Evidence for disulfide bonds in SR Protein Kinase 1 (SRPK1) that are required for activity and nuclear localization

Abstract

Serine/arginine protein kinases (SRPKs) phosphorylate Arg/Ser dipeptide-containing proteins that play crucial roles in a broad spectrum of basic cellular processes. The existence of a large internal spacer sequence that separates the bipartite kinase catalytic core is a unique structural feature of SRPKs. Previous structural studies on a catalytically active fragment of SRPK1, which lacks the main part of the spacer domain, revealed that SRPK1 remains in an active state without any post-translational modifications or specific intra-protein interactions, while the spacer domain is depicted as a loop structure, outside the kinase core. Using systematic mutagenesis we now provide evidence that replacement of any individual cysteine residue in the spacer, apart from Cys414, or in its proximal flaking ends of the two kinase catalytic domains has an impact on kinase activity. Furthermore, the cysteine residues are critical for nuclear translocation of SRPK1 in response to genotoxic stress and SRPK1-dependent splicing of a reporter gene. While replacement of Cys207, Cys502 and Cys539 of the catalytic domains is predicted to distort the kinase active structure, our findings suggest that Cys356, Cys386, Cys427 and Cys455 of the spacer domain and Cys188 of the first catalytic domain are engaged in disulfide bridging. We propose that such a network of intramolecular disulfide bonds mediates the bending of the spacer region thus allowing the proximal positioning of the two catalytic subunits which is a prerequisite for SRPK1 activity.

Fig 1. Structural features of SRPK1.

(A) Domain organization of SRPK1 as presented in ref. 8. The N- and C-terminal conserved kinase subdomains are colored purple and magenta, respectively. The N- and C-terminal spacer regions are colored yellow and green, respectively, while the remaining spacer domain is colored gray. SRPK1(FL): full-length SRPK1; SRPK1 ΔNS1: the crystallized fragment of SRPK1. (B) Overall structure of SRPK1 ΔNS1 as presented in refs 8 and 16. The coloring of the domains is as in (A). Note that the spacer domain is depicted as a loop outside the kinase core.

Fig 2. Effect of reducing agents on SRPK1 electrophoretic mobility.

(A) Extracts from K562, MEL, MOLT-4, JM1, 293T and HeLa cells were analyzed under non-reducing conditions (i.e., without DTT) or reducing conditions (in the presence of 90 mM DTT) on 10% SDS-polyacrylamide gels. The proteins were then transferred to nitrocellulose, and SRPK1 was detected with an anti-SRPK1 monoclonal antibody. (B) GST-SRPK1 was analyzed in the absence or in the presence of 90 mM DTT on 10% SDS-polyacrylamide gels and stained with Coomassie Blue.

Fig 3. Effect of reducing agents on SRPK1 activity.

(A) GST-SRPK1 was incubated with 0, 150, 250 and 350 mM DTT at room temperature for 14 h, then analyzed on 10% SDS-PAGE under reducing conditions and stained with Coomassie Blue. No degradation of GST-SRPK1 was observed following its overnight incubation with DTT. (B) GST-SRPK1 was incubated with 0, 150, 250 and 350 mM DTT at room temperature for 2, 5 and 14 h respectively and then used in kinase assays with GST-LBRNT(62–92) as substrate. Phosphorylated proteins were separated by 12% SDS-PAGE, stained with Coomassie Blue and autoradiographed. Only the relevant part of the autorad corresponding to phosphorylated GST-LBRNt(62–92) is shown. (C) Signals were quantified by excising the radioactive bands from the gel and scintillation counting. Measurements were done in triplicate. Error bars designate standard error.

Fig 4. Evaluation of disulfide bond(s) location in the SRPK1 molecule.

(A) Lysates from 293T cells overexpressing FLAG-SRPK1 or FLAG-SRPK1Δspacer were analyzed by SDS-PAGE, under non-reducing or reducing conditions, and Western blotting. SRPK1 was detected using the M5 anti-FLAG monoclonal antibody. (B) Amino acid sequence of SRPK1. The two catalytic domains are highlighted by grey shadows. All cysteines are marked in red; underlined cysteines were mutated to alanine or glycine.

Fig 5. Influence of cysteine mutations on SRPK1 activity.

Phosphorylation of GST-LBRNt(62–92) by 0.5 μg wild-type GST-SRPK1, GST-SRPK1 188A, GST-SRPK1 207G, GST-SRPK1 356G, GST-SRPK1 386G, GST-SRPK1 414G, GST-SRPK1 427G, GST-SRPK1 455G, GST-SRPK1 502G and GST-SRPK1 539A, respectively. (A) On top of the figure we show a Coomassie Blue staining of the recombinant GST-SRPK1 proteins (1.5 μg of each) used in the phosphorylation assays. (B) The samples were analyzed by SDS-PAGE on 12% gels, stained with Coomassie Blue and autoradiographed. The radioactive bands corresponding to GST-LBRNt(62–92) were excised and scintillation counted. Data represent the means ± SE of three independent experiments.

Fig 6. Effect of reducing agents on SRPK1 cysteine mutants electrophoretic mobility.

Lysates from 293T cells transfected with wild-type SRPK1 and its mutant forms were analyzed under non-reducing conditions (i.e., without DTT) or reducing conditions (in the presence of 90 mM DTT) on 10% SDS-polyacrylamide gels. The proteins were then transferred to nitrocellulose and epitope-tagged wild-type or mutant SRPK1 was detected with the M5 anti-FLAG monoclonal antibody.

Fig 7. Nuclear translocation of SRPK1 and derived cysteine mutants in response to genotoxic stress.

(A) Fluorescent pattern of wild-type FLAG-SRPK1 and mutant FLAG-SRPK1 188A, SRPK1 356G, SRPK1 386G, SRPK1 427G and SRPK1 455G in 5-FU-treated HeLa cells. SRPKs were detected using the M5 anti-FLAG monoclonal antibody, while nuclei were stained with propidium iodide (PI). Scale bar, 10 μm. (B) The ratio of average fluorescence intensity in the cell nucleus versus average intensity in the cell cytoplasm (N/C ratio) was quantified using ImageJ software. Each column represents the means ± SE of measurements from 20–30 cells.

Fig 8. Impact of overexpressed wild-type and mutant SRPK1 on insulin reporter splicing.

(A) Schematic representation of the insulin expression construct containing 2 introns and the respective flanking exons I1, I2 and I3. SV40 promoter/enhancer regions and insulin transcription terminators are shown in black. Primers used for PCR are indicated. (B) HeLa cells were transfected with increasing concentrations (0, 0.25, 0.3, 0.35, 0.5 and 0.7 μg) of plasmids expressing wild-type and mutant SRPK1 along with 1.65 μg of the reporter gene. Cells were treated with 50 μg/ml 5-FU for 24 h, prior harvesting. RNA was isolated 48 h following transfection, and RT-PCR was carried out. The spliced and unspliced products have a size of 0.3 and 1 kb respectively. (C) The ratio of the upper and lower bands was quantified using ImageJ software. Data represent the means ± SE of two independent experiments. (D) Western blots showing the levels of wild-type and mutated SRPK1.