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Figure 1. DYRK1A phosphorylates SF3b1 within the TP-domain. A, Phosphorylation of His6-SF3b1304–493 by DYRK1A. – Data from a representative experiment were evaluated by linear regression analysis of the Lineweaver-Burke plot. B, Schematic representation of human SF3b1 and the recombinant fusion proteins used in this study. The carboxyterminal part of SF3b1 consists of 22 nonidentical repeats related to the regulatory subunit A of protein phosphatase 2A (PP2A-like) . Numbers indicate amino acids. TP-rich, region rich in Thr-Pro dipeptides; His, hexahistidine tag.
Figure 2. Phosphorylation of SF3b1 by DYRK1A in COS-7 cells. COS-7 cells were transiently transfected with expression plasmids for GFP-SF3b1-NT and the protein kinases DYRK1A or CLK3. Two days after transfection, cells were lysed under denaturing conditions and the recombinant proteins were immunoprecipitated with polyclonal GFP antiserum (A, B, C) or monoclonal SF3b1 antibody (D). Immunoprecipitates were subjected to Western blot analysis with antibodies specific for phosphoThr-Pro (anti pTP), GFP, or SF3b1. A, COS-7 cells were transfected with 1 μg of pEGFP-SF3b1-NT and increasing amounts of pEGFP-DYRK1A (0 μg, 0.2 μg, 1 μg and 2 μg of DNA per 6-cm plate). B, Cells were transfected with pEGFP-SF3b1-NT and either pEGFP-DYRK1A-K188R, pEGFP-DYRK1A (WT), pEGFP (Co), or pEGFP-CLK3. C, Cells were transfected pEGFP-SF3b1-NT and either pEGFP-DYRK1A (DIA) or pEGFP (Co). Cells from one plate were lysed in buffer lacking phosphatase inhibitors, and the lysate was incubated for 1 h at 37°C with 2000 u of calf intestinal phosphatase (CIP) before immunoprecipitation. D, Cells were transfected pEGFP-SF3b1-NT (left lane) or were not transfected. Migration of the immunoprecipitating antibody is indicated (IgG). The asterisk marks a slowly migrating form of SF3b1-NT (see text).
Figure 3. Kinetic analysis of SF3b1 phosphorylation by DYRK1A and CDK2. SF3b1-NT-His6 was phosphorylated with GST-DYRK1A-ΔC or cyclin E/CDK2 at two different substrate concentrations (0.7 and 7 μM). Phosphate incorporation was measured at different time points (2, 4 and 8 minutes). The slope of the straight line reflects the velocity of phosphorylation at the different substrate concentrations. The calculated Km values are indicated. The experiment was repeated with similar results.
Figure 4. DYRK1A, but not cyclin E/CDK2 phosphorylates SF3b1 in vitro on phosphopeptides comigrating with the endogenous phosphopeptides from COS-7 cells. SF3b1-NT-His6 was labelled in vitro by GST-DYRK1A-ΔC (A) or cyclin E/CDK2 (B). GFP-SF3b1-NT was immunoprecipitated from COS-7 cells after metabolic labelling with 32PO4 (C). Recombinant proteins were subjected to two-dimensional phosphopeptide mapping. To verify that spots detected in different experiments are identical, samples were mixed and analysed on the same plate (A+B, A+C, B+C). Numbers label those spots that were both present in panels A and C. The panels show only the relevant area of the plates.
Figure 5. SF3b1 is phosphorylated at Thr434 by endogenous kinases in COS-7 cells and in vitro by DYRK1A. A, SF3b1-NT-His6 (wt) and point mutants of Thr273 (T273A) or Thr434 (T434A) were phosphorylated by GST-DYRK1A-ΔC in vitro. B, GFP-SF3b1-NT (wt) or the alanine mutant of Thr434 (T434A) were expressed in COS-7 cells, metabolically labelled with 32P and purified by immunoprecipitation with a GFP-specific antiserum. Phosphopeptide maps were generated as described above. Arrows point to phosphopeptides that are absent in the mutants. X marks a spot apparently superimposing spot1 (see text).
Figure 6. Overexpression of DYRK1A increases the phosphorylation of endogenous sites in COS-7 cells. GFP-SF3b1-NT was expressed in COS-7 cells either alone (A) or coexpressed with GFP-DYRK1A (B) and metabolically labelled with 32P. SF3b1 fusion proteins were subjected to phosphopeptide mapping as above. Intensities of peptide maps A and B were adjusted to spot X/1.
Figure 7. Detection of Thr434 phosphorylation with the help of a phosphorylation site-specific antibody. A, Verification of antibody specificity. – Recombinant SF3b1-NT-His6 (WT) or the T434A point mutant thereof were phosphorylated in vitro by GST-DYRK1A-ΔC or incubated under the same conditions in the absence of GST-DYRK1A-ΔC. The indicated amounts of SF3b1-NT-His6 (500 ng, 100 ng, 20 ng, 4 ng) were separated by SDS-PAGE and subjected to Western blot analysis with antibodies specific for pT434, pThrPro (pTP), and SF3b1. B, Phosphorylation of Thr434 in vivo. – COS-7 cells seeded in 6-well plates were co-transfected with expression plasmids for wild type GFP-SF3b1-NT (WT) or the T434A mutant (0.6 μg/well) and either GFP (Co) or GFP fusion constructs (0.1 μg, 0.2 μg or 0.4 μg/well) of the indicated protein kinases (wild type DYRK1A or the K188R point mutant, DYRK1B, CLK3, HIPK2). The total amount of transfected DNA was kept constant by addition of vector DNA where appropriate. Two days after transfection, total cellular lysates were prepared and subjected to Western blot analysis with antibodies specific for pT434, pThrPro (pTP), SF3b1, or GFP. A shorter exposure of the top panel is shown below because the most intense signals exceeded the linear range of the detection camera. A slowly migrating form of SF3b1-NT is marked by an asterisk (*). C, Nuclei were purified from COS-7 cells transfected with expression plasmids for wild type GFP-DYRK1A (1 μg, 2 μg or 4 μg/6-cm plate) or the point mutant K188R. Nuclear proteins were subjected to Western blot analysis with the indicated antibodies.
Figure 8. Downregulation of endogenous DYRK1A reduces phosphorylation of Thr434 in SF3b1. A, Test of shRNA vectors for DYRK1A knockdown. – HEK293T cells seeded in 6-well plates were co-transfected with expression plasmids for GFP-DYRK1A (0.2 μg/well) and either empty vector (Ctrl) or plasmids expressing to different small hairpin RNAs directed against DYRK1A (673 or 2060; 0.8 μg DNA/well). Two days after transfection, nuclear extracts were prepared and subjected to Western blot analysis with a DYRK1A-specific antibody. B, Effect of DYRK1A knockdown on Thr434 phosphorylation. – HEK293T cells or HepG2 cells were co-transfected with the expression plasmid for GFP-SF3b1-NT (0.5 μg/well) and the pSUPER constructs (0.8 μg/well). Total cellular lysates were subjected to Western blot analysis with antibodies specific for pThr434 or GFP. The asterisks mark unspecific bands (*).
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