SRF phosphorylation by glycogen synthase kinase-3 promotes axon growth in hippocampal neurons

Abstract

The growth of axons is an intricately regulated process involving intracellular signaling cascades and gene transcription. We had previously shown that the stimulus-dependent transcription factor, serum response factor (SRF), plays a critical role in regulating axon growth in the mammalian brain. However, the molecular mechanisms underlying SRF-dependent axon growth remains unknown. Here we report that SRF is phosphorylated and activated by GSK-3 to promote axon outgrowth in mouse hippocampal neurons. GSK-3 binds to and directly phosphorylates SRF on a highly conserved serine residue. This serine phosphorylation is necessary for SRF activity and for its interaction with MKL-family cofactors, MKL1 and MKL2, but not with TCF-family cofactor, ELK-1. Axonal growth deficits caused by GSK-3 inhibition could be rescued by expression of a constitutively active SRF. The SRF target gene and actin-binding protein, vinculin, is sufficient to overcome the axonal growth deficits of SRF-deficient and GSK-3-inhibited neurons. Furthermore, short hairpin RNA-mediated knockdown of vinculin also attenuated axonal growth. Thus, our findings reveal a novel phosphorylation and activation of SRF by GSK-3 that is critical for SRF-dependent axon growth in mammalian central neurons.

Keywords: GSK-3; axon growth; filopodia; neurite outgrowth; serum response factor.

Figure 1.

SRF interacts with and is phosphorylated by GSK-3 on ser224. A, Protein alignment shows that ser224 in mouse SRF (ser228 in human) is highly conserved among different species. The boxed region represents the suggested GSK-3 consensus sequence, S/T-x-x-x-S/T. B, Analysis of the solved x-ray crystal structure of the N-terminus 1–223 amino acids of human SRF bound to DNA shows that the aspartate residue at 223 is far away from the DNA binding region of SRF. Therefore, the target serine 228 (human), located five amino acids further c terminus, is unlikely to interfere with DNA binding. C, SRF and GSK-3 interact in the brain. SRF was immunoprecipitated from whole-brain lysate of P0.5 wild-type mice and immunoblotted for GSK-3 and SRF (top). GSK-3α and GSK-3β were efficiently pulled down only with the SRF antibody and not with anti-IgG control antibody. Likewise, GSK-3 was able to pull down SRF only from wild-type but not from Srf-NestinCKO brain lysate (bottom). Pull down using IgG alone served as control. D, SRF is efficiently phosphorylated by GSK-3β in vitro. Kinase assays ware performed using bacterially generated GST, GST-SRF, or GST-SRF^S224A along with either GSK-3β-CA, GSK-3β-R96A mutant kinases or commercial recombinant wild-type GSK-3β in the presence of [γ-³²P]ATP. GST-SRF alone was phosphorylated by GSK-3β-CA, GSK-3β-R96A, and wild-type GSK-3β. In contrast, SRF^S224A mutant and GST alone showed no signal suggesting that there are no other GSK-3β sites on SRF. E, SRF is also phosphorylated by GSK-3β in vivo. HEK293T cells were transfected with HA-tagged SRF^WT or HA-SRF^S224A along with empty Myc vector or with each of the different Myc-tagged GSK-3β kinases. SRF was pulled down with anti-HA antibody and immunoblotted using anti-phosphoserine (anti-MPM2) and anti-SRF antibodies. The level of phosphorylation of HA-SRF^WT increased in the presence of active GSK-3β kinases (WT and CA), whereas phosphorylation is attenuated in the presence of GSK-3β-KD mutant kinase. SRF^S224A mutant did not exhibit any changes in phosphorylation levels. F, Densitometry analysis of results from E shows changes in phosphorylation levels relative to that for SRF^WT or SRF^S224A protein levels. Error bars indicate SEM. ***p < 0.001 (one-way ANOVA, Tukey post-test analysis). n.s., Not significant. G, SRF is phosphorylated by GSK-3 in neurons. Hippocampal neurons from neonatal pups were grown for 3 d in the presence of either DMSO (vehicle) or 500 nm 6-BIO to inhibit GSK-3. Endogenous SRF was immunoprecipitated with anti-SRF antibody and immunoblotted with anti-phosphoserine antibody. The level of serine phosphorylation decreased in the presence of 6-BIO compared with DMSO-treated control. H, Densitometric analysis of phospho-SRF to total SRF levels from G shows significant decrease in SRF serine phosphorylation in the presence of 6-BIO relative to DMSO control. Error bars indicate SEM. I, Changes in SRF and GSK-3 phosphorylation during brain development. SRF was immunoprecipitated from whole-brain lysates of wild-type embryos and pups at the indicated stages and immunoblotted with pSer antibody. GSK-3β activity was measured by immunoblotting for GSK-3β-Ser9 phosphorylation. SRF expression increased during development, whereas the level of pSer phosphorylation peaked ∼e15.5-e18.5. GSK-3β-Ser9 phosphorylation decreased between e15.5 and e18.5 and postnatally, suggesting GSK-3β activation during this time period. J, Densitometric analysis of pSer levels and GSK-3β-pSer9 from I shows increase in pSer phosphorylation of SRF and GSK-3β ∼e15.5 to e18.5. Error bars indicate SEM. ***p < 0.001 (one-way ANOVA, Tukey post-test analysis). **p < 0.01 (one-way ANOVA, Tukey post-test analysis).

Figure 2.

Serine 224 is required for SRF function. A, SRF^S224A mutant cannot rescue axonal growth deficits of SRF-deficient neurons. P0.5 hippocampal neurons from Srf-NestinCKO mice were transfected with IRES-EGFP empty vector, HA-SRF^WT, or HA-SRF^S224A. Cells were fixed and immunostained for GFP or HA (green) to visualize transfected cells and anti-Tuj1 (red) at 4 DIV. Axonal growth deficits exhibited by SRF-deficient neurons were rescued by SRF^WT alone and not by empty vector or SRF^S224A (arrows). Arrowhead indicates an untransfected cell. Scale bar, 50 μm. B, Quantitation of axon length from A. n = 4 mice; “n” in the bars indicates the number of cells measured for statistics. Error bars indicate SEM. ***p < 0.001 (one-way ANOVA, Tukey post-test analysis). n.s., Not significant. C, SRF^S224A functions as a dominant mutant when expressed in wild-type hippocampal neurons. Cells were transfected with HA-SRF^WT or HA-SRF^S224A and grown for 4 DIV. Cells were fixed and immunostained with anti-HA (green) and anti-Tau (red) antibodies. HA-SRF^S224A expression attenuated axonal growth (arrows). Scale bar, 50 μm. D, Data quantified from C. Error bars indicate SEM. ***p < 0.001 (one-way ANOVA, Tukey post-test analysis). E, Inhibiting GSK-3 activity or mutating serine 224 to alanine abolishes SRF transcriptional activity. HEK293T cells were transfected with 5×-SRE luciferase and Renilla luciferase (for transfection efficiency) and treated with either 6-BIO or cotransfected with control vector, SRF^WT, SRF-S224A, or SRF-VP16. 6-BIO treatment blocked serum-induced SRF-transcriptional activity. Whereas vector and SRF^WT transfected cells showed normal activation of an SRE reporter, the SRF^S224A mutant was severely compromised in activating transcription. Expression of constitutively active SRF-VP16 served as the control. Error bars indicate mean ± SEM.

Figure 3.

Serine 224 phosphorylation is required for SRF interaction with MKL-family cofactors and not with TCF-family cofactor ELK1. A, HEK293T cells were cotransfected with HA-SRF^WT or HA-SRF^S224A along with Flag-MKL1 or Myc-MKL2. Immunoprecipitation with anti-HA antibody pulled down significantly less MKL1 (left) and MKL2 (right) from HA-SRF^224A-transfected cells. The expression levels of Flag-MKL1, Myc-MKL2, and HA-SRF were the same in both groups of transfected cells. β-Tubulin levels in equal amounts of lysates used for IP served as loading control. B, Densitometric analysis of immunoblot in A. Error bars indicate SEM. C, Serine 224 to alanine mutation does not affect SRF interaction with the TCF-family member ELK1. HEK293T cells were transfected with HA-SRF^WT or HA-SRF^S224A. Immunoprecipitation with anti-HA antibody and immunoblotting for endogenous activated ELK1 using anti-phosphoELK1 (pELK1) antibody showed that a similar amount of pELK1 was immunoprecipitated with HA-SRF^WT and HA-SRF^S224A. Levels of SRF in the immunoprecipitates were similar and β-tubulin levels in equal amounts of lysates used for IP served as loading control. D, Densitometric analysis of immunoblot in C. Error bars indicate SEM. n.s., Not significant. E, MKL1 and MKL2 are required for axon growth. Neonatal wild-type hippocampal neurons were transfected with IRES-EGFP vector or DN-MKL2-IRES-EGFP. Expression of DN-MKL2 blocked axonal growth (arrow) as visualized with anti-Tau (red) and anti-GFP (green). Scale bar, 50 μm. F, Quantitation of D. Error bars indicate SEM; n = 3 mice. ***p < 0.001 (two-tailed t test analysis). G, Inhibition of TCF-family cofactors does not affect axonal growth. Hippocampal neurons from wild-type neonatal pups were transfected with dominant-negative Elk1-En (bottom) or empty vector (top). Cells were stained for Tuj1 (red) and Flag epitope (green) at 4 DIV. Expression of Elk1-En, which inhibits all three members of the TCF family, does not affect axonal growth. Scale bar, 50 μm. H, Quantitation of axon length in G. Error bars indicate SEM; n = 3 mice. p = 0.8087 (two-tailed t test analysis).

Figure 4.

SRF can rescue axonal growth deficits caused by GSK-3 inhibition. A, P0.5 hippocampal neurons from wild-type mice were transfected with either empty IRES-EGFP vector or constitutively active SRF (SRF-VP16-IRES-EGFP) or constitutively active CREB (VP16-CREB) and grown in the presence of the GSK-3-specific inhibitor 6-BIO (500 nm) or DMSO (vehicle). Cells were fixed and immunostained using anti-Tau (red) and GFP or VP16 (green) at 4 DIV. GSK-3 inhibition by 6-BIO results in attenuation of axonal growth (top, arrowhead), and this was rescued by expression of SRF-VP16 (middle). Expression of a constitutively active CREB (VP16-CREB) was unable to promote axonal growth under similar growth conditions (bottom). Arrows indicate dead cells/debris. Scale bar, 50 μm. B, Quantitation of A. Error bars indicate SEM. **p < 0.01 (two-tailed t test analysis). ***p < 0.001 (two-tailed t test analysis). n.s., Not significant. C, Hippocampal neurons were cotransfected with GSK-3β constructs and SRF-VP16 as indicated above and grown for 4 d. Expression of GSK-3β-KD attenuated axon growth (middle). This attenuation was rescued by SRF-VP16 expression (bottom). Expression of GSK-3β-WT alone had no influence on axon outgrowth. Arrows indicate dead cells/debris. Scale bar, 50 μm. D, Axon length quantified from C. Error bars indicate SEM. ***p < 0.0001 (one-way ANOVA, Tukey post-test analysis).

Figure 5.

SRF-deficient neurons are deficient in Stage 1 neurite growth and exhibit attenuated axonal growth in vitro. A, Hippocampal neurons from Srf-NestinCKO mice exhibit delayed and attenuated axonal growth as visualized by immunostaining with Tuj1 (red) and MAP2 (green). SRF-deficient neurons produced fewer neurites at 12 h (arrows, left second row panels) and, at 5 d, had shorter axons and enlarged growth cone (right bottom, arrow). Control neurons extended longer axon (right middle, arrow) and multiple dendrites. Arrowheads indicate dead cells. Scale bar, 50 μm. B, Axonal growth in A was studied by measuring the length of the Tuj1⁺/MAP2⁻ process at the indicated time points. Error bars indicate SEM. C, Srf-NestinCKO neurons are deficient in filopodia formation. Rhodamine-conjugated phalloidin reveals several filopodia (12 h, arrows) and distinct neurites (48 h) in control neurons. In contrast, mutant neurons had fewer or no filopodia (arrow) and an enlarged cell body. Scale bar, 12.5 μm. D, Histograms of the total filopodia counted from C. p < 0.0001 (two-tailed t test analysis). E, Immunostaining with axon-specific PanNa_V (green) and Tuj1 (red) antibodies shows normal polarization of Srf-NestinCKO neurons as observed for Srf-f/f neurons (n = 32). Scale bar, 25 μm.

Figure 6.

Putative SRF target genes identified from forebrain. A, Table represents Srf target genes identified from forebrain of neonatal Srf-f/f and Srf-NestinCKO mice. The genes in bold were further analyzed for their ability to rescue axonal growth deficits of Srf-NestinCKO mutant neurons. B, mRNA expression of Srf target genes in A. Relative mRNA expression levels were assessed by real-time qRT-PCR. The expression level in control sample was set to 1 (n = 3 experiments).

Figure 7.

MAP1B expression is attenuated in SRF-deficient neurons. A, Lack of expression of MAP1B in SRF-deficient neurons. SemiqRT-PCR of total RNA isolated from brains of Srf-NestinCKO mice and Srf-f/f control littermates (n = 3). RPS29 expression served as the control. B, MAP1B expression partially promotes axon growth in SRF-null neurons. Hippocampal neurons from neonatal Srf-NestinCKO mice were transfected with either control myc vector or myc-tagged MAP1B along with farnesylated GFP (F-GFP) and grown for 4 d on poly-d-lysine and laminin. MAP1B was able to partially increase axonal length in SRF-deficient cells. However, MAP1B was not efficient in rescuing the enlarged growth cone (arrows) and abnormal cell soma (arrowheads) morphologies observed in SRF-deficient neurons. Expression of MAP1B did not increase axonal growth in WT neurons. Scale bar, 50 mm. C, Quantitation of axon length and growth cone area from B. ***p < 0.001 (one-way ANOVA and Tukey post-test analysis). n.s., Not significant.

Figure 8.

Vinculin is sufficient to promote axonal growth in SRF-deficient neurons. A, Western blot of total brain lysate showing reduced Vcl expression in Srf-NestinCKO mice compared with control littermates. B, P0.5 hippocampal neurons from Srf-NestinCKO and Srf-f/f mice were cotransfected with empty vector (pMyc) or pMyc-VCL and cultured for 4 DIV on poly-d-lysine and laminin. Cells were fixed and immunostained for Tuj1 (red) and Myc (green). Expression of Vcl but not empty vector was able to promote axon growth. Scale bar, 25 μm. C, Quantitation of B. Error bars indicate SEM. ***p < 0.001 (one-way ANOVA, Tukey post-test analysis). n.s., Not significant. D, Vinculin only partially rescues axonal growth in SRF-deficient neurons in the absence of laminin. P0.5 hippocampal neurons from Srf-NestinCKO and Srf-f/f mice were cotransfected with empty vector (pMyc) or pMyc-VCL along with farnesylated-GFP (F-GFP) and cultured for 4 DIV on poly-d-lysine alone without laminin. Cells were fixed and immunostained for GFP (green) to visualize neuronal morphology and Myc (red) for transfected cells. Expression of VCL, but not empty vector, could partially rescue axonal growth in the absence of laminin, suggesting a role for laminin–integrin signaling downstream of VCL in promoting axon growth. Scale bar, 25 μm. E, Quantitation of axon length in D. ***p < 0.001 (one-way ANOVA, Tukey post-test analysis).

Figure 9.

Vinculin can partially rescue axonal growth in the absence of GSK-3 signaling. A, Vcl mRNA expression is attenuated in neurons when GSK-3 is inhibited. Vcl mRNA expression was assessed by real-time qRT-PCR from total RNA isolated from hippocampal neurons grown in the presence of DMSO (con), 500 nm 6-BIO or 6-BIO+SRF-VP16 at 3 DIV. Vcl mRNA levels decreased in the presence 6-BIO and expression of SRF-VP16 rescued Vcl levels. ***p < 0.001 (one-way ANOVA, Tukey post-test analysis). n.s., Not significant. B, VCL expression was also able to promote axonal growth when GSK-3 is blocked by 6-BIO. Wild-type hippocampal neurons were transfected with either empty Myc vector or Myc-VCL and cultured in the presence of DMSO (vehicle) or 500 nm 6-BIO for 4 DIV on poly-d-lysine and laminin. Cells were fixed and immunostained for Myc (green) and Tuj1 (red). VCL expression alone promoted axonal growth (arrow). Scale bar, 25 μm. C, Quantitation of B. Error bars indicate SEM. **p = 0.005 (6-BIO+Myc vs 6-BIO+Myc-VCL) (two-tailed t test analysis). ***p = 0.0002 (6-BIO+Myc vs 6-BIO+Myc-VCL) (two-tailed t test analysis). D, shRNA knockdown of VCL expression in hippocampal neurons attenuates axonal growth. Wild-type hippocampal neurons were transfected with shRNA targeting mouse Vcl (Vcl-shRNA-CMV-mCherry) or a control shRNA (shRNA-CMV-mCherry) along with YFP and grown for 4 d. Cells were fixed and stained for mCherry encoded from within the shRNA vector and YFP. For rescue experiment, neurons were transfected with Vcl-shRNA along with the chicken homolog, venus-chicken Vcl (Ch-Vcl). Ch-Vcl is resistant to mouse Vcl-shRNA, and its expression rescued axonal growth (arrow). Scale bar, 25 μm. E, Quantitation of D. Error bars indicate SEM. **p < 0.01 (one-way ANOVA, Tukey post-test analysis). ***p < 0.001 (one-way ANOVA, Tukey post-test analysis).

Figure 10.

Summary model. Model of GSK-3-SRF signaling pathway in regulation of axon growth. Phosphorylation by GSK-3 on serine 224 results in the activation of SRF and subsequent recruitment of MKL-family cofactors. The SRF-MKL transcriptional complex activates genes, such as VCL and MAP1B. MAP1B regulates microtubule dynamics, whereas VCL tethers F-actin to focal adhesion junctions to promote neurite growth downstream of the GSK-3-SRF pathway. Absence of SRF or GSK-3 activity results in inhibition or attenuation of neurite growth.