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. 2002 Sep 2;21(17):4491-9.
doi: 10.1093/emboj/cdf409.

Suppression of the STK15 oncogenic activity requires a transactivation-independent p53 function

Shih-Shun Chen  1 , Pi-Chu Chang, Yu-Wen Cheng, Fen-Mei Tang, Young-Sun Lin
Affiliations

Suppression of the STK15 oncogenic activity requires a transactivation-independent p53 function

Shih-Shun Chen et al. EMBO J. .

Abstract

Using a transactivation-defective p53 derivative as bait, STK15, a centrosome-associated oncogenic serine/threonine kinase, was isolated as a p53 partner. The p53-STK15 interaction was confirmed further by co-immunoprecipitation and GST pull-down studies. In co-transfection experiments, p53 suppressed STK15-induced centrosome amplification and cellular transformation in a transactivation-independent manner. The suppression of STK15 oncogenic activity by p53 might be explained in part by the finding that p53 inhibited STK15 kinase activity via direct interaction with the latter's Aurora box. Taken together, these findings revealed a novel mechanism for the tumor suppressor function of p53.

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Figures

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Fig. 1. Characterization of the p53–STK15 interaction in yeast. Left column: GAL4 DNA-binding domain (amino acids 1–147) hybrids. Middle column: GAL4 activation domain (amino acids 768–881) hybrids. The STK15 fragment fused to GAL4 activation domain is indicated on the right of the diagrams. The relative positions of the Aurora box and kinase domains of STK15 are also depicted. Right column: yeast colony color after transformation; the relative color is in parentheses. No colony indicates that no yeast colony was observed after incubation for a standard period of time.
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Fig. 2. In vivo interaction of p53 with STK15. (A) Interaction of endogenous p53 and STK15 in 293 cells. Panels I and II: co-immunoprecipitation of p53 and STK15 using lysates prepared from 293 cells. The antibody used is indicated on the top of each lane. The position of p53 or STK15 is shown. (B) Interaction of p53 and STK15 derivatives in H1299 cells. Panel I: co-immunoprecipitation experiments using lysates prepared from H1299 cells transfected with HA-tagged STK15 alone (lanes 5 and 9), and plus p53 (lanes 6 and 10), p53(R175H) (lanes 7 and 11) or p53(R249S) (lanes 8 and 12), and then precipitated with pre-immune serum (lanes 5–8) or with an anti-HA antibody (lanes 9–12) followed by immunoblotting with an anti-p53 antibody. Cell extracts: one hundredth of the cell lysates used in the co-immunoprecipitation was loaded directly onto the gel. Panel II: as in panel I, except that HA-tagged STK15 was replaced with HA-tagged STK15(121–403) for transfection. Panel III: as in panel I, except that p53(R175H) and p53(R249S) were replaced with p53(R273H) and p53(R248W) for transfection.
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Fig. 3. GST pull-down assay. (A) In vitro translated STK15 was incubated with GST (lane 2) and derivatives of GST–p53 (lanes 3–7). Input: one-tenth of the input protein directly loaded onto the gel. (B) As in (A), except that a series of STK15 derivatives were incubated with GST–p53 (1–318). Schematic diagrams of the human p53 and STK15 domain structure are shown below each of the autoradiograms.
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Fig. 4. Blocking of STK15-mediated centrosome amplification by a transactivation-defective p53 mutant. (A) Transactivation assay. Panel I: a luciferase reporter driven by three p53-binding sites was co-transfected with vector alone (lane 1), p53 (lane 2), p53M (lane 3) or p53M(1–318) (lane 4). Panel II: as in panel I, except that the reporter was driven by the p21 promoter. Panel III: protein expression levels of p53 derivatives. Lysates from NIH-3T3 cells transfected with vector alone (lane 1), p53 (lane 2), p53M (lane 3) or p53M(1–318) (lane 4) were blotted with an anti-p53 antibody. (B) Inhibition of STK15-mediated centrosome amplification by p53M(1–318). Panel I: centrosome numbers in 900 NIH-3T3 cells transfected with the specified plasmids were counted by immunostaining with an anti-γ-tubulin monoclonal antibody. The combination of plasmids transfected is indicated. Experiments were repeated three times. Panel II: protein expression levels of STK15. Lysates from NIH-3T3 cells transfected with vector alone (lane 1), HA-STK15 or HA-STK15 plus p53M(1–318) were blotted with an anti-HA antibody.
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Fig. 5. Inhibition of STK15-mediated cellular transformation by a transactivation-defective p53 mutant. NIH-3T3 cells were transfected with vector alone (lane 1), HA-tagged STK15 (lane 2), HA-tagged STK15 plus p53M(1–318) (lane 3), HA-tagged STK15 plus p53(101–318) (lane 4), HA-tagged STK15(121–403) (lane 5), HA-tagged STK15(121–403) plus p53M(1–318) (lane 6), HA-tagged STK15(121–403) plus p53(101–318) (lane 7), HA-tagged H-ras(Q61L) (lane 8), HA-tagged H-ras(Q61L) plus p53M(1–318) (lane 9) or HA-tagged H-ras(Q61L) and p53(101–318) (lane 10). Colonies were counted from three independent experiments.
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Fig. 6. p53 inhibits the STK15 kinase activity in vitro. Myelin basic protein (MBP) was used as a substrate for STK15 (panel I) or STK15(121–403) (panel II). The absence (–) or presence of GST derivatives is indicated.
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