PMC

A, a schematic shows the GST fusion C terminus of SSRP1 (bottom of the panel). GST-C-SSRP1 was subjected to SDS-PAGE and stained with Coomassie Brilliant Blue (Coom). B, the purified GST-C-SSRP1 fragment bound to DNA. The EMSA was conducted using increasing amounts of GST-C-SSRP1 (50, 100, and 200 ng). C, phosphorylation of GST-C-SSRP1 by CK2 inhibited its DNA-binding activity. The same kinase/EMSA assay was conducted as described in the legend to using 100 ng of GST-C-SSRP1.

A, a schematic shows the positions of S510A, S657A, and S688A on GST-C-SSRP1 (upper panel). GST-C-SSRP1, GST-C-SSRP1-S3A, and GST-C-SSRP1-S2A (S657A/ S688A) were subjected to SDS-PAGE and stained with Coomassie Brilliant Blue (Coom, lower panel). The amounts of proteins used are indicated on top. B, comparison between GST-C-SSRP1, GST-C-SSRP1-S3A, and GST-C-SSRP1-S2A in EMSA. 25, 50, and 100 ng of GST-C-SSRP1, GST-C-SSRP1-S3A, or GST-C-SSRP1-S2A were used in the EMSA. C, CD analysis of GST-C-SSRP1, GST-C-SSRP1-S3A, and GST-C-SSRP1-S2A. CD spectra of the wild type, triple, and double mutant GST-C-SSRP1 proteins were recorded in the wavelength range of 190–260 nm.

A, phosphorylation of GST-C-SSRP1 by CK2 inhibits its DNA-binding activity. The same kinase/EMSA assay was conducted as described in the legend to , and 50 ng of GST-C-SSRP1 was used. B, phosphorylation of GST-C-SSRP1-S3A by CK2 failed to inhibit its DNA-binding ability. The kinase/EMSA assay on the triple mutant of GST-C-SSRP1 was conducted under the same conditions as those for wild type GST-C-SSRP1 in A. C, phosphorylation of GST-C-SSRP1-S2A by CK2 still inhibited its DNA-binding ability. The kinase/EMSA assay on the double mutant of GST-C-SSRP1 was conducted under the same conditions as those for wild type GST-C-SSRP1 in A. D, GST-C-SSRP1-S3A is partially phosphorylated by CK2. In vitro radioactive kinase assays were carried out under the same conditions as used for the EMSA reactions in A and B. The ³²P-labeled SSRP1 fragment signals were quantified and plotted in a graph shown in E using imaging software.

A panel of designed SSRP1 10mer peptides was spotted on a nitrocellulose membrane. In vitro CK2 kinase reactions were conducted on the membrane using [γ-³²P]ATP. After extensive washing, radioactive signals were detected by autoradiography. The asterisks denote potential phosphorylation sites, and the underlined asterisks indicate the identified CK2 sites. Only serines or threonines and their corresponding mutant alanines are shown here. The corresponding positions of these amino acids in the C terminus of SSRP1 are approximately marked on the schematic at the bottom. The sequences for peptide pairs 10, 15, and 19 are shown in corresponding positions, and CK2 target sites are underlined.

A, phosphorylation of SSRP1 in cells was induced by UV irradiation. Hela cells were labeled in vivo by ³²P-labeled inorganic phosphate 3 h after irradiation by UVB or γ rays, followed by an additional 3 h of incubation before being harvested for immunoprecipitation (IP). 200 µg of protein was used for the immunoprecipitation. Immunoprecipitates were subjected to SDS-PAGE and labeled SSRP1 was detected by autoradiography. B, phosphoamino acid analysis was carried out as described under “Experimental Procedures,” revealing that both serines and threonines are phosphorylated on SSRP1. Samples (1720 cycles/min) were loaded onto a cellulose thin-layer chromatography plate, which was subjected to two-dimensional electrophoresis and autoradiography. Only the UVB irradiation-treated sample is shown in the figure, although nontreated or γ-treated samples show the same pattern. WB, Western blot.

A, phosphorylation of FACT by CK2 inhibited its DNA-binding ability. The FACT complex was purified from FLAG-SSRP1-expressing HEK293 cells, as described under “Experimental Procedures.” The 0.5 m fraction, which contains the FACT complex, is shown in the left panel. 4 µl of this fraction was used in an in vitro kinase assay followed by EMSA. 10-µl CK2 kinase reactions were carried out with the FACT complex for 1 h at 30 °C and then incubated with 3′ end-labeled DNA (87 bp) probes for 20–25 min. Also, antibodies specific against SSRP1 (lane 6) and Spt16 (lane 7) were used. B, the purified recombinant His-SSRP1 protein bound to DNA. The purity of the recombinant His-SSRP1 is shown in the right panel. The EMSA was done using 250 and 500 ng of purified His-SSRP1. The His·SSRP1·DNA complex was supershifted by an anti-SSRP1 antibody (lanes 4 and 5). C, phosphorylation of SSRP1 by CK2 inhibited its DNA-binding activity. The same kinase/EMSA assay was conducted as in A, except that 50 ng of His-SSRP1 was used. D, the purified recombinant His-SSRP1 was phosphorylated by CK2. In vitro kinase reactions were conducted for 30 min using [γ-³²P]ATP and 50 ng of His-SSRP1 as substrates. Coom, Coomassie.