SRPK2 provokes neuronal apoptotic through up-regulating cyclin D1-Cdk4. A, wild-type SRPK2 and T492D provoke cyclin D1and Cdk4 expression and trigger apoptosis in cortical neurons. Various lentivirus expressing SRPK2 wild type and mutants were infected into cortical neurons and incubated for 4 days. The lysates were assessed by immunoblotting using antibodies against indicated proteins. Tubulin was included as a loading control. DNA fragmentation was also conducted to verify apoptosis (bottom panel). B, 14-3-3 suppresses cyclin D1-Cdk4 expression and prevents apoptosis in neurons. Various SRPK2 were co-infected with 14-3-3β into cortical neurons and incubated for 4 day. The lysates were assessed by immunoblotting using antibodies against indicated proteins. DNA fragmentation was also conducted to verify apoptosis (bottom panel). C, INKp16 blocks cyclin D1-Cdk4 kinase activity and prevents apoptosis. Various SRPK2 lentiviruses were co-infected with INKp16 into cortical neurons and incubated for 4 days. The lysates were assessed by immunoblotting using antibodies against indicated proteins. DNA fragmentation was also conducted to verify apoptosis (bottom panel).

Characterization of Akt/SRPK2/cyclin D1 cascade in ischemia-attacked brain. A, SRPK2 depletion blocks Akt-provoked cyclin D1 expression in neurons. Primary cortical neurons were infected with adenovirus expressing Akt-CA or KD followed by infection with lentivirus expressing short hairpin RNA of SRPK2. The cell lysates were analyzed by immunoblotting with indicated antibodies. Interestingly, SRPK2 knocking down alone initiated caspase-3 activation (sixth panel). B, SRPK2 depletion decreases glutamate-provoked cyclin D1 expression. Control and short hairpin RNA-SRPK2 lentivirus-infected cortical neurons were treated with 50 μm glutamate and then harvested at the indicated time. Extracts were separated by SDS-PAGE and analyzed by Western blotting using antibodies against indicated proteins. C, stroke triggers Akt/SRPK2/cyclin D1 signaling cascade. The stroke-attacked mouse brain tissues at the indicated times were analyzed by Western blotting using antibodies against indicated proteins.

SRPK2 phosphorylates SC35, inhibits p53 activation, and up-regulates cyclin D1. A, in vitro SRPK2 kinase assay with SC35 recombinant protein. The reaction mixture was resolved on 10% SDS-PAGE. Wild-type SRPK2 and T492D mutant strongly phosphorylated SC35, but T492A and KD mutants were unable to phosphorylate it. B, SRPK2 phosphorylates SC35 in neurons. Various SRPK2 lentivirus were infected into cortical neurons and followed by metabolic labeling in [³²P]H₃PO₄. SC35 was pulled down and resolved on SDS-PAGE. Wild-type SRPK2 and T492D mutant strongly phosphorylated SC35, whereas other mutants displayed weak effect. C, SRPK2 redistributes SC35 in the nucleus. Cortical neurons were infected with various SRPK2 lentivirus. Localization of various SRPK2 and endogenous SC35 was visualized by indirect immunofluorescent staining using anti-Myc polyclonal antibody and anti-SC35 antibody. Wild-type SRPK2 and T492D mutant strongly redistributed SC35 from the nuclear speckles into the nucleoplasm. D, depletion of SC35 abolishes SRPK2-regulated cyclin D1 expression. Various SRPK2 constructs co-transfected with si-control, si-SC35, or si-p53 into cortical neurons. The lysates were analyzed by Western blotting using antibodies against indicated proteins. E, 14-3-3 inhibits the SRPK2 effect on p53 activation. Various SRPK2 constructs were co-infected with control and 14-3-3 into cortical neurons. The lysates were analyzed by immunoblotting with various antibodies. Wild-type SRPK2 and T492D repressed p53 phosphorylation and increased cyclin D1 expression, which was abolished by 14-3-3. F, a schematic model for Akt/SRPK2/sc35/p53/cyclin D1 signaling in neuronal cell cycle and cell death.

14-3-3 inhibits the biological activities of SRPK2. A, in vitro SRPK2 kinase assay. Purified His-SRPK2 recombinant proteins were incubated with acinus wild type and acinus S422A in the presence of [γ-³²P]ATP. Wild-type SRPK2 and T492D strongly phosphorylated wild-type acinus and weakly phosphorylated S422A. By contrast, the kinase activity was significantly decreased in SRPK2 T492A. B, Akt-phosphorylated SRPK2 strongly phosphorylates acinus in cells. Lysates prepared from cortical neurons were infected with various lentiviruses encoding various SRPK2 proteins and analyzed by immunoblotting with various antibodies. Wild type and T492D SRPK2 provoked strong phosphorylation in endogenous acinus, but T492A and KD mutants failed to phosphorylate acinus (middle panel). Verification of SRPK2 and acinus expression (top and bottom panels). C, 14-3-3β inhibits SRPK2 kinase activity. Cortical neurons were co-infected with14-3-3β, and various SRPK2 proteins and acinus phosphorylation was monitored by immunoblotting. D, SRPK2 increases BrdUrd incorporation in neurons. Cortical neurons were infected with various SRPK2 proteins expressing lentiviruses. The infection efficiency was about 80∼90%. The cortical neurons were labeled with BrdUrd and stained 4 days after infection. The numbers under the figures are BrdUrd-positive percentage. E, 14-3-3 but not K50E blocks BrdUrd incorporation in neurons by SRPK2. Quantitative analysis of BrdUrd incorporation in cortical neurons by SRPK2 in the presence of wild-type 14-3-3β and K50E. Results are expressed as the mean ± S.D. from three independent experiments. p < 0.05 () and p < 0.001 (**), multiple comparison versus control group by analysis of variance with post hoc test.

14-3-3 interacts with Akt-phosphorylated SRPK2 and inhibits its nuclear translocation. A, SRPK2 binds to 14-3-3. HEK293 cells were transfected with Myc-SRPK2 and a variety of HA-14-3-3 isoforms. 14-3-3 was immunoprecipitated using HA antibody and was detected with Myc antibody (top panel). Expression of Myc-SRPK2 and HA-14-3-3s was verified (middle and bottom panels). IP, immunoprecipitate; NS, nonspecific. B, PI 3-kinase signaling regulates the interaction between endogenous SRPK2 and 14-3-3β. HEK293 cells were pretreated with PD98059, wortmannin, LY294002 for 30 min before EGF was introduced. Endogenous SRPK2 was immunoprecipitated and analyzed using anti-14-3-3β-specific antibody. The relative signal strength is labeled underneath of each blot. Equal amounts of SRPK2 were immunoprecipitated (second panel). Confirmation of Akt and SRPK2 phosphorylation status (third and fifth panels). C, SRPK2 binding to 14-3-3 is Akt phosphorylation-dependent. Myc-SRPK2 T492A and T492D were cotransfected into 293 cells with HA-14-3-3β, -σ, and -ϵ isoforms. 14-3-3 was immunoprecipitated using HA antibody and detected with Myc antibody. 14-3-3σ bound to both SRPK2 T492A and T492D proteins.14-3-3β and -ϵ isoforms selectively bound to SRPK2/T492D but not T492A (top panel). The expression of Myc-SRPK2 and HA-14-3-3s were examined (middle and bottom panels). D, 14-3-3 K50E mutant fails to interact with SRPK2. SRPK2 construct alone or together with HA-14-3-3 wild type or K50E mutant was transfected into HEK293 cells. 14-3-3 was immunoprecipitated using HA antibody and detected using Myc antibody (top panel). The expression of Myc-SRPK2, HA-14-3-3 wild type and mutant was examined (middle and bottom panels). E, Akt phosphorylation regulates SRPK2 subcellular location. Various HA-Akt constructs (WT, CA (constitutively active), KD) were cotransfected with SRPK2 wild type into HEK293 cells and monitored by immunofluorescent staining. F, effects of 14-3-3 on intracellular localization of SRPK2. Myc-SRPK2 wild type, T492A, and T492D were cotransfected with HA-14-3-3β into 293 cells, and indirect immunofluorescent staining was performed. Cotransfection with HA-14-3-3 inhibited SRPK2 T492D from translocating into the nucleus. The experiments described in E and F have been done three times. In total, 100 cells in E and 150 cells in F, were counted.

SRPK2 regulates cyclin D1 expression and its localization. A, SRPK2 mediates cyclin D1 promoter activity, which is regulated by Thr-492 phosphorylation. HEK293 cells were transfected various SRPK2 constructs and/or14-3-3β and 14-3-3 K50E mutant. Empty vector was used to normalize the same total amount of DNA in all experiments. Values are the means (±S.D.) of three independent experiments. SRPK2 T492A and KD mutants lost the activity, whereas T492D strongly elevated cyclin D1 promoter activity (top panel). Coexpression of14-3-3β with SRPK2 abolished SRPK2 activity (bottom left panel). The 14-3-3 K50E mutant displayed a negligible effect on SRPK2 activity (bottom right panel). B, SRPK2 mediates cyclin D1 nuclear location in cortical neurons. Cortical neurons were infected with various SRPK2 constructs. After 4 days, neurons were fixed with 3.7% paraformaldehyde and stained with anti-cyclin D1 and anti-MAP2 antibodies. SRPK2 wild type and T492D mutant strongly elevated cyclin D1 nuclear location (data represent the mean ± S.D. of three independent experiments; *, p < 0.05, multiple comparison versus control group by analysis of variance with post hoc test). C, SRPK2 controls cyclin D1 expression in cortical neurons. SRPK2 wild type, T492A, T492D, KD, and control lentivirus were co-infected with control or 14-3-3 adenovirus into cortical neurons. Reverse transcription-PCR was conducted. SRPK2 wild type and T492D mutant increased cyclin D1 expression (top left panel), whereas coexpressed 14-3-3 blocked it (top right panel). D, quantitative apoptotic assay. A variety of SRPK2 constructs were infected into cortical neurons and incubated for 4 days. The infection efficiency was about ∼80–90%. The numbers under the figures are MR(DEVD)2-positive percentage. The apoptotic neurons were analyzed with MR(DEVD)2, a fluorescent dye turning to red upon caspase-3 cleavage. E, 14-3-3 prevents SRPK2-provoked apoptosis. A variety of SRPK2 constructs were co-infected with 14-3-3 wild type and mutant into cortical neurons and incubated for 4 days. The apoptotic neurons were analyzed with MR(DEVD)2. Coexpression of14-3-3β with SRPK2 abolished its pro-apoptotic activity (left panel). The 14-3-3 mutant almost had no effect (right panel). p < 0.05 (*) and p < 0.001 (**) multiple comparison versus control group by analysis of variance with post hoc test.

Akt phosphorylates SRPK2 on Thr-492 in vitro and in vivo. A, diagram of SRPK2. SRPK2 has three putative Akt phosphorylation motifs (RXRXX(S/T)) as indicated (▾). B, C, and D, in vitro Akt kinase assay. Purified recombinant GST fusion proteins were incubated with active Akt and monitored by immunoblotting with anti-phospho-Akt substrate antibody. Fragment 439–688 was robustly phosphorylated, whereas other fragments were not. B, Akt kinase assay. In vitro kinase assay with various SRPK2 fragments and active Akt in the presence of [γ-³²P]ATP. The radiolabeled SRPK2 fragments were resolved on SDS-PAGE. Fragment 439–539 displayed the strongest phosphorylation activity. C and D, Thr-492 residue in SRPK2 is phosphorylated by Akt. Wild type (WT), T494A, and T634A but not T492A mutant were strongly phosphorylated (left panel). An equal amount of His proteins was employed (right panel). E, phospho-Thr-492 antibody selectively recognizes phosphorylated SRPK2. Although Thr-492 site was markedly phosphorylated in wild type, S494A, and T634A, no Thr-492 phosphorylation was detected in T492A or T492AS494A mutant (second panel). Expression of Myc-SRPK2 WT, mutants, and Akt was verified (third and fourth panels). Immunoblotting with phospho-Akt substrate antibody verified phospho-Thr-492 antibody results (top panel). HRP, horseradish peroxidase; IP, immunoprecipitate. F, PI 3-kinase inhibitors block SRPK2 phosphorylation on Thr-492. HEK293 cells were pretreated with 20 nm wortmannin, 10 μm LY294002, or 2 μm GF109203X for 30 min. The cells were then treated with EGF for 10 min. EGF stimulated SRPK2 phosphorylation, which was diminished by PI 3-kinase but not protein kinase C inhibitor pretreatment (top panel). Akt phosphorylation was verified (second panels). The relative signal strength of the blots was labeled underneath the blots. G, Akt is required for SRPK2 phosphorylation on Thr-492. HEK293 cells were infected with adenovirus expressing active myristoylated Akt or short hairpin RNA of Akt1. Myristoylated Akt provoked strong phosphorylation in endogenous SRPK2, and depletion of Akt abolished SRPK2 phosphorylation (top panel). Verification of Akt and SRPK2 expression (second and fourth panels). Akt phosphorylation was verified (third panel). H, glutamate triggers both Akt and SRPK2 phosphorylation in cortical neurons.