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

Abstract

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.

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). DmAtr-II, Drosophila melanogaster activin type II receptor; Daf4, cell-surface receptor DAF-4 precursor; ActRIIA, activin type II A receptor; PhK-γ, phosphorylase kinase γ; PKC-α, protein kinase C α; HSVK, herpes simplex virus kinase. The amino acids that are often found in the serine/threonine kinases are listed in the first row. In the bottom row, the same region of TIP30M is shown and the substituted amino acids are boxed. (B and C) Bacterially expressed recombinant TIP30 is autophosphorylated. The recombinant proteins purified as described (Xiao et al., 1998) were incubated with [γ-³²P]ATP for 30 min, resolved on 12.5% SDS—PAGE and visualized with Coomassie Blue staining in (B) and autoradiography in (C). Arrows indicate the positions of TIP30 protein.

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 of the proteins were resolved on SDS–PAGE and stained with Coomassie Blue. (B) In vitro phosphorylation of GST–CTD by TIP30. The purified proteins GST–CTD (lane 1) plus TIP30 (lane 2) or TIP30M (lane 3) were subjected to kinase assays followed by SDS—PAGE and autoradiography. (C) Phosphoamino acid analysis. [γ-³²P]ATP GST—CTD labeled with TIP30 was separated on SDS—PAGE and transferred into a nitrocellulose filter by electrophoresis. The labeled band was excised from the filter, hydrolyzed in 6 M HCl, and phosphoamino acids with unlabeled standard phosphorylated amino acids were separated by electrophoresis on a thin-layer cellulose plate in one dimension. The positions of ³²P-labeled amino acids were determined by autoradiography (right panel). The amino acid standards are visualized by ninhydrin staining (left panel). (D) Tat stimulates phosphorylation of the CTD by TIP30. Wild-type TIP30 was incubated with GST (lane 1) or GST–CTD (lane 2) and increasing concentrations of wild-type Tat (lanes 3 and 4) or mutant Tat (lanes 6 and 7) or GAL4-VP16 (lane 9). As controls, the kinase reactions contained GST–CTD and either wild-type Tat (lane 5) or mutant Tat (lane 8).

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), a control pSV-β-Gal (0.5 μg) and pSVTat (40 ng), in the presence of either pCIN4-TIP30 (2 μg) or pCIN4-TIP30M (2 μg) or pCIN4 (2 μg). Results represent the averages of three experiments. (B) TIP30 cooperates with CycT1 and CDK9 to stimulate Tat-dependent transcription. NIH 3T3 cells were co-transfected with plasmids pGL/HIV-1lLuc (200 ng), control pSV-β-Gal (200 ng) directing β-galactosidase expression and plasmid expressing Tat (20 ng), CycT1 (200 ng), CDK9 (200 ng), TIP30 (200 ng) or TIP30M (200 ng) as indicated. Results represent averages of two experiments. (C) Immunoblot analysis of wild-type and mutant TIP30 expression. Aliquots of lysates (10 μg) from NIH 3T3 cells transfected with empty vector, pCIN4-TIP30 or pCIN4-TIP30M were analyzed by Western blotting with anti-TIP30 antibody (Xiao et al., 1998).

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 line. (B) TIP30 expression inhibits NIH 3T3 (upper panel) and N417 (bottom panel) cell growth. NIH 3T3 (2 × 10⁴) or N417 cells (2 × 10⁵) expressing TIP30 or TIP30M, or containing an empty vector, were seeded on six-well plates in DMEM or RPMI medium supplemented with 10% FCS. After 24 h, cells were washed with serum-free medium and grown in medium supplemented with 0.1% bovine calf serum for 3 days. Cells were then trypsinized and stained with Trypan blue. Trypan blue-negative cells were counted. The average numbers represent three independent experiments. (C) Inhibition of TIP30-mediated cell death by the caspase inhibitor zVAD-FMK. NIH 3T3 cell lines containing an empty vector or TIP30-expressing plasmid were assayed for cell death with or without 50 μM zVAD-FMK in the medium during serum deprivation as described in Figure 4B. Trypan blue-positive and -negative cells were counted. (D) DNA fragmentation analysis. Fragmented DNA was purified from different NIH 3T3 cells that were grown in DMEM medium supplemented with 10% FCS or 0.1% bovine calf serum for 3 days as described (Shtivelman, 1997). Aliquots of DNA were separated on a 1.5% agarose gel and then stained with ethidium bromide.

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

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 (C). The two housekeeping genes and the TIP30-responsive genes are listed in (D). (E) Northern blot analyses of Bad, Siva and NM23-H2 mRNA expressions in N417 clones. Total RNAs from different N417 clones were analyzed by Northern blotting with probes as indicated.