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EMBO J. Mar 1, 2000; 19(5): 956–963.
PMCID: PMC305635

TIP30 has an intrinsic kinase activity required for up-regulation of a subset of apoptotic genes


CC3 is a metastasis suppressor that inhibits metastasis of the variant small cell lung carcinoma (v-SCLC) by predisposing cells to apoptosis. The same protein was also reported as a cellular cofactor, TIP30, which stimulates HIV-1 Tat-activated transcription by interacting with both Tat and RNA polymerase II. We report here that TIP30/CC3 is a novel serine/threonine kinase. It phosphorylates the heptapeptide repeats of the C-terminal domain (CTD) of the largest RNA polymerase II subunit in a Tat-dependent manner. Amino acid substitutions in the putative ATP binding motif that abolish the TIP30 kinase activity also inhibit the ability of TIP30 to enhance Tat-activated transcription or to sensitize NIH 3T3 and v-SCLC cells to apoptosis. Furthermore, ectopic expression of TIP30/CC3 in v–SCLC cells induces expression of a number of genes that include the apoptosis-related genes Bad and Siva, as well as metastasis suppressor NM23-H2. These data demonstrate a molecular mechanism for TIP30/CC3 function and suggest a novel pathway for regulating apoptosis.

Keywords: apoptosis/metastasis suppressor/SCLC/Tat/transcription


Metastasis is a complex process that involves differential expression of a number of genes whose products act as positive or negative regulators of metastasis in malignant tumor cells (Steeg, 1989). Recently, the CC3 gene was identified by differential display analysis of mRNAs from the highly metastatic human small cell lung carcinoma (SCLC) versus less metastatic SCLC cell lines (Shtivelman, 1997). Introduction of the CC3 gene into a variant SCLC (v-SCLC) line that does not express the CC3 gene results in significant suppression of its metastatic potential. This suppression appears to result, at least in part, in a propensity of the cells to undergo apoptosis. Therefore, CC3 was proposed to function as a metastasis suppressor that links the control of apoptosis to metastasis. The anti-tumor activity of CC3 was further confirmed by the finding that intravenous delivery of the CC3 gene to melanoma-bearing mice via a cationic liposome–DNA complex significantly reduced pulmonary and extrapulmonary metastases (Liu et al., 1999). Nevertheless, the molecular mechanism by which the CC3 gene mediates apoptosis has remained largely unknown (Shtivelman, 1997).

We have recently identified a transcription cofactor, called TIP30, that binds to the human immunodeficiency virus (HIV) Tat protein (Xiao et al., 1998). TIP30 is essential for Tat-mediated transcription in vitro and potentiates Tat-activated transcription in transient transfection assays. Other cellular factors that include elongation factor P-TEFb (Mancebo et al., 1997; Zhu et al., 1997) and coactivators CBP/p300 (Hottiger and Nabel, 1998; Marzio et al., 1998) and PCAF (Benkirane et al., 1998) have also been found to interact with Tat and to potentiate transcription from the HIV-1 long terminal repeat (LTR). Ectopic expression of either the CycT1 component of P–TEFb or the histone acetyltransferase PCAF increases Tat-mediated transcription from the HIV-1 LTR in rodent cells (Benkirane et al., 1998; Wei et al., 1998). Although CycT1 appears to be a human-specific cofactor for Tat, as indicated by the inability of the mouse homologue to support Tat-mediated transcription (Bieniasz et al., 1998; Garber et al., 1998), P-TEFb appears to have a general role in the regulation of transcription elongation (Marshall and Price, 1995; Peng et al., 1998). In contrast, TIP30 appears not to be required either for basal level transcription or for activated transcription by GAL4-SP1 and GAL4-VP16, suggesting that it is a gene-specific cofactor (Xiao et al., 1998). While it is evident that TIP30 can be usurped by the viral Tat protein to enhance transcription elongation from the HIV-1 LTR, cellular genes that are targeted by TIP30 remain to be identified. Significantly, however, the finding that TIP30 is identical to CC3 indicates that TIP30 might facilitate suppression of metastasis by regulating the transcription of genes involved in apoptosis.

In this study, we have employed in vivo analyses to delineate further the molecular mechanisms whereby TIP30 potentiates Tat-mediated transcription and apoptosis. We demonstrate that TIP30 contains an intrinsic kinase activity that is capable of phosphorylating the C-terminal domain (CTD) of the largest subunit of RNA polymerase II and, further, that phosphorylation of the CTD by TIP30 is enhanced specifically by Tat. We further show that the kinase activity of TIP30 is required not only for Tat-mediated transcription from the HIV-1 LTR, but also for predisposing cells to apoptotic signals. By using a cDNA microarray analysis, we have identified a number of TIP30-responsive genes that are involved in the regulation of apoptosis, metastasis and DNA repair.


TIP30 displays an intrinsic kinase activity

To understand better the underlying mechanism by which TIP30 stimulates Tat-activated transcription and predisposes v-SCLC cells to apoptosis, we initially performed DDBJ/EMBL/GenBank database searches with the predicted TIP30 protein sequence. These analyses revealed sequence relationships (46% similarity and 22% identity) between TIP30 and a Caenorhabditis elegans cAMP-dependent protein kinase catalytic subunit (accession No. gi125214). A weak homology of TIP30 with a reductase was also reported, but such an activity for TIP30 remains elusive (Baker, 1999). However, the N-terminal region of TIP30 contains a sequence motif that is similar to a putative ATP-binding motif often found in serine/threonine kinases (Hanks et al., 1988; Bossemeyer, 1994), raising the possibility that TIP30 might be a kinase. Alignments of the putative ATP-binding motif of TIP30 with that of selected kinases are shown in Figure Figure1A.1A. To investigate this possibility, we tested the ability of bacterially expressed TIP30 to catalyze autophosphorylation. In order to exclude the possibility of TIP30 phosphorylation by a contaminating kinase in the preparation, control kinase assays were performed with an identically expressed and purified mutant TIP30 containing Val and Ala substitutions for two Gly residues (Gly28 and Gly31) within the ATP-binding motif (Figure (Figure1A1A and B). As shown in Figure Figure1C,1C, wild-type TIP30 was phosphorylated, whereas mutant TIP30 was not, even at higher concentrations of the mutant protein. This result clearly indicates autophosphorylation of TIP30.

figure e051201
Fig. 1. TIP30 has an intrinsic kinase activity. (A) Alignment of the putative TIP30 ATP-binding motif with other kinases. The ATP- binding motif sequences of the known kinases were derived from the Protein Kinase Resource (http://www.sdsc.edu/kinases ...

TIP30 phosphorylates the CTD of RNA polymerase II

To ascertain whether TIP30 is a kinase that is capable of phosphorylating other protein substrates, epitope-tagged wild-type and mutant TIP30 proteins were expressed via baculovirus vectors in Sf9 cells, purified to near homogeneity (Figure (Figure2A),2A), and tested in an in vitro kinase assay. Since we previously showed an association of TIP30 with an RNA polymerase II–SRB complex (Xiao et al., 1998), we first tested the ability of TIP30 to phosphorylate the CTD of the largest subunit of RNA polymerase II. The analysis in Figure Figure2B2B shows that wild-type TIP30, but not mutant TIP30, is able to phosphorylate the CTD of the largest subunit of RNA polymerase II. A phosphoamino acid analysis indicated that both Ser and Thr residues within the CTD were phosphorylated (Figure (Figure2C).2C). Other experiments also showed that TIP30 could not phosphorylate GST (Figure (Figure2D,2D, lane 1), but that it could phosphorylate the largest subunit of a purified native RNA polymerase II (data not shown). TIP30, like the TFIIH-associated CTD kinase (Ohkuma and Roeder, 1994), can also phosphorylate other substrates that include TFIIEα and RAP74, but not TBP, TFIIB and histones (including histone H1), although the significance of these phosphorylations is not clear (data not shown).

figure e051202
Fig. 2. TIP30 phosphorylates the CTD of RNA polymerase II. (A) TIP30 and TIP30M expressed with baculovirus expression system. Wild-type Flag-tagged TIP30 and mutant TIP30 were expressed in Sf9 cells and purified with M2–agarose beads. Aliquots ...

Although we cannot yet rule out the formal possibility that the observed CTD phosphorylation is mediated by a contaminating kinase (from Sf9 cells) in the purified TIP30 preparation, this is unlikely in view of the observation that mutation of the presumptive ATP-binding site in TIP30 results in the loss of both CTD phosphorylation by TIP30 purified from Sf9 cells and autophosphorylation by TIP30 purified from bacteria. On the other hand, TIP30 kinase activity might be stimulated by trace amounts of associated proteins from insect cells, consistent with preliminary results indicating associated proteins in affinity-purified TIP30 from HeLa cells (H.Xiao and R.G.Roeder, unpublished data) and with many examples of kinase regulation by associated factors.

The results above suggest that TIP30 is a serine/threonine kinase and the observed phosphorylation of the CTD by TIP30 raised the possibility that HIV-1 Tat might interact with TIP30 and enhance its intrinsic kinase activity. We examined this possibility by asking whether Tat might stimulate phosphorylation of GST–CTD by TIP30. As shown in Figure Figure2D,2D, wild-type Tat stimulated phosphorylation of the CTD by TIP30 (lanes 3 and 4 versus lane 2), whereas an equivalent amount of the transactivation-defective mutant TatK41T had no effect on CTD phosphorylation (lanes 6 and 7 versus lane 2). As a control, GAL4-VP16, which does not interact with TIP30, had no effect on CTD phosphorylation by TIP30 (Figure (Figure2D,2D, lane 9 versus lane 2). Given the importance of CTD phosphorylation in both transcription elongation and Tat-mediated transactivation (Dahmus, 1995; Chun and Jeang, 1996; Okamoto et al., 1996), this result suggests that the interaction of Tat with TIP30 may increase phosphorylation of the CTD of RNA polymerase II in Tat-mediated transcription.

The kinase activity of TIP30 is essential for its ability to stimulate transcription

Our previous study has shown that TIP30 acts as a coactivator to increase Tat-mediated transcription. To determine the role of the TIP30 kinase in its coactivator activity, we tested whether alterations in the ATP binding motif of TIP30 that inhibit its kinase activity also abolish its ability to stimulate Tat-mediated transcription. Plasmids expressing Flag-tagged wild-type and kinase-defective TIP30 were co-transfected with or without a plasmid expressing Tat into Jurkat cells. Consistent with our previous results, wild-type TIP30 stimulated Tat-activated transcription whereas the mutant TIP30 did not (Figure (Figure3A).3A). Instead, it slightly inhibited Tat-activated transcription. Furthermore, in rodent cells in which vectors expressing CDK9 and the human-specific Tat coactivator CycT1 elevated the Tat response from 9- to 30-fold, ectopically expressed wild-type TIP30, but not the mutant TIP30, cooperated with CycT1 and CDK9 to effect a larger (140-fold) Tat response (Figure (Figure3B).3B). An immunoblot analysis showed that both wild-type and mutant TIP30 were expressed in transfected cells at similar levels (Figure (Figure3C).3C). These results suggest that the kinase activity of TIP30 is essential for its observed coactivator function.

figure e051203
Fig. 3. The kinase activity of TIP30 is required for enhancing Tat-activated transcription. (A) Inhibition of Tat-activated transcription by overexpression of TIP30M in Jurkat cells. Jurkat cells were co-transfected with a reporter pGL/HIV-1Luc (1 μg), ...

The kinase activity of TIP30 is essential for mediating apoptosis in serum-deprived cells

To confirm further that TIP30 is able to predispose cells to apoptosis, we generated NIH 3T3 and v-SCLC cell lines that stably overexpress either the wild-type TIP30 or the kinase-defective TIP30 and control cell lines containing an empty vector. A Western blot analysis of the levels of ectopically expressed TIP30 in these cell lines is shown in Figure Figure4A.4A. We then examined proliferation of cells overexpressing TIP30 by counting live cells (Trypan blue negative) after growth in low-serum medium for 3 days. Expression of the mutant TIP30 had no effect on cell growth in comparison with cells containing an empty vector; however, expression of TIP30 resulted in a significantly lower number of live cells (Figure (Figure4B).4B). Since TIP30 was implicated previously in the regulation of apoptosis, it is conceivable that inhibition of growth in cells overexpressing TIP30 results from cell death. Indeed, as demonstrated in Figure Figure4C,4C, cultures of cells expressing TIP30 showed a significantly higher proportion (50%) of dead cells than did cultures of control cells (15%) in low serum. Importantly, this TIP30 effect on cell death was inhibited by addition of the caspase-specific inhibitor VAD-FMK to the cell cultures (Figure (Figure4C).4C). DNA fragmentation (Figure (Figure4D)4D) and TUNEL (data not shown) analyses confirmed that the observed cell death resulted from apoptosis. NIH 3T3-TIP30 cells showed the DNA fragmentation pattern that is characteristic of apoptotic cells, whereas the NIH 3T3-Vector and NIH 3T3-TIP30M cells did not (Figure (Figure4D).4D). Furthermore, and as expected, N417-TIP30 cells displayed higher sensitivity to the apoptotic effects of serum deprivation than did the N417-TIP30M and N417-Vector cells (data not shown). Taken together, these results indicate that TIP30 requires its kinase activity for sensitizing cells to apoptosis under low-serum growth conditions.

figure e051204
Fig. 4. (A) Immunoblot analysis of TIP30 and TIP30M expression in stable cell lines. Aliquots (100 μg) of lysates from each cell line as indicated were analyzed by Western blotting with anti-TIP30 antibody. H146 is a less metastatic SCLC cell ...

The kinase activity of TIP30 is required for suppression of colony formation under anchorage-independent conditions

Since it has been demonstrated previously that ectopic expression of TIP30 can inhibit colony formation of v–SCLC cells on soft agar (Shtivelman, 1997), we examined whether the kinase activity of TIP30 also plays a role in this event. As shown in Figure Figure5,5, cells expressing wild-type TIP30 resulted in significantly fewer colonies when grown on soft agar medium than did cells expressing mutant TIP30 or containing an empty vector. This result suggests that the kinase activity of TIP30 could also be involved in suppression of metastasis.

figure e051205
Fig. 5. Growth of N417 clones in semi-solid medium. The numbers represent average numbers of colonies from three independent experiments.

Expression of apoptotic genes Bad and Siva is induced by overexpression of wild-type, but not kinase-defective, TIP30

Having demonstrated that the kinase activity of TIP30 is required both for enhancing Tat-mediated transcription and for sensitizing cells to apoptosis, we hypothesized that TIP30 might directly regulate transcription of genes that facilitate apoptosis. To identify potential TIP30-targeted genes, we examined the expression of known apoptosis-related genes, as well as cancer-related genes, in v-SCLC cells. Poly(A) mRNAs were isolated from the v-SCLC-TIP30 and v-SCLC-TIP30M cell lines and used to generate probes for cDNA microarray analysis. The human cancer cDNA array contained 588 genes that are known to be involved in the regulation of tumor growth, metastasis, cell cycle, DNA repair and apoptosis, as well as nine control genes for normalization. Of the 597 genes tested, only ~12 genes were expressed at visibly higher levels (relative to the control genes) in v-SCLC-TIP30 cells (Figure (Figure6A6A versus B; indicated in Figure Figure6C)6C) than in v-SCLC-TIP30M cells (Figure (Figure6B).6B). As summarized in Figure Figure6D,6D, these included the apoptotic genes Bad (Yang et al., 1995) and Siva (Prasad et al., 1997), as well as genes involved in DNA repair and tumor invasion. The elevated expression of Bad, Siva and NM23-H2 (Postel et al., 1993) genes was also confirmed by Northern blot analysis (Figure (Figure66E).

figure e051206
Fig. 6. Identification of TIP30-responsive genes. The human cancer cDNA array membranes that were hybridized with probes derived from N417-TIP30 (A) or N417-TIP30M (B) were examined with Molecular Dynamic Storm 840. The positions of cDNAs are shown in ...


An interesting and potentially important feature of TIP30 is that it displays a serine/threonine kinase activity. Although it is possible that TIP30 may have other physiological substrates, we speculate that one target for TIP30 is the CTD of the largest subunit of RNA polymerase II. Besides TIP30, other cellular kinases that include the SRB10 and 11 components of the RNA polymerase II holoenzyme/mediator (Liao et al., 1995), c-ABL tyrosine kinase (Baskaran et al., 1997), TFIIH (Lu et al., 1992), P-TEFb (Marshall et al., 1996) were previously reported to be capable of phosphorylating the CTD on the largest subunit of RNA polymerase II, suggesting multiple pathways for regulation of RNA polymerase II phosphorylation. Among these kinases, TFIIH, P-TEFb and TIP30 are able to interact with the activation domain of Tat (Blau et al., 1996; Cujec et al., 1997; Mancebo et al., 1997; Parada and Roeder, 1997; Zhu et al., 1997; Xiao et al., 1998) and participate in Tat-mediated transcription. However, both TFIIH and P-TEFb have been implicated, by immunodepletion studies, in basal transcription of the HIV-1 and adenovirus major late promoters in nuclear extracts (Parada and Roeder, 1997; Zhu et al., 1997). These observations are consistent with previous findings that TFIIH serves as a basal transcription factor and P-TEFb as a general elongation factor in systems reconstituted with partially purified factors. In contrast, TIP30 appears not to be necessary for basal transcription from the adenovirus major late promoter or for activation by GAL4-SP1 or GAL4-VP16 in nuclear extracts, suggesting that TIP30 might be more of a gene-specific cofactor (Xiao et al., 1998). This is also consistent with our observation that ectopic expression of TIP30 only induces expression of a subset of genes. It is possible that phosphorylation of RNA polymerase II by TIP30 would increase expression of this subset of genes, whereas phosphorylation of RNA polymerase II by P-TEFb and TFIIH is required for constitutive or basal expression of most, if not all, cellular genes. Therefore, TIP30 might represent one of the first examples of the regulation of apoptosis by a mechanism involving the kinase activity of a transcriptional coactivator.

Apoptosis is facilitated by the cell death machinery that acts downstream of the death signals in the cytosol (Green and Reed, 1998). Growing evidence has indicated that there are multiple genetically programmed pathways that can be triggered by a variety of physiological death signals, as well as pathological cellular insults that activate those death molecules to execute apoptosis (Dragovich et al., 1998; Vaux and Korsmeyer, 1999). While pro- and anti-apoptotic molecules may be regulated via mechanisms involving post-translation modification and/or conformation changes, as well as translocations of these molecules, they may also be regulated at the level of transcription. Biochemical and genetic studies have suggested that some of the transcription factors do not induce apoptosis per se, but instead maintain appropriate expression of pro- and anti-apoptotic genes in order to establish the sensitivity of cells to apoptosis (Vaux and Korsmeyer, 1999). Thus, an obvious prediction that can be made from this scenario is that loss or inactivation of those transcription factors might reduce the sensitivity of cells to death signals and decrease their ability to undergo apoptosis, thus leading in turn to tumor progression.

Consistent with this idea, it was demonstrated previously that the SCLC cells lacking TIP30 were more resistant to death-inducing signals such as growth factor withdrawal and chemotherapeutic drugs than the TIP30-expressing SCLC cells (Shtivelman, 1997). Our present data clearly support a role for TIP30 in apoptosis, as proposed by Shtivelman (1997). It appears that ectopic expression of TIP30 elevates the mRNA levels of pro-apoptotic genes, as exemplified by Bad and Siva. Since Bad is associated with BCL-2 and induces apoptosis (Yang et al., 1995) and since Siva participates in the CD27-mediated apoptosis (Prasad et al., 1997), it is likely that TIP30 sensitizes cells to apoptosis, at least in part, by elevating expression of Siva and Bad. Higher levels of these two proteins would presumably alter cell growth and commit cells to an apoptotic pathway. Regardless of whether these genes are direct or indirect targets of TIP30-mediated transcriptional activation, our data have provided a plausible explanation for TIP30-induced apoptosis and implicated a novel mechanism for regulation of apoptosis in cells.

Our present data also raise a number of intriguing questions. First, with which cellular transcriptional activator(s) does TIP30 interact? Since TIP30 was isolated by virtue of an interaction with Tat, it is possible that a cellular transcriptional activator, possibly analogous in function to Tat, might also interact with TIP30 to activate transcription. Thus, identification of such an activator would contribute to a better understanding of TIP30-mediated apoptosis. Secondly, what are the genes and signals that are upstream of TIP30? To date, many transcription factors have been demonstrated to participate in the regulation of apoptosis. Among these, the tumor suppressor p53 has been shown to delay cell division in order to repair DNA damage or to induce apoptosis and to eliminate cells and, when overexpressed, to induce a number of genes that include Bax (Miyashita and Reed, 1995) and pigs 1–12 (Polyak et al., 1997). Although TIP30 induces one p53-targeted gene, pig 12, p53 clearly regulates the expression of many other genes that are different from TIP30-targeted genes. Myc is another important transcription factor that can control the decision of a cell to undergo apoptosis (Evan et al., 1992). While the mechanism underlying Myc-mediated apoptosis remains unclear, Myc was reported to affect p53-mediated apoptosis and to cooperate with Ras in inducing apoptosis (Hermeking and Eick, 1994; Wagner et al., 1994; Kauffmann-Zeh et al., 1997). Similar to TIP30-mediated apoptosis, Myc-mediated apoptosis is also promoted by serum deprivation (Evan et al., 1992). v-SCLC N417 cells were reported to have higher level of Myc but no expression of wild-type p53 (Adachi et al., 1996; Shtivelman, 1997). It is tempting to speculate that p53 and Myc might act as upstream regulators in a TIP30-mediated signal pathway. If so, TIP30-mediated transcription regulation would provide a novel mechanism for p53- and Myc-mediated apoptosis. Nevertheless, the validation of this proposal requires further experimentation. Finally, since two malignant tumors, v-SCLC and neuroblastoma, do not express TIP30 and since introduction of TIP30 into v-SCLC cells inhibits their metastatic potential in mice (Shtivelman, 1997), identification of the kinase activity of TIP30 and its downstream target genes provides clues for further studies of the mechanism of metastasis suppression. It is conceivable that TIP30 somewhat directly induces apoptotic pathways and thereby results in inhibition of metastasis. However, the finding that several DNA repair and metastasis-related genes (including RPA70, NM23-H2 and RAP) are induced by TIP30 (Figure (Figure6D)6D) indicates that other cellular responses may also be involved in the inhibition of tumor growth and metastasis by TIP30. Unraveling TIP30-mediated signal pathway(s) might provide novel connections between networks of signal transduction pathways for controlling apoptosis, metastasis, DNA repair response and proliferation within cells.

Materials and methods

Plasmids and cell lines

The bacterial expression plasmid pRSET-his-TIP30M was generated by directly changing DNA sequences of the TIP30 gene on pREST-his-TIP30 (Xiao et al., 1998) with the site-directed mutagenesis kit (Stratagene). The oligonucleotide (5′-TTGGGCGCCAGCGTAGAAACCGCTAGAGTGCTCTTA-3′) was used for mutagenesis according to the manufacturer's instructions (Stratagene).

The insect cell expression vectors pVL-Flag-TIP30 and pVL-Flag-TIP30M were generated by inserting DNA fragments encoding Flag-TIP30 and Flag-TIP30M (isolated from pFlag-TIP30 and pFlag-TIP30M) into the vector pVL1392 at the EcoRI–BamHI site. pFlag7-TIP30 and pFlag7-TIP30M were constructed by insertion of DNA fragments encoding TIP30 and TIP30M (from pRSET-his-TIP30 and pRSET-his-TIP30M) into pFlag-7 (Chiang and Roeder, 1993) at NdeI and BamHI sites. Recombinant baculoviruses were generated according to the manufacturer's instructions (Novagen). The mammalian expression vectors pCIN4-TIP30 and pCIN4-TIP30M were created by cloning EcoRI–BamHI fragments of Flag-TIP30 into pCIN4 (Invitrogen). NIH 3T3, H146 and N417 were obtained from the American Tissue Culture Collection. All cells were grown in Dulbecco's modified Eagle's medium (DMEM) or RPMI supplemented with 10% fetal calf serum (FCS) and antibiotics. TIP30-expressing and control cell lines were established as described previously (Shtivelman, 1997; Xiao et al., 1998). pCIN4-CycT1 was created by insertion of PCR-amplified human CycT1 DNA encoding the entire open reading frame into pCIN4 at EcoRI and BamHI sites. pGST-CycT1 (Wei et al., 1998) was used as a DNA template for PCR. Oligonucleotides for PCR were 5′-GGAATTCCATGGAGGGAGAGAGGAAGAACA-3′ and 5′-CGGGGATCCTTACTTAGGAAGGGGTGGAAG-3′. The plasmid for expressing CDK9 has been described (Mancebo et al., 1997). pGL2/HIV-1Luc was described previously (Bassuk et al., 1997)

Kinase assay

Recombinant proteins were mixed in 20 μl of reaction buffer containing 10 mM Tris pH 7.9, 50 mM KCl, 1 mM EDTA, 1 mM dithiothreitol, 5 mM MgCl2, 10% glycerol and 5 μCi [γ-32P]ATP (3000 Ci/mmol). Reactions were incubated at 37°C for 30 min and then stopped by adding 10 μl of 3× SDS sample buffer. The kinase products were analyzed by SDS—PAGE followed by autoradiography. GST–CTD was expressed and purified as described (Peterson et al., 1992). CTD peptides were purchased from Research Genetic Inc. (Huntsville, AL). Phosphoamino acid analysis was performed as described (Ausubel et al., 1995).

Transient transfection and luciferase assay

Jurkat cells at a density of 2 × 106 per well in six-well dishes were co-transfected with plasmid DNAs with Superfectin according to the manufacturer's instructions (Qiagen). NIH 3T3 cells in six-well dishes were transfected with lipofectamine as described (Xiao et al., 1998). All transfections were balanced to an equal amount of DNA with the vector pCIN4. Forty-eight hours after transfection, cells were collected and assayed for luciferase activity with Promega's luciferase assay kit and for β-galactosidase activity as described previously (Xiao et al., 1998).

Soft agar assays

The N417 transfectants (5 × 103) were grown in RPMI medium containing 0.53% agarose and 10% FCS, on top of a layer containing 0.4% agarose for 21 days. The clones whose diameters were >1 mm were counted.

Microarray and Northern blot analyses

Poly(A) mRNA was purified from the N417-TIP30 and N417-TIP30M cell lines that were cultured in RPMI containing 10% FCS with the RNA/DNA Midi and oligo direct mRNA mini kits according to the manufacturer's instructions (Qiagen). The cDNA probes were generated and hybridized with the atlas human cancer cDNA expression array filters according to the manufacturer's instructions (Clontech). For Northern blot analyses, total RNA was isolated from cells cultured in RPMI containing 10% FCS by using an RNA purification kit (Qiagen). Ten micrograms of RNA were electrophoresed on a 1% agarose–formaldehyde gel and transferred to nitrocellulose filters as described (Ausubel et al., 1995). The filters were hybridized with probes in ExpressHyb hybridization buffer and washed according to the manufacturer's instructions (Clontech). The cDNAs encoding human Bad and Siva were amplified by RT–PCR from human placental poly(A) mRNA and subcloned into pRSET-His Vector (Hoffmann and Roeder, 1996) at NdeI and BamHI sites. NM23-H2 cDNA probe was derived from the plasmid (IMAGE: 1651303) containing the NM23-H2 gene, which was obtained from the American Tissue Culture Collection. Probes were labeled with [α-32P]dCTP with the Megaprime DNA labeling system RPN1606 (Amersham).


We thank Zhengxing Wang for affinity-purified RNA polymerase II and valuable suggestions. This work was supported by a grant to R.G.R. from the National Institutes of Health (AI37327) and by the Tebil Foundation. H.X. was supported in part by a fellowship from the National Institutes of Health.


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