Journal: The Journal of Biological Chemistry

Article Title: Dual-specificity Tyrosine Phosphorylation-regulated Kinase 1A (Dyrk1A) Modulates Serine/Arginine-rich Protein 55 (SRp55)-promoted Tau Exon 10 Inclusion

doi: 10.1074/jbc.M112.355412

Figure Lengend Snippet: SRp55 promotes Tau exon 10 inclusion. A, SRp55 promoted Tau exon 10 inclusion in various cell lines. Cultured cells were co-transfected with pCEP4/SRp55 and Tau mini-gene pCI/SI9-LI10 for 48 h. The alternative splicing products of Tau exon 10 were measured by RT-PCR. The ratio of inclusion and exclusion of Tau exon 10 is presented under each lane; the ratio varies not only from cell line to cell line but also in different passages of the same cell line. B, knockdown of SRp55 suppressed Tau exon 10 inclusion. HEK-293FT cells were co-transfected with siRNA of human SRp55 and pCI/SI9-LI10 for 48 h, and then the splicing products of Tau exon 10 were measured by RT-PCR. The ratio of inclusion and exclusion of Tau exon 10 is presented as the mean ± S.D. **, p < 0.01. C, knockdown of SRp55 suppressed endogenous Tau exon 10 inclusion. SH-SY5Y cells were transfected with siRNA of human SRp55 and differentiated with 10 μm retinoid acid for 72 h, and then the splicing products of endogenous Tau exon 10 were measured by RT-PCR. The ratio of inclusion and exclusion of Tau exon 10 is presented as the mean ± S.D. **, p < 0.01.

Article Snippet: TRITC-conjugated goat anti-rabbit IgG, FITC-conjugated goat anti-mouse IgG, and siRNA of human SRp55 were from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Cell Culture, Transfection, Reverse Transcription Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: Dual-specificity Tyrosine Phosphorylation-regulated Kinase 1A (Dyrk1A) Modulates Serine/Arginine-rich Protein 55 (SRp55)-promoted Tau Exon 10 Inclusion

doi: 10.1074/jbc.M112.355412

Figure Lengend Snippet: SRp55 requires RS domain to promote Tau exon 10 inclusion. A, schematic presentation of SRp55 deletion mutants. B, deletion mutants of SRp55 did not promote Tau exon 10 inclusion. HEK-293FT cells were co-transfected with pCEP4/SRp55 and its deletion mutants and pCI/SI9-LI10 for 48 h. The splicing products of Tau exon 10 were measured by RT-PCR. The ratio of inclusion/exclusion of Tau exon 10 is presented as the mean ± S.D. **, p < 0.01 as compared with mock transfection. C, SRp55 and its deletion mutants were localized in the nucleus. SRp55 and its deletion mutants tagged with HA were overexpressed in HeLa cells for 48 h. The cells were fixed and immunostained by anti-HA, followed by Cy3 anti-rabbit IgG. TO-PRO-3 was used for nuclear staining.

Article Snippet: TRITC-conjugated goat anti-rabbit IgG, FITC-conjugated goat anti-mouse IgG, and siRNA of human SRp55 were from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Transfection, Reverse Transcription Polymerase Chain Reaction, Staining

Journal: The Journal of Biological Chemistry

Article Title: Dual-specificity Tyrosine Phosphorylation-regulated Kinase 1A (Dyrk1A) Modulates Serine/Arginine-rich Protein 55 (SRp55)-promoted Tau Exon 10 Inclusion

doi: 10.1074/jbc.M112.355412

Figure Lengend Snippet: SRp55 interacts with Dyrk1A. A, Dyrk1A was pulled down from rat brain extracts by GST-SRp55. GST-SRp55 or GST coupled onto glutathione-Sepharose was incubated with rat brain extracts. After washing, the bound proteins were subjected to Western blots using anti-GST and anti-Dyrk1A. B, Dyrk1A was co-immunoprecipitated by HA-SRp55 using anti-HA. SRp55 tagged with HA and Dyrk1A was co-expressed in HEK-293T cells for 48 h. The cell extracts were incubated with anti-HA prebound onto protein G beads. The bound proteins were subjected to Western blots using antibodies indicated under each blot. HC, heavy chain of IgG. C, Dyrk1A was co-immunoprecipitated by anti-SRp55. HEK-293FT cell extracts were incubated with anti-SRp55 prebound onto protein G beads. The bound proteins were subjected to Western blots using antibodies indicated under each blot. D, co-localization of SRp55 with Dyrk1A in the nucleus. HA-SRp55 and Dyrk1A were co-transfected into HepG2 cells. After a 48-h transfection, the cells were fixed and immunostained by anti-HA and anti-Dyrk1A and followed by TRITC anti-rabbit IgG and FITC anti-mouse IgG. Hoechst 33342 was used for nuclear staining.

Article Snippet: TRITC-conjugated goat anti-rabbit IgG, FITC-conjugated goat anti-mouse IgG, and siRNA of human SRp55 were from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Incubation, Western Blot, Immunoprecipitation, Transfection, Staining

Journal: The Journal of Biological Chemistry

Article Title: Dual-specificity Tyrosine Phosphorylation-regulated Kinase 1A (Dyrk1A) Modulates Serine/Arginine-rich Protein 55 (SRp55)-promoted Tau Exon 10 Inclusion

doi: 10.1074/jbc.M112.355412

Figure Lengend Snippet: SRp55 interacts with Dyrk1A through RRM domain. A, co-immunoprecipitation of Dyrk1A by SRp55 required RRM. HA-tagged SRp55FL or its deletion mutants and Dyrk1A were co-expressed in HEK-293FT cells for 48 h. The cell extracts were incubated with anti-HA prebound onto protein G beads. The bound proteins were subjected to Western blots using antibodies indicated under each blot. B, co-localization of SRp55FL and its deletion mutants with Dyrk1A in the nucleus. HA-SRp55 and Dyrk1A were co-transfected into HeLa cells. After 48 h of transfection, the cells were fixed and immunostained by anti-HA or anti-Dyrk1A, followed by Cy3 anti-rabbit IgG or Alexa 488 anti-mouse IgG. IP, immunoprecipitation.

Article Snippet: TRITC-conjugated goat anti-rabbit IgG, FITC-conjugated goat anti-mouse IgG, and siRNA of human SRp55 were from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Immunoprecipitation, Incubation, Western Blot, Transfection

Journal: The Journal of Biological Chemistry

Article Title: Dual-specificity Tyrosine Phosphorylation-regulated Kinase 1A (Dyrk1A) Modulates Serine/Arginine-rich Protein 55 (SRp55)-promoted Tau Exon 10 Inclusion

doi: 10.1074/jbc.M112.355412

Figure Lengend Snippet: Dyrk1A phosphorylates SRp55 in vitro and in cultured cells. A, SRp55 was phosphorylated by Dyrk1A in vitro. Recombinant GST-SRp55 or GST was incubated with various concentrations of Dyrk1A or Dyrk1A alone (last lane) in the presence of [32P]ATP at 30 °C for 30 min, and the reaction mixture was then separated by SDS-PAGE and visualized with Coomassie Blue staining or autoradiography (upper panel). Quantitation of 32P incorporation is shown in the lower panel. PLS, photostimulated luminescence. B and C, SRp55 was phosphorylated by Dyrk1A in cultured cells. HEK-293FT cells were transfected with pCEP4/SRp55-HA for 45 h and then treated with 10 μm Tg003 and/or 20 μm harmine (B) or co-transfected with pCEP4/SRp55-HA and pcDNA3/Dyrk1A or pcDNA/Dyrk1AK188R for 48 h (C). After 45 h of transfection, [32P]phosphate was added to label the phosphoproteins for 3 h, and then the cell lysates were subjected to immunoprecipitation with anti-HA. The immunoprecipitated HA-SRp55 was analyzed by autoradiography. Quantitation of 32P incorporation is shown in the lower panels of B and C after normalization by SRp55 level detected by Western blots developed with anti-HA. D, schematic of SRp55 deletion mutants. E, GST fused deletion mutants of SRp55 were incubated with Dyrk1A in vitro for 30 min at 30 °C. 32P incorporation into GST-SRp55 mutants was determined by autoradiography after the separation of the phosphorylation products by SDS-PAGE. Quantitation of the 32P incorporation after normalization by the protein level is shown in the right panel. The results represent the means ± S.D. *, p < 0.05; **, p < 0.01. Con, control.

Article Snippet: TRITC-conjugated goat anti-rabbit IgG, FITC-conjugated goat anti-mouse IgG, and siRNA of human SRp55 were from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: In Vitro, Cell Culture, Recombinant, Incubation, SDS Page, Staining, Autoradiography, Quantitation Assay, Transfection, Immunoprecipitation, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Dual-specificity Tyrosine Phosphorylation-regulated Kinase 1A (Dyrk1A) Modulates Serine/Arginine-rich Protein 55 (SRp55)-promoted Tau Exon 10 Inclusion

doi: 10.1074/jbc.M112.355412

Figure Lengend Snippet: Dyrk1A suppresses SRp55-promoted Tau exon 10 inclusion. A, effects of Dyrk1A or dominant negative Dyrk1A, Dyrk1AK188R, on SRp55-mediated Tau exon 10 splicing. Dyrk1A or Dyrk1AK188R was co-expressed with SRp55 in pCI/SI9-LI10 transfected HEK-293FT cells for 48 h. The alternative splicing products of Tau exon 10 were measured by RT-PCR. The ratio of inclusion and exclusion of Tau exon 10 is presented in the lower panel. B, syngeneic effect of Dyrk1A and siRNA of SRp55 (siSRp55) on Tau exon 10 splicing. Dyrk1A or Dyrk1AK188R was co-expressed with siRNA of human SRp55 in pCI/SI9-LI10 transfected HEK-293FT cells for 48 h. The alternative splicing products of Tau exon were measured with RT-PCR. The ratio of inclusion and exclusion of Tau exon 10 is presented in the lower panel. C, mutation of SRp55 at Ser-303 to Ala enhanced its promotion in Tau exon 10 inclusion. Wild type SRp55 (SRp55WT) or its mutants (SRp55S303A, SRp55S316A, and SRp55S280A) were expressed in pCI/SI9-LI10-transfected HEK-293FT cells for 48 h. The alternative splicing products of Tau exon 10 were measured by RT-PCR. The ratio of inclusion and exclusion of Tau exon 10 is presented in the lower panel. D, Dyrk1A or Dyrk1AK188R differently affected the subcellular localization of SRp55. HA-SRp55 and Dyrk1A or Dyrk1AK188R were co-transfected into HeLa cells. After 48 h transfection, the cells were fixed and double-immunostained by anti-HA and anti-Dyrk1A, followed by Cy3 anti-rabbit IgG and Alexa 488 anti-mouse IgG. The results represent the means ± S.D. *, p < 0.05; **, p < 0.01. Con, control.

Article Snippet: TRITC-conjugated goat anti-rabbit IgG, FITC-conjugated goat anti-mouse IgG, and siRNA of human SRp55 were from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Dominant Negative Mutation, Transfection, Reverse Transcription Polymerase Chain Reaction, Mutagenesis