1. Volume 7, Issue 2, Pages 283-292 (February 2001)
A DNA Damage–Induced p53 Serine 392 Kinase Complex Contains CK2, hSpt16, and SSRP1 
David M Keller, Xiaoya Zeng, Yun Wang, Qing Hong Zhang, Mini Kapoor, Hongjun Shu, Richard Goodman, Guillermina Lozano, Yingming Zhao, Hua Lu 
Molecular Cell 
Volume 7, Issue 2, Pages (February 2001)
DOI: /S (01)

2. Figure 1 Ser-392 Is Evolutionarily Conserved
Sequence alignment of carboxy-terminal amino acids of p53, showing that Ser-392 of human p53 is conserved through mammals, amphibians, bony fishes, and birds. Numbers in parentheses refer to the last amino acid residue of p53 for each species
Molecular Cell 2001 7, DOI: ( /S (01) )

3. Figure 2 p53 Ser-392 Kinase Activity Is Stimulated by UV
(A) p53 Ser-392 kinase activity is present in the 0.5 M KCl fraction. F9 cells were treated with 20 J/m2 UVC for 8 hr and harvested for nuclear extract preparation. Extracts (from 109 cells) were loaded onto a phosphocellulose (P11) column, and fractions were collected at the indicated KCl concentrations. In vitro cold kinase assays were performed using 1 μg of the indicated fraction and unlabeled ATP and 100 ng p53 C-terminal peptide (aa 311–393) as substrates. Phosphorylated products were detected by Western blot with a phospho-specific Ser-392 antibody (top panel), while WB with PAb421 was used to show equal loading of substrates (bottom panel). Endogenous murine p53 is denoted by mp53, and the phosphorylated form is denoted by pi-mp53.
(B) p53 Ser-392 kinase activity in the P M KCl fraction is stimulated after UV irradiation. F9 nuclear extracts from both UV-treated and untreated cells were run on a P11 column. A cold kinase assay using the various fractions (1 μg protein) was performed identically as in (A)
Molecular Cell 2001 7, DOI: ( /S (01) )

4. Figure 3 Purification Scheme of p53 Ser-392 Kinase
(A) p53 Ser-392 kinase purification procedure. To scale up the kinase purification, HeLa cells (1010 cells) were employed. p53 Ser-392 kinase activity was monitored through each fractionation step by kinase assays as described in Experimental Procedures. See the legend for Table 1 for details.
(B) Anti-Ser-392 antibodies selectively detected p53 Ser-392 kinase activity in the MonoQ fractions. Column fractions (3 μl) were used in a radioactive kinase assay (top panel) and a WB kinase assay using the anti-Ser-392 antibody (middle panel). C-terminal p53 311–393 (100 ng) detected by Pab421 was used as a substrate in each reaction (bottom panel)
Molecular Cell 2001 7, DOI: ( /S (01) )

5. Figure 4
p53 Ser-392 Kinase Is Associated with a High Molecular Weight Protein Complex
(A) Chromatograph of molecular weight standards loaded on a Superdex 200 column.
(B) p53 Ser-392 kinase activity elutes at a native molecular weight of 700 kDa as judged by both a radioactive kinase assay using [γ32P]ATP (top) and a Western blot kinase assay (middle). Column fractions (3 μl) were used in each lane with 100 ng C-terminal p53 311–393 (detected by PAb421 in bottom panel).
(C) Fractions 9–11 (7.5 μg protein) were pooled and subjected to SDS–PAGE and stained with collodial blue, followed by digestion for mass spectrometry. Corresponding proteins identified by MS analysis were labeled on the right of the panel. Asterisk denotes the possible contaminating protein, as it did not show in a separate purification
Molecular Cell 2001 7, DOI: ( /S (01) )

6. Figure 5 hSpt16 and SSRP1 Associate with CK2
(A) Representative column from the purification (MonoQ) showing coelution of CK2α′ and β subunits with hSpt16 and SSRP1. Column fractions (15 μl) were run on SDS–PAGE for Western blot with the indicated antibodies. A Western blot kinase assay was performed as in the bottom panel and demonstrates that p53 Ser-392 kinase activity peak coelutes with these proteins.
(B) Immunoprecipitation (IP) of CK2 and p53 Ser-392 kinase activity with hSpt16 and SSRP1. IPs were done using either normal rabbit IgG, αhSpt16, or αSSRP1 antibodies (1 μg each) to pull down the members of the complex as indicated from the P M KCl fraction. These same IPs were used in a Western blot kinase assay to pull down p53 Ser-392 kinase activity using 311–393 as a substrate. Asterisk indicates IgG heavy chain
Molecular Cell 2001 7, DOI: ( /S (01) )

7. Figure 6
Comparison of the Purified p53 Ser-392 Kinase with CK2 Alone Reveals Different Substrate Specificities
(A) Both the purified p53 Ser-392 kinase complex and CK2 alone phosphorylate C-terminal p53 (aa 311–393, 100 ng) equally well in an in vitro radioactive kinase assay, but p53 Ser-392 kinase does not efficiently phosphorylate casein (1 μg), an N-terminal deleted, GST-fusion MDM2 (GΔMDM2, 50 ng), or histone H1 (100 ng). CK2 (0.5 U) (Promega) and 300 ng of the purified p53 Ser-392 kinase (pool of fractions 9–11) were used in kinase reactions.
(B) The substrate specificity of the p53 Ser-392 kinase complex is reconstituted in vitro. A radioactive kinase assay was done as above using casein and 311–393, along with his-SSRP1 (4 and 100 ng) and/or Flag-hSpt16 (4 and 100 ng). rhSpt16 and rSSRP indicate recombinant proteins.
(C) The substrate switching as seen above is not due to a nonspecific protein–protein interaction because N-terminal SSRP1 (aa 1–240) does not affect casein phosphorylation. A radioactive kinase assay was done as above with 10 ng ΔNSSRP1, 25 ng his-SSRP1, 25 ng Flag-hSpt16, 100 ng his-p53, and 1 μg casein. The asterisk denotes the migration of p53 in lanes 12 and 13 changes due to cleavage of the histidine tag by contaminating thrombin in the ΔNSSRP1 preparation
Molecular Cell 2001 7, DOI: ( /S (01) )

8. Figure 7
The Purified p53 Ser-392 Kinase Complex Enhances p53's Sequence-Specific DNA Binding and Transcriptional Activity In Vitro
(A) Cold kinase assays were performed, followed by an electro-mobility shift assay (EMSA) using full-length his-p53 (50 ng) that was phosphorylated by increasing amounts of purified p53 Ser-392 kinase as indicated. Phosphorylated his-p53 was incubated with a 3′ end-labeled DNA probe harboring two copies of the p53RE sequence derived from the p21waf1/cip1 promoter. S392K indicates the purified p53 Ser-392 kinase.
(B) The DNA/protein complex formed was specific to p53. The same kinase-EMSA assay was conducted as in (A). Two unlabeled oligomers were used, poly(dIdC) (100 ng, lane 4) and p53RE (100 ng, lane 5). Also, antibodies (0.1 μg each) were used that were specific against p53 (PAb 1801, lane 6) and CK2α′ subunit (lane 7).
(C) The p53 Ser-392 kinase stimulates transcription mediated by wild-type (WT) but not mutant (MT) p53. A reconstituted transcription system was used containing purified RNA pol II (50 ng), general transcription factors, and a DNA template containing a p53RE motif upstream from the TATA box derived from adenovirus major late promoter. One hundred nanograms of WT or MT p53 and 4 μl fraction 12 (S392K, Ser-392 kinase) from Superdex 200 column were preincubated in the presence or absence of ATP prior to being added into the transcription reaction. RNA transcripts were detected by autoradiography (top), and Ser-392 phosphorylation of p53 under the same reaction condition was detected by Western blot using the anti-Ser-392 antibody (bottom).
(D) Ser-392 is critical for p53-dependent transcription in vivo following UV treatment. H1299 cells (105/well, six-well plate) were transfected with plasmids (making up to 1 μg DNA in total with a control vector) encoding no gene, murine wild-type p53, or S389A mutant (400 ng), together with a luciferase reporter plasmid (100 ng) driven by the p53RE motif derived from the p21 promoter and a β-galactosidase reporter plasmid (200 ng) driven by the CMV promoter. Forty-eight hours posttransfection, cells were UV-treated for 0, 1, or 2 hr and then harvested for luciferase and β-gal assays. Luciferase activity was normalized by the internal β-gal activity. Fold increase in luciferase activity was calculated and is presented in the graph (each column is from three independent assays, and bars show standard deviation). The bottom panels show p53 expression as detected by immunoprecipitation with PAb 421 and Western blot with either αSer-392 or αp53 polyclonal antibodies
Molecular Cell 2001 7, DOI: ( /S (01) )