilin. GST-Htt exon 1(Q25) interacted with His-profilin, whereas GST and GST-ARN127(Q25) YFP did not. (B) Sepharose-bound GST, GST-Htt exon 1(Q20), or GST-Htt exon 1(Q53) was mixed with HEK293 cell lysate containing profilin. GST-Htt exon 1(Q53) bound 85% less profilin than GST-Htt exon 1(Q20). Asterisks indicate two bands nonspecifically reacting with the anti-profilin antibody. (C) Sepharose-bound GST or GST-Htt exon 1(Q25) was mixed with HEK293 cell lysate containing profilin(wt) or the polyproline binding mutants (H133S and W31F). Profilin(wt) bound GST-Htt exon 1(Q25), and the two mutants did not. (D) HEK293 cells were transfected with untagged (first lane) or Myc-tagged profilin (wt, H133S, or W31F). Cell lysate was immunoprecipitated using an anti-Myc antibody, followed by Western blotting against actin. Polyproline binding-deficient profilin mutants bound amounts of actin similar to those of profilin(wt). (E) Profilin (wt, H133S, or W31F) was cotransfected into HEK293 cells with Htt exon 1(Q72) CFP/YFP or ARN127(Q65) CFP/YFP. Relative aggregation was measured by FRET. Both mutants inhibited ARN127(Q65) aggregation comparably to profilin(wt) but were significantly less effective in inhibiting Htt exon 1(Q72) aggregation (***, P < 0.0005; **, P < 0.005 [unpaired t test]). Error bars represent the SEM.

ROCK1 phosphorylates profilin at Ser-137 in vitro and in vivo. (A) Immunoprecipitated Myc-tagged ROCK1(wt) or ROCK1(KDIA) was mixed with profilin(wt) or profilin(S137A) in an in vitro kinase assay and analyzed by autoradiography and Western blotting. While the kinase-dead ROCK1(KDIA) caused little ³²P incorporation into profilin, ROCK1(wt) phosphorylated profilin(wt) and, to a 40% lesser extent, profilin(S137A) (average of three experiments) (*, P < 0.05 by unpaired t test). Error bars represent the SEM. (B) Myc-tagged profilin(wt) or profilin(S137A) was cotransfected into HEK293 cells with ROCK1, ROCK2, or pcDNA3 and treated with or without 50 μM Y-27632 for 24 h. Immunoprecipitation was performed with a phosphoserine-specific antibody (P-Ser) or normal IgG (control [Ctrl]), followed by Western blotting for the Myc tag. No profilin(wt) bound to the P-Ser antibody without ROCK1, whereas ROCK1 overexpression increased binding, which was inhibited by Y-27632. Profilin(S137A) failed to bind the P-Ser antibody regardless of ROCK1 overexpression. ROCK2 overexpression did not increase the binding of profilin(wt) to the P-Ser antibody but increased the binding of profilin(S137A).

Phosphorylation at Ser-137 inhibits profilin activities. (A) Myc-tagged profilin(wt) or mutants (S137A or S137D) were immunoprecipitated (IP) from HEK293 cells. In 50 mM NaCl, all three profilin mutants bound a similar amount of actin; in 500 mM NaCl, profilin(S137A) bound more actin than profilin(wt), and profilin(S137D) bound less (significant difference based on three experiments) (P < 0.01 by unpaired t test). (B) Nickel resin-bound His₆-profilin (wt, S137A, or S137D) was mixed with bacterial lysate containing GST-Htt exon 1(Q25). GST-Htt exon 1(Q25) bound profilin(wt) and profilin(S137A) but not profilin(S137D) or nickel resin alone (first lane). (C) Profilin (wt, S137A, or S137D) was cotransfected into HEK293 cells with Htt exon 1(Q72) CFP/YFP or ARN127(Q65) CFP/YFP. Relative aggregation was measured by FRET. Profilin(S137A) inhibited aggregation as effectively as did profilin(wt). Profilin(S137D) partially failed to inhibit Htt exon 1(Q72) aggregation (***, P < 0.0005 by unpaired t test) and completely failed to inhibit ARN127(Q65) aggregation. Error bars represent the SEM.

Profilin is required for the aggregation-inhibitory activity of Y-27632. (A) Control (ctrl), profilin-1 (Prof-1), or profilin-1/profilin-2a-targeting siRNAs were transfected into HEK293 cells, and lysate was evaluated for profilin-1 or profilin-2a by Western blotting. Profilin-1 siRNAs knocked down profilin-1 by 56% and had no effect on profilin-2a; profilin-1/profilin-2a siRNAs knocked down both isoforms by more than 90%. (B) HEK293 cells were transfected with control, profilin-1, or profilin-1/profilin-2a siRNAs along with Htt exon 1(Q72) CFP/YFP or ARN127(Q65) CFP/YFP. Relative effects on the aggregation of profilin-1 or profilin-1/profilin-2a siRNAs versus control siRNAs were measured by FRET. Profilin-1 siRNAs had little effect on aggregation; profilin-1/profilin-2a siRNAs increased the aggregation of both Htt exon 1(Q72) (**, P < 0.005) and ARN127(Q65) (***, P < 0.0001 by unpaired t test). Error bars represent the SEM. (C) HEK293 cells were transfected with control, profilin-1, or profilin-1/profilin-2a siRNAs along with Htt exon 1(Q72) CFP/YFP or ARN127(Q65) CFP/YFP with or without Y-27632 (50 μM) treatment. Note that the relative effects of Y-27632 on aggregation in the presence of different siRNAs, not the direct effects of siRNAs on aggregation, as in B, are shown here. Profilin knockdown by both pools of siRNA partially blocked the effect of Y-27632 on ARN127(Q65) aggregation (***, P < 0.0005 by unpaired t test) and nearly eliminated (profilin-1 siRNAs) or reversed (profilin-1/profilin-2a siRNAs) the effect on Htt exon 1(Q72) aggregation (**, P < 0.005; ***, P < 0.0005 [unpaired t test]). Error bars represent the SEM.