Cofilin phosphorylation in PC12 cells is Rho dependent

To obtain more direct evidence for changes in LIMK activity upon serum stimulation of PC12 cells, we used two-dimensional (2D) gel electrophoresis and immunoblotting to examine cofilin phosphorylation. In both cycling or serum-starved PC12 cell extracts, cofilin migrated as two predominant species of 19 kD, presumably representing cofilin and actin depolymerizing factor, which should cross react with the antiserum used (Fig. 2 A, top, arrows). Serum stimulation induced the appearance of an additional spot, which was sensitive to treatment of the cells with Y27632 (Fig. 2 A, middle, arrow). This species was also sensitive to phosphatase treatment, indicating that it is a phosphorylated derivative of cofilin (Fig. 2 A, bottom, arrow).

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Figure 2.

Cofilin phosphorylation via the Rho pathway in PC12 cells. The 2D gels are shown with acidic isoelectric point to the right, SDS-PAGE top to bottom. (A) Serum-induced phosphorylation of endogenous cofilin requires ROCK activity. PC12 cells were maintained in 0.5% serum before stimulation with 15% serum with or without Y27632 (Y) pretreatment. Cell extracts were prepared and fractionated by 2D gel electrophoresis; cofilin was detected by immunoblotting. Where indicated, extracts were treated with alkaline phosphatase (P'tase) before analysis. (B) FLAG–cofilin reporter assay. PC12 cells transfected with the indicated FLAG–cofilin expression plasmids (0.2 μg) were analyzed by 2D gel electrophoresis and immunoblotting with anti-FLAG antibody; phosphatase treatment was performed on anti-FLAG immunoprecipitates. (C) Phosphorylation status of FLAG–cofilin under different growth conditions. Cells transfected with wild-type FLAG–cofilin expression plasmids were maintained as indicated and analyzed as in part B. Only the cofilin species are shown; the positions of unphosphorylated and phosphorylated forms are indicated above the figure. (D) Serum-induced phosphorylation of FLAG–cofilin requires RhoA and ROCK activity. Cells transfected with expression plasmids encoding wild-type FLAG–cofilin and C3 transferase (50 ng), as indicated, were serum starved and restimulated with 15% serum for 5 min; Y27632 treatment (Y) was as indicated.

To allow investigation of the role of RhoA and its effectors in signaling to SRF via LIMK, we set up a simple cofilin reporter assay. Extracts from transfected cells expressing FLAG epitope–tagged cofilin were subjected to 2D gel electrophoresis followed by immunoblotting with FLAG antibody. Wild-type FLAG–cofilin generated two species in this assay. The more acidic of these represents the phosphorylated form, because it is not generated by the nonphosphorylatable mutant cofilin S3A, and is sensitive to phosphatase treatment (Fig. 2 B, arrows). The ratio of phosphorylated to nonphosphorylated FLAG–cofilin observed in serum-starved cells varied between experiments, increasing as cofilin expression increased (Fig. 2 D, middle and bottom), perhaps reflecting homeostatic adjustment of the treadmilling machinery to maintain the F-actin level. We therefore only compared FLAG–cofilin phosphorylation levels within a given experiment. Serum starvation caused a reduction in the amount of phosphorylated FLAG–cofilin relative to the unphosphorylated form (Fig. 2 C, top and middle). Upon serum stimulation, increased phosphorylation of FLAG–cofilin was detectable within 5 min and persisted for at least 30 min (Fig. 2 C, bottom). Serum-induced FLAG–cofilin phosphorylation did not occur in cells expressing C3 transferase or treated with Y27632, indicating that it depends on RhoA–ROCK signaling (Maekawa et al., 1999; Ohashi et al., 2000) (Fig. 2 D, top and middle); C3 or Y27632 treatment also slightly but consistently reduced the proportion of phosphorylated FLAG–cofilin in serum-starved cells, suggesting that the basal level of LIMK activity is also significantly dependent on RhoA (Fig. 2 D, bottom).

SRF activation via ROCK and LIMK requires functional RhoA

The data presented above show that ROCK–LIMK signaling is required for serum-induced SRF activation in PC12 cells. We used C3 transferase expression to test whether this is the only RhoA-dependent event required for signaling to SRF in these cells. In parallel, we examined the effects of C3 transferase upon cofilin phosphorylation using the FLAG–cofilin transfection assay. As expected, C3 transferase expression abolished SRF activation by RhoA.V14 (Fig. 3 A). Surprisingly, C3 expression also abolished SRF activation by overexpressed LIMK1 (Fig. 3 A), although it did not inhibit induction of FLAG–cofilin phosphorylation by LIMK1 (Fig. 3 B, top). This result suggests that LIMK activation of SRF requires an additional RhoA-dependent event, even in starved cells with only basal levels of RhoA activity.

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Figure 3.

LIMK1 requires an additional RhoA-dependent event to activate SRF. (A) Sensitivity of SRF activation to C3 transferase. PC12 cells were transfected with the SRF reporter and expression plasmids encoding RhoA.V14 (1.0 μg), LIMK1 (1.0 μg), or ROCKΔ3 (60 ng), and C3 transferase (50 ng) as indicated, and reporter activity was measured. (B) FLAG–cofilin phosphorylation does not require RhoA. Cells were transfected with expression plasmids encoding wild-type FLAG–cofilin (0.2 μg) alone or with LIMK1 (1.0 μg), ROCKΔ3 (30 ng), LIMK1 kinase domain K1.C (0.1 μg), or ROCK-insensitive LIMK1 kinase domain mutant K1.C/T508V (1.0 μg), and C3 transferase (50 ng) as indicated, before processing by 2D gel electrophoresis followed by anti-FLAG immunoblot. Only the FLAG–cofilin species are shown; the positions of unphosphorylated and phosphorylated forms are indicated above the figure. (C) SRF activation by LIMK1 T508V requires RhoA. Cells were transfected with the SRF reporter and expression plasmids encoding LIMK1 kinase domain K1.C (0.1 μg) or ROCK-insensitive LIMK1 kinase domain mutant K1.C/T508V (0.1, 0.3, or 1.0 μg), with C3 transferase (50 ng) as indicated, and reporter activity was measured. (D) LIMK1 derivatives. The LIMK1 coding region is indicated at the top with LIM, PDZ, and kinase domains shown as boxes; the K1.C kinase domain (residues 293–646) is shown below and its ROCK-insensitive mutant T508V is also indicated.

The RhoA effector kinase ROCK activates LIMK1 by phosphorylating it at a single site, T508 (Maekawa et al., 1999; Ohashi et al., 2000), and we therefore tested whether the sensitivity of LIMK1 to C3 transferase reflected the involvement of RhoA–ROCK signaling in LIMK activation. First, we examined whether functional RhoA is required for SRF activation induced by the activated ROCK derivative, ROCKΔ3, which should activate LIMK1 independently of RhoA (Maekawa et al., 1999; Ohashi et al., 2000). ROCKΔ3 activated SRF with an efficiency comparable to that of RhoA.V14, but as observed with LIMK1, this was sensitive to C3 transferase (Fig. 3 A). Expression of ROCKΔ3 also induced phosphorylation of FLAG–cofilin, which was not blocked by C3 expression although it was somewhat reduced (Fig. 3 B, middle). Next, we examined SRF activation by LIMK T508V, in which the target site for ROCK phosphorylation is replaced with a nonphosphorylatable valine residue (Fig. 3 D); such mutants retain some catalytic activity both in vitro and in vivo (Edwards and Gill, 1999; Ohashi et al., 2000). Expression of the wild-type LIMK1 kinase domain efficiently activated SRF, and this was again substantially RhoA dependent (Fig. 3 C), although at high expression levels some C3-resistant SRF activation was observed (unpublished data; see Discussion). Overexpression of the LIMK1-T508V kinase domain also activated SRF, although substantially less efficiently than wild type; however, activation was still RhoA dependent (Fig. 3 C). Under transfection conditions in which expression levels of wild-type and T508V LIMK were adjusted to give similar levels of SRF activation, both proteins induced a similar degree of FLAG–cofilin phosphorylation, which was not impaired by coexpression of C3 transferase (Fig. 3 B, bottom). Taken together, these results show that to activate SRF in PC12 cells, LIMK requires a second Rho-dependent signal, which must regulate a process distinct from cofilin phosphorylation.

mDia is required for SRF activation in PC12 cells

The RhoA effector mDia promotes F-actin accumulation and is required for serum-induced SRF activation in NIH3T3 cells (Tominaga et al., 2000; unpublished data). We therefore investigated its role in signaling to SRF in PC12 cells. An activated form of mDia1, mDia1*, which lacks the RhoA-binding domain (Watanabe et al., 1999), efficiently activated SRF in PC12 cells (Fig. 4 A). In contrast to SRF activation by LIMK, activation of SRF by mDia1* was substantially unaffected by coexpression of C3 transferase, indicating that it did not require functional Rho (Fig. 4 B). Expression of activated mDia1 did not alter the proportion of phosphorylated FLAG–cofilin generated in the cofilin reporter assay (Fig. 4 C, top), indicating that it does not activate SRF by regulating LIMK activity. Thus, mDia provides an alternative route to the ROCK–LIMK pathway for signaling to SRF downstream of RhoA.

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Figure 4.

mDia activity is required for SRF activation in PC12 cells. (A) mDia1 derivatives. The mDia1 coding region is indicated at the top with RhoA-binding domain as a circle and formin homology regions 1–3 as boxes; below are shown the activated mDia1 derivative Dia1* (residues 256–1255), the inactive interfering derivative dnDia1 (residues 571–1181, lacking 750–771), and the control inactive noninterfering derivative ΔDia1 (residues 256–567). (B) SRF activation by mDia1 in PC12 cells. Cells were transfected with the SRF reporter and expression plasmids encoding Dia1* (0.3 μg) with C3 transferase (50 ng), as indicated, and reporter activity was measured. The effect of C3 transferase on reporter activation by the synthetic activator SRF-VP16 (50 ng) is shown for comparison. Error bars show half range (n = 2). (C) mDia1 does not promote cofilin phosphorylation. Cells were transfected with expression plasmids encoding wild-type FLAG–cofilin (0.2 μg) alone or with Dia1* (0.3 μg), dnDia1 (2.0 μg), or LIMK1 (1.0 μg). After maintenance in 0.5% serum with serum stimulation as indicated, cells were analyzed for FLAG–cofilin phosphorylation by 2D gel electrophoresis followed by anti-FLAG immunoblot. Only the FLAG–cofilin species are shown; the positions of unphosphorylated and phosphorylated forms are indicated above the figure. (D) Serum-induced SRF activation requires functional mDia. Left, cells were transfected with the SRF reporter and an expression plasmid encoding dnDia1 (2.0 μg), as indicated, and either maintained in 0.5% serum or serum stimulated before measurement of reporter activity. Serum-stimulated reporter activity is taken as 100%. Error bars show SEM (n = 4). (E) Cofilin S3A potentiates the effect of interfering mDia. Cells were transfected with the SRF reporter together with expression plasmids encoding dnDia1 (2.0 μg) and/or cofilin S3A (0.5 or 2.0 μg), as indicated, and either was maintained in 0.5% serum or was serum stimulated before quantitation of reporter activity. Serum-induced reporter activity is taken as 100%. Error bars show half range (n = 2).

To evaluate the contribution of mDia proteins to serum-induced SRF activation, we used an inactivated mDia1 derivative that can interfere with SRF activation when overexpressed in NIH3T3 cells. This mutant, dnDia1, lacks the NH₂-terminal RhoA-binding domain of mDia1 and sequences within the COOH-terminal domain (Fig. 4 A; unpublished data). In PC12 cells, dnDia1 expression did not activate SRF, but instead substantially inhibited SRF activation after serum stimulation (Figs. 4 D and 5 A). Importantly, expression of dnDia1 did not prevent the increase in FLAG–cofilin phosphorylation that occurred upon serum stimulation (Fig. 4 C, middle), indicating that its effect on SRF is specific and does not result from a nonspecific impairment of Rho signaling. Expression of dnDia1 was not toxic, as it affected neither the number of GFP-positive cells nor the thymidine kinase–renilla control reporter (unpublished data). Expression of cofilin S3A and dnDia1 inhibited reporter induction more effectively than dnDia1 alone (Fig. 4 E). Taken together, these data show that in addition to the ROCK–LIMK pathway, functional mDia is required for serum-induced SRF activation in these cells, and activation of SRF by mDia occurs independently of cofilin phosphorylation.

Cell culture, transfection, and gene expression assays

PC12 cells were maintained on collagen-coated plates in DME with 6% FCS and 6% horse serum and seed at 6 × 10⁵ cells per 6-cm dish before transfection using FuGene reagent (Roche) according to the manufacturer's instructions. Each transfection mix (4 μg total plasmid DNA) included SRF reporter plasmid 3D.ALuc (firefly luciferase; 0.1 μg), reference standard pRLTK (thymidine kinase promoter controlling renilla luciferase; 0.4 μg), EF-eGFP (0.2 μg) to monitor transfection efficiency, and EFplink expression vector or derivatives encoding activator proteins or FLAG–cofilin (3.3 μg). For phosphorylation studies, cofilin expression plasmids were used at 0.2 μg per transfection. From control immunoblotting experiments with transfection efficiency estimated using GFP, we estimate that FLAG–cofilin was overexpressed by between two- and fivefold (unpublished data). Transfected cells were maintained in 0.5% serum for 18 h before stimulation with 15% serum; lysates were prepared 8 h later. For reporter experiments, activities were normalized to the activity of a parallel control transfection in which reporter activation was achieved using the constitutively active SRF derivative SRFVP16 (50 ng; Dalton and Treisman, 1992), taken as 100. Each reporter figure shows mean ± SEM for at least three independent transfection experiments. RNA analysis and transfection of mouse NIH3T3 cells were performed as previously described (Sotiropoulos et al., 1999); total RNA (10 μg) was analyzed using mouse GAPDH and vinculin probes with RNase T1 digestion (8 U per hybridization). Drug treatments were as follows: cytochalasin D (2 μM; Calbiochem); jasplakinolide (0.5 μM; Molecular Probes); pretreatments for 45 min with Y27632 (10 μM; gift from Yoshitomi Corp, Osaka, Japan) or latrunculin B (0.5 μM; Calbiochem).

2D . gel electrophoresis assay for cofilin phosphorylation

Cells were washed with ice cold phosphate-buffered saline and fixed with 10% TCA for 15 min on ice before scraping and collection. The acetone-washed cell pellet was solubilized with sonication in 2D sample buffer (8 M urea, 4% CHAPS, 40 mM Tris base, 0.5% IPG buffers, pH 3–10; Amersham Pharmacia Biotech), and cleared by centrifugation before loading Immobilon strips with a linear pH gradient from 3 to 10 (Amersham Pharmacia Biotech). First dimension electrophoresis was performed using the IPGphor isoelectric focusing system (Amersham Pharmacia Biotech); second dimension was by 18% SDS-PAGE. Immunoblotting was performed by standard procedures with detection by anti-FLAG antibody (M2; Sigma-Aldrich) for transfected cells expressing FLAG–cofilin or the anti-cofilin antibody (Cytoskeleton).

Phosphatase treatment

Cells were washed with ice cold PBS and lysed directly on the plate (6-cm dish) with 100 μl of SDS lysis buffer (1% SDS, 10 mM EDTA, 5 mM NaF, 10 mM KH₂PO₄, 50 mM Tris, pH 8.1, with protease inhibitors) for 10 min on ice and transferred to a test tube. The lysate was then diluted with 900 μl of IP dilution buffer (0.01% SDS, 1.1% triton, 1.2 mM EDTA, 167 mM NaCl, 5 mM NaF, 10 mM KH₂PO₄, 16.7 mM Tris, pH 8.0, with protease inhibitors) and clarified by centrifugation before immunoprecipitation using anti-FLAG beads (25 μl) for 2 h at 4°C. Beads were then washed twice using IP buffer and dispersed in 100 μl of phosphatase buffer (50 mM Tris-HCl, pH 8.5, 1 mM EDTA) containing 20 U of alkaline phosphatase (Boehringer); control beads were dispersed in 100 μl phosphatase buffer containing 5 mM NaF and 10 mM KH₂PO₄. Incubation was at 37°C for 1 h; beads were then collected and cofilin was eluted using 2D sample buffer. For phosphatase treatment of endogenous cofilin, lysates were prepared as above in IP buffer lacking NaF and KH₂PO₄ and treated directly with 40 U alkaline phosphatase for 1 h at 37°C. Proteins were recovered by TCA precipitation and processed for 2D gel analysis as above.

We thank the following colleagues from the London Research Institute of Cancer Research UK for advice, technical assistance, and helpful comments on the manuscript: David Frith (2D gel facility); Cathy Simpson and Derek Davies (FACS^® facility); and Caroline Hill, Gipi Schiavo, Nassia Sotiropoulos, Sharon Tooze, and Michael Way.

O. Geneste was supported by postdoctoral fellowships from the Human Frontier Science Program and the ICRF. J. Copeland was supported by a postdoctoral fellowship from the Hitchings-Elion Program of the Burroughs Wellcome Fund.