p53 Serine 392 Phosphorylation Increases after UV through Induction of the Assembly of the CK2·hSPT16·SSRP1 Complex
2002; Elsevier BV; Volume: 277; Issue: 51 Linguagem: Inglês
10.1074/jbc.m209820200
ISSN1083-351X
Autores Tópico(s)Epigenetics and DNA Methylation
ResumoPreviously, we purified a UV-responsive p53 serine 392 kinase from F9 and HeLa cells and found that its activity is attributed to a high molecular weight protein complex containing the protein kinase CK2, along with the chromatin-associated factors hSPT16 and SSRP1. Here we determine that these proteins interact in vitro and in cells via non-overlapping domains and provide evidence consistent with the idea that hSPT16 and SSRP1 change the conformation of CK2 upon binding such that it specifically targets p53 over other substrates. Also, UV irradiation apparently induces the association of the complex, thereby increasing the specificity of CK2 for p53 at the expense of other cellular CK2 substrates and leading to an overall increase in p53 serine 392 phosphorylation. Previously, we purified a UV-responsive p53 serine 392 kinase from F9 and HeLa cells and found that its activity is attributed to a high molecular weight protein complex containing the protein kinase CK2, along with the chromatin-associated factors hSPT16 and SSRP1. Here we determine that these proteins interact in vitro and in cells via non-overlapping domains and provide evidence consistent with the idea that hSPT16 and SSRP1 change the conformation of CK2 upon binding such that it specifically targets p53 over other substrates. Also, UV irradiation apparently induces the association of the complex, thereby increasing the specificity of CK2 for p53 at the expense of other cellular CK2 substrates and leading to an overall increase in p53 serine 392 phosphorylation. The tumor suppressor protein p53 is a highly connected cellular sensor of DNA damage and aberrant cell growth and serves a protective role by inhibiting the cell cycle or inducing apoptosis once damage occurs (1Vogelstein B. Lane D. Levine A.J. Nature. 2000; 408: 307-310Crossref PubMed Scopus (5905) Google Scholar). Cellular insults that activate p53 include DNA-damaging agents such as radiation and chemical mutagens (2Tishler R.B. Calderwood S.K. Coleman C.N. Price B.D. Cancer Res. 1993; 53: 2212-2216PubMed Google Scholar, 3Kapoor M. Lozano G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2834-2837Crossref PubMed Scopus (195) Google Scholar, 4Lu H. Taya Y. Ikeda M. Levine A.J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6399-6402Crossref PubMed Scopus (152) Google Scholar, 5Hirao A. Kong Y.Y. Matsuoka S. Wakeham A. Ruland J. Yoshida H. Liu D. Elledge S.J. Mak T.W. Science. 2000; 287: 1824-1827Crossref PubMed Scopus (1063) Google Scholar), hypoxia (6Graeber T.G. Peterson J.F. Tsai M. 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Science. 1999; 286: 2528-2531Crossref PubMed Scopus (775) Google Scholar) prevents binding of the MDM2 oncoprotein, an E3 1The abbreviations used are: E3, ubiquitin-protein ligase; CHD1, chromodomain-helicase-DNA binding; CK2, casein kinase 2; DTT, dithiothreitol; FACT, facilitates chromatin transcription; GST, glutathione S-transferase; SSRP1, structure-specific recognition protein; hSPT16, human ortholog of yeast suppressor of Ty insertion mutations; IP, immunoprecipitation; MDM2, a protein encoded by a gene amplified in mouse double-minute chromosome; P11, phosphocellulose; WB, Western blot; WT, wild-type; aa, amino acid(s) ubiquitin ligase that targets p53 for degradation by the proteasome pathway (22Momand J. Zambetti G.P. Olson D.C. George D. Levine A.J. Cell. 1992; 69: 1237-1245Abstract Full Text PDF PubMed Scopus (2850) Google Scholar, 23Honda R. Tanaka H. Yasuda H. FEBS Lett. 1997; 420: 25-27Crossref PubMed Scopus (1627) Google Scholar, 24Fuchs S.Y. Adler V. Buschmann T. Wu X. Ronai Z. Oncogene. 1998; 17: 2543-2547Crossref PubMed Scopus (213) Google Scholar). On the C terminus, phosphorylation of Ser-392 (corresponding to murine Ser-389, for simplicity, Ser-392 will be used) enhances DNA sequence-specific binding and transcription activityin vitro (25Hupp T.R. Meek D.W. Midgley C.A. Lane D.P. Cell. 1992; 71: 875-886Abstract Full Text PDF PubMed Scopus (887) Google Scholar, 26Keller D.M. Zeng X. Wang Y. Zhang Q.H. Kapoor M. Shu H. Goodman R. Lozano G. Zhao Y. Lu H. Mol. Cell. 2001; 7: 283-292Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar), possibly by stabilizing p53 tetramerization (27Sakaguchi K. Sakamoto H. Lewis M.S. Anderson C.W. Erickson J.W. Appella E. Xie D. Biochemistry. 1997; 36: 10117-10124Crossref PubMed Scopus (230) Google Scholar). In cells, the importance of Ser-392 phosphorylation for p53 function appears to be situation-specific. For example, overexpression of a p53 Ser-392 > Ala mutant suppressed cell growth equal to wild-type p53 in human osteosarcoma SAOS2 cells, but instead impaired the ability of p53 to suppress ras-mediated transformation in rat embryonic fibroblasts (28Crook T. Marston N.J. Sara E.A. Vousden K.H. Cell. 1994; 79: 817-827Abstract Full Text PDF PubMed Scopus (224) Google Scholar). Also, transient transfection of p53 with a Ser-392 > Glu substitution, but not with six other phosphorylation mutants, constitutively activated p53 as a transcription factor in NIH 3T3 mouse fibroblasts after cell growth arrest by contact inhibition (29Hao M. Lowy A.M. Kapoor M. Deffie A. Liu G. Lozano G. J. Biol. Chem. 1996; 271: 29380-29385Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Taken together, these results suggest that p53 Ser-392 is important for full p53 function. In multiple cell types, p53 Ser-392 is phosphorylated specifically after UV but not γ irradiation or etoposide (3Kapoor M. Lozano G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2834-2837Crossref PubMed Scopus (195) Google Scholar, 4Lu H. Taya Y. Ikeda M. Levine A.J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6399-6402Crossref PubMed Scopus (152) Google Scholar). Casein kinase 2 (CK2) was originally identified as the kinase that targets this sitein vitro (30Meek D.W. Simon S. Kikkawa U. Eckhart W. EMBO J. 1990; 9: 3253-3260Crossref PubMed Scopus (252) Google Scholar). However, it was unclear what the true kinase that targets this site is in cells. Previously, we used a biochemical fractionation to purify UV-responsive p53 Ser-392 kinase activity from murine testicular carcinoma F9 cells (26Keller D.M. Zeng X. Wang Y. Zhang Q.H. Kapoor M. Shu H. Goodman R. Lozano G. Zhao Y. Lu H. Mol. Cell. 2001; 7: 283-292Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar). Our results showed that indeed the kinase was CK2, but it eluted from gel filtration chromatography in a high molecular weight fraction corresponding to ∼700 kDa. We identified two other proteins that eluted with CK2 as hSPT16 and SSRP1. Together, these molecules are known in mammals as the chromatin associated factor, FACT (31Orphanides G. Wu W.H. Lane W.S. Hampsey M. Reinberg D. Nature. 1999; 400: 284-288Crossref PubMed Scopus (450) Google Scholar). Interestingly, when hSPT16 and SSRP1 are complexed with CK2, they change the substrate specificity of CK2 to phosphorylate p53 over all other tested substrates. However, it remains unclear how CK2 preferentially targets p53 as a substrate after association with hSPT16 and SSRP1 and how this kinase complex is activated by DNA damaging signals. To determine the mechanism by which the p53 Ser-392 kinase complex is activated by DNA damage, we have further characterized this complexin vitro and in cells. First, we have mapped the interaction domains between CK2, hSPT16, and SSRP1 in vitro and in cells, demonstrating that these proteins interact with each other via non-overlapping regions, consistent with the idea that they form a complex. Second, steady-state kinetic analysis of the kinase activity of CK2 shows that binding of hSPT16 and SSRP1 to CK2 inhibits casein phosphorylation while having no effect upon p53 phosphorylation and that hSPT16 and SSRP1 apparently do not bind to the substrate binding pocket of CK2 but instead are inhibiting casein phosphorylation in an indirect fashion. Furthermore, we find that hSPT16, SSRP1, and CK2 protein levels are dramatically increased in the column fraction that contains the UV-responsive p53 Ser-392 kinase activity. Thus these results demonstrate that the association of CK2 with SSRP1 and hSPT16 in cells is induced by DNA damage signals leading to specific targeting of p53 at Ser-392. Casein was purchased from Sigma. CK2 was purchased from Promega. Baculovirus expressing FLAG-WT-hSPT16 was as described (26Keller D.M. Zeng X. Wang Y. Zhang Q.H. Kapoor M. Shu H. Goodman R. Lozano G. Zhao Y. Lu H. Mol. Cell. 2001; 7: 283-292Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar). Buffer C 100 (BC100) contains 20 mm Tris/HCl (pH 7.9), 0.1 mm EDTA, 15% glycerol, 100 mm KCl, 1 mm DTT, and protease inhibitors including 0.2 mm phenylmethylsulfonyl fluoride, 4 μm pepstatin A, 1 μg/ml leupeptin, and 1 μg/ml aprotinin. BC100 buffer was used for IP assays and included phosphatase inhibitors NaF (100 μm) and sodium orthovanadate (100 μm). Kinase buffer (1×) is 20 mm Tris/HCl (pH 7.5), 10 mm MgCl2, and 1 mmDTT. Lysis buffer consists of 50 mm Tris/HCl (pH 8.0), 0.5% Nonidet P-40, 1 mm EDTA, 150 mm NaCl, 1 mm DTT, and protease inhibitors as above. Radioimmune precipitation assay buffer is 50 mm Tris/HCl (pH 7.4), 150 mm NaCl, 1% Triton X-100, 0.1% SDS, 1% sodium deoxycholate, 1 mm DTT, and protease inhibitors as above. The His-p53 and pET-311–393 expression vectors were as described previously (26Keller D.M. Zeng X. Wang Y. Zhang Q.H. Kapoor M. Shu H. Goodman R. Lozano G. Zhao Y. Lu H. Mol. Cell. 2001; 7: 283-292Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar). The pRc/CMV/CK2α′-HA and pRc/CMV/CK2β-myc were generous gifts from David Litchfield (32Vilk G. Saulnier R.B. St. Pierre R. Litchfield D.W. J. Biol. Chem. 1999; 274: 14406-14414Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar), and the CK2-encoding cDNAs were subcloned into pET24a expression vectors (Novagen). pET28-antisense hSPT16 was from Danny Reinberg (University of Medicine and Dentistry in New Jersey, Robert Wood Johnson Medical School, NJ) and was used in PCR to generate N-terminal (aa 1–329) and mid-hSPT16 (aa 321–640) followed by subcloning into pET24a and pGEX-KG (Amersham Biosciences) expression vectors. pKK233-3+SSRP1 plasmid was as described previously (26Keller D.M. Zeng X. Wang Y. Zhang Q.H. Kapoor M. Shu H. Goodman R. Lozano G. Zhao Y. Lu H. Mol. Cell. 2001; 7: 283-292Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar) and SSRP1 was then subcloned into pET24a and pGEX-KG expression vectors. N-terminal SSRP1 (aa 1–242), mid-SSRP1 (aa 235–475), and C-SSRP1 (aa 471–709) were generated by PCR and subcloned into pGEX-KG. N- and C-SSRP1 were also subcloned into a FLAG-modified pCDNA3.1 mammalian expression vector (Invitrogen). Polyclonal anti-CK2α′ and anti-p53 antibodies were from Santa Cruz Biotechnology, Inc, monoclonal anti-CK2β antibody was from Transduction Laboratory, Inc., and monoclonal anti-FLAG and anti-γ-tubulin antibodies were from Sigma. Anti-Ser-392 phosphospecific p53 antibody was prepared as previously described (3Kapoor M. Lozano G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2834-2837Crossref PubMed Scopus (195) Google Scholar, 18Shieh S.Y. Ikeda M. Taya Y. Prives C. Cell. 1997; 91: 325-334Abstract Full Text Full Text PDF PubMed Scopus (1783) Google Scholar) as was PAb421 (33Harlow E. Crawford L.V. Pim D.C. Williamson N.M. J. Virol. 1981; 39: 861-869Crossref PubMed Google Scholar). Polyclonal anti-SSRP1 antiserum was generated against full-length histidine-tagged SSRP1 and polyclonal anti-hSPT16 antiserum was made against the middle portion of the protein (aa 321–640). Histidine-tagged proteins were purified on nickel-nitrilotriacetic acid agarose as per the manufacturer's instructions (Qiagen). GST fusion proteins were bound to glutathione-agarose beads (Sigma) and then were either left on the beads or were digested with thrombin protease (Amersham Biosciences) to remove the GST tag. The anti-SSRP1 antibody was purified as described previously (26Keller D.M. Zeng X. Wang Y. Zhang Q.H. Kapoor M. Shu H. Goodman R. Lozano G. Zhao Y. Lu H. Mol. Cell. 2001; 7: 283-292Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar). Murine embryonic testicular carcinoma F9 cells containing wild-type p53 were grown on plates in Dulbecco's modified Eagle medium with 4.5g/liter glucose and with l-glutamine (Invitrogen), supplemented with 5% fetal bovine serum and penicillin/streptomycin. Human colorectal carcinoma RKO cells were grown on plates in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum. All cells were grown at 37 °C in a 5% CO2 atmosphere. GST fusion proteins overexpressed in bacteria were purified on glutathione-agarose beads (Sigma) as described by the manufacturer. The GST fusion protein levels were then equalized by loading onto SDS-PAGE and visualized with Coomassie Brilliant Blue. 1 μg of fusion proteins were combined with 1 μg of soluble His-WT-SSRP1, FLAG-WT-hSPT16, His-CK2α′, or His-CK2β and incubated at room temperature for 40 min with light vortexing. The samples were washed once with lysis buffer, once with 1:3 diluted lysis buffer in water, once with radioimmune precipitation assay buffer, and once with lysis buffer. They were then run on SDS-PAGE and transferred to a polyvinylidene difluoride membrane for Western blotting (WB). GST-pull-down kinase assays were performed as above except that GST fusion proteins were combined with 1 unit of CK2 followed by washing three times with lysis buffer and once with 1× kinase buffer. WB-kinase reactions were carried out as previously described (26Keller D.M. Zeng X. Wang Y. Zhang Q.H. Kapoor M. Shu H. Goodman R. Lozano G. Zhao Y. Lu H. Mol. Cell. 2001; 7: 283-292Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar, 34Lu H. Fisher R.P. Bailey P. Levine A.J. Mol. Cell. Biol. 1997; 17: 5923-5934Crossref PubMed Scopus (139) Google Scholar) using ATP and 100 ng of His-p53 as substrates for 30 min at 30 °C. RKO cells were transfected with 3 μg of either pCDNA3.1, pCDNA3.1 FLAG-N-SSRP1, or pCDNA-FLAG-C-SSRP1 expression constructs using LipofectAMINE reagent (Invitrogen). 24 h post-transfection, cells were trypsinized and transferred to 10-cm plates at low density. 0.5 mg/ml G418 was added to the media as a selectable marker, and the cells were maintained for 2–3 weeks until colonies became visible. Individual colonies were expanded into 12-well plates and screened for FLAG-N- or C-SSRP1 protein expression by WB with anti-FLAG and anti-SSRP1 antibodies. WB, co-IP, and IP kinase assays were carried out as previously described (26Keller D.M. Zeng X. Wang Y. Zhang Q.H. Kapoor M. Shu H. Goodman R. Lozano G. Zhao Y. Lu H. Mol. Cell. 2001; 7: 283-292Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar). The WB in Fig.6 B was analyzed by a Bio-Rad Model GS700 imaging densitometer. FLAG-N- and FLAG-C-SSRP1 RKO cell lysates were used to perform co-IPs with the anti-FLAG antibody. hSPT16, SSRP1, and CK2α′ were immunoprecipitated with anti-SSRP1 from the P11 0.5 mKCl fractions of the F9 cell nuclear extract preparations as previously described. Radioactive in vitro kinase assays were performed with [γ-32P]ATP, in which the total ATP concentration (cold and hot) was 40 μm. Substrates were either 100 ng of His-p53 or 1 μg of casein. In Fig. 4 C, WT-SSRP1, N-hSPT16, and mid-hSPT16 proteins are histidine-tagged. N-SSRP1, mid-SSRP1, and C-SSRP1 are thrombin-cleaved from GST while bound to the glutathione-agarose. WT-hSPT16 is FLAG-tagged. Alternatively, kinase assays were done using unlabeled ATP (1 mm) followed by SDS-PAGE and then phosphorylated His-p53 was detected by WB using the anti-Ser-392 antibody.Figure 4SSRP1 and hSPT16 influence the substrate specificity of CK2. A, the p53 family member p63γ, but not p73α, is phosphorylated by CK2, although its phosphorylation is inhibited by SSRP1 and hSPT16. In vitro kinase reactions were done for 30 min using [γ-32P]ATP and using either 50 ng of His-p53, 150 ng of His-p63γ, or 500 ng of His-p73α as substrates. rFACT indicates recombinant SSRP1 and hSPT16 incubated together, titrated at 15 ng, and 30 ng of total protein.B, Coomassie-stained SDS-PAGE of the substrates used inpanel A (∼1 μg of each protein was loaded). Theasterisks indicate the proteins, and His-p73α exists as two polypeptides. C, a radioactive kinase assay was done as above with either His-p53 (100 ng, top panel) or casein (1 μg, bottom panel). CK2 was incubated along with the various SSRP1 and hSPT16 proteins as described under "Experimental Procedures." The dots indicate that casein and the mid-SSRP1 construct are both phosphorylated by CK2 and have equal migration on SDS-PAGE. Thus, although casein phosphorylation inlane 6 is inhibited, the signal is actually due to mid-SSRP1 (see lane 6, compare the top and bottom panels).View Large Image Figure ViewerDownload Hi-res image Download (PPT) CK2 (0.5 unit) was incubated with or without 4 pmol of FACT (2 pmol of FLAG-hSPT16 and 2 pmol of His-SSRP1) on ice for 1 h. In vitro radioactive kinase assays were then carried out in the presence of 250 μm ATP (including 375 μCi of [γ-32P]ATP) for 0.5 h while titrating substrates casein and His-p53. Casein concentrations ranging from 0.25 to 64 μm and His-p53 ranging from 0.037 to 9.4 μm were titrated into the CK2-FACT-ATP mixture. Reactions were analyzed by SDS-PAGE, the bands were cut out, and radioactivity was quantified using a Beckman model LS 6500 scintillation counter. Reaction velocities were obtained by measuring the picomoles of ATP incorporated into substrate per second and plotted against substrate concentration. Data points were then fit to the Michaelis-Menten equation using Kaleidagraph (Synergy Software) to obtain values ofV max, K m, andV/K. In our previous study, the purified p53 Ser-392 kinase complex from murine testicular carcinoma F9 cells eluted from gel filtration chromatography at ∼700 kDa (26Keller D.M. Zeng X. Wang Y. Zhang Q.H. Kapoor M. Shu H. Goodman R. Lozano G. Zhao Y. Lu H. Mol. Cell. 2001; 7: 283-292Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar). Seven polypeptides co-eluted with the kinase activity, five of which were identified as SSRP1, hSPT16, or the subunits of the CK2 heterotetramer. To determine whether hSPT16, SSRP1, and CK2 together can form a complexin vitro, we incubated these recombinant proteins together and loaded them onto a Superdex 200 size exclusion column (Fig.1). When CK2 was run on the column alone, it eluted at close to the predicted molecular mass of the heterotetramer, 140 kDa (top panel). Interestingly, when recombinant hSPT16 and SSRP1 were mixed with CK2 and then run on the column, the p53 kinase activity of CK2 shifted to a high molecular mass fraction, at ∼670 kDa, and at a similar molecular weight to the native p53 Ser-392 kinase complex (middle panel, and see Ref. 26Keller D.M. Zeng X. Wang Y. Zhang Q.H. Kapoor M. Shu H. Goodman R. Lozano G. Zhao Y. Lu H. Mol. Cell. 2001; 7: 283-292Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar). The bottom panel is a Western blot for hSPT16 and SSRP1 when combined with CK2. The combined molecular masses of the proteins in the FACT·CK2 complex is predicted to be 360 kDa; thus we speculate that the recombinant complex contains multiple copies of some or all the components. Also, hSPT16, SSRP1, and CK2 appear to be the primary components of our previously purified native kinase complex, although it is still likely that other proteins may associate with this complex in cells. To characterize the interactions between members of the p53 Ser-392 kinase complex, we performedin vitro glutathione S-transferase (GST) binding assays. GST-SSRP1 fusion proteins were made with either WT SSRP1 or with three deletion mutants that spanned the length of the protein (Fig. 2 A). We generated mutants for both the N terminus and middle region of hSPT16 (Fig.2 A) but were unable to generate either the WT or the C-terminal hSPT16 fusion proteins, because the C terminus of hSPT16 is apparently toxic to bacteria (our observations). 2D. Reinberg, personal communication. The Coomassie-stained SDS-PAGE gel in Fig. 2 A shows that equal levels of the proteins were used, including a GST only (GST-0) control. As shown in Fig. 2 B, FLAG-WT-hSPT16 generated in baculovirus binds to GST-WT-SSRP1 in vitro and also to GST-N-SSRP1 (amino acids 1–242), although with an apparent decrease in affinity (left panel, compare lanes 7 and 8). In the reverse experiment, His-WT-SSRP1 binds to GST-mid-hSPT16 (aa 321–640) (Fig. 2 B, right panel, lane 6). CK2 binding to SSRP1 and hSPT16 was tested by GST-pull-down followed by a kinase assay. Fig. 2 C shows that CK2 binds to GST-WT-SSRP1 and to GST-C-SSRP1 (aa 471–709) (Fig. 2 C,left panel, compare lanes 2 and 5) and that there is also diminished binding to GST-mid-SSRP1 (aa 235–475) (compare lanes 2 and 4). CK2 also binds hSPT16 directly in vitro via the N terminus (aa 1–329) (Fig.2 C, right panel, lane 2). Thus, from these experiments we conclude that these proteins bind to each other via non-overlapping regions, consistent with the idea that they form an SSRP1·hSPT16·CK2 protein complex. The data are summarized in Fig.2 D. Protein kinase CK2 exists as a heterotetramer with catalytic subunits α and α′ and regulatory subunit β, the stoichiometry being α2β2, α2′β2, or αα′β2 (35Guerra B. Boldyreff B. Sarno S. Cesaro L. Issinger O.G. Pinna L.A. Pharmacol. Ther. 1999; 82: 303-313Crossref PubMed Scopus (87) Google Scholar). To determine which of these subunits bind to SSRP1 and hSPT16, we used either GST-WT-SSRP1 or GST-N-hSPT16, the region that interacts with the CK2 heterotetramer (Fig. 2 C). We tested the catalytic α′ subunit and found that it binds to both GST-WT-SSRP1 and GST-N-hSPT16 (Fig.3 A, compare lanes 5and 6), whereas the regulatory β subunit apparently binds much more strongly to GST-WT-SSRP1 (Fig. 3 A, comparelanes 8 and 9). A Coomassie Blue-stained gel is shown of the purified His-tagged CK2 proteins (Fig. 3 B). To test whether these protein-protein interaction domains are also true in cells, we made stable cell lines with FLAG-tagged N- and C-SSRP1 in human colorectal carcinoma RKO cells and performed co-immunoprecipitations (co-IPs) with the FLAG antibody. Using these cell lines, we reproduced the results seen in the in vitroGST pull-down assay exactly. That is, FLAG-N-SSRP1 bound exclusively to endogenous hSPT16 (Fig. 3 C, top panel, lane 2) and FLAG-C-SSRP1 bound exclusively to the endogenous CK2α′ subunit and p53 Ser-392 kinase activity (Fig. 3 C,bottom two panels, lane 3). Therefore, these cellular data confirm the SSRP1 interactions with hSPT16 and CK2 that were observed in the in vitro GST pull-down assay. Interestingly, although recombinant GST-C-SSRP1 migrates on SDS-PAGE faster than GST-N-SSRP1 (Fig. 2 A, compare lanes cand e), FLAG-C-SSRP1 stably expressed in RKO cells migrates slower than FLAG-N-SSRP1 (Fig. 3 C, compare lanes 2 and 3). One possibility for the slower migration of FLAG-C-SSRP1 is that the C terminus of SSRP1 is highly modified in cells by post-translational modifications; this is supported by the fact that this region has a high serine content and that this region is phosphorylated in vitro by CK2 (see Fig.4). Based upon our protein-protein interaction experiments, we can present a model for the binding of hSPT16 and SSRP1 to the CK2 heterotetramer (Fig. 3 D). The CK2 crystal structure is solved (36Niefind K. Guerra B. Ermakowa I. Issinger O.G. EMBO J. 2001; 20: 5320-5331Crossref PubMed Scopus (347) Google Scholar) and resembles a butterfly, with the two regulatory β subunits making contacts along a 2-fold axis of symmetry, and the catalytic α and α′ subunits situated like the butterfly wings making contacts only with one β subunit. Because the molecular weights of the recombinant and native complexes are similar, we speculate that there are two FACT heterodimers bound per CK2 heterotetramer, which would be a predicted size of 580 kDa, close to the 670 kDa estimated size from gel filtration chromatography. Previously, we discovered that SSRP1 and hSPT16 could modulate the kinase activity of CK2 such that it phosphorylated p53 but inhibited its activity toward other substrates such as casein, histone H1, and MDM2 (26Keller D.M. Zeng X. Wang Y. Zhang Q.H. Kapoor M. Shu H. Goodman R. Lozano G. Zhao Y. Lu H. Mol. Cell. 2001; 7: 283-292Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar). Here we identify the p53 family member p63γ as anin vitro substrate for CK2 (Fig. 4 A, lane 4), although p63γ and p53 do not share sequence conservation in the C-terminal domain and there is no p63γ equivalent of Ser-392. We also tested p73α, another p53 family member, but found that CK2 does not phosphorylate this protein (Fig. 4 A, lanes 7–9). Surprisingly, hSPT16 and SSRP1 inhibit the CK2-induced phosphorylation of p63γ (compare lane 4 with lanes 5 and 6), providing more evidence for the specificity of the p53 Ser-392 kinase complex. As described above, we identified the regions of SSRP1 and hSPT16 that directly bind to CK2 and could now test whether these truncation mutants were sufficient to modulate the kinase activity of CK2 also. Kinase assays were performed using either p53 or casein as substrates, with the addition of the various WT and mutant SSRP1 and hSPT16 proteins. As shown in the bottom panel of Fig.4 C, and as seen previously (26Keller D.M. Zeng X. Wang Y. Zhang Q.H. Kapoor M. Shu H. Goodman R. Lozano G. Zhao Y. Lu H. Mol. Cell. 2001; 7: 283-292Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar), casein phosphorylation was strongly inhibited by His-WT-SSRP1 (compare lanes 1 and2), although p53 phosphorylation was not affected (Fig. 4,top panel, compare lanes 1 and 2). N-SSRP1, which does not bind CK2, also did not affect CK2 activity toward casein or p53 (compare lanes 3 and 4). In contrast, both mid-SSRP1 and C-SSRP1 inhibited casein phosphorylation (compare lanes 5–8), although mid-SSRP1 inhibited to a greater degree. However, this
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