Depletion of endogenous KAP1 expression by RNAi stimulates p53 transcriptional activity, sensitizes p53 response to DNA damage, and increases apoptosis

Depletion of endogenous KAP1 expression by RNAi stimulates p53 transcriptional activity, sensitizes p53 response to DNA damage, and increases apoptosis. p53 was determined by Ni-NTA purification of proteins conjugated to His6-ubiquitin and p53 Western blot. As expected, ubiquitination of p53 was stimulated by MDM2, and expression of KAP1 further enhanced p53 ubiquitination level in an MDM2-dependent fashion (Figure 4A). The ability of KAP1 to stimulate p53 ubiquitination required the MDM2 RING domain. The stable MDM2-457S E3 ligase mutant did not cooperate with KAP1, and possibly acted in a dominant-negative fashion over the endogenous MDM2 due to high-level expression (Figure 4A). Open in a separate window Figure 4 KAP1 promotes p53 ubiquitination by MDM2. (A) H1299 cells were transfected with His6-ubiquitin and indicated plasmids. The level of p53 ubiquitination was determined by Ni-NTA purification and p53 Western blot. KAP1 stimulated p53 ubiquitination in the presence of full-length MDM2. The MDM2-457S mutant contains an inactivating point mutation in the RING domain. (B) The ability of KAP1 mutants to promote p53 ubiquitination in the presence or absence of MDM2 was determined in MDM2-null 174.1 MEFs using the same assay as in panel A. The KAP20C419 RBCC fragment promoted p53 ubiquitination independent of MDM2. (C) H1299 cells were transfected with p53 in combination with indicated plasmids and p53 expression level was determined by Western blot after 48 h. KAP1 promoted degradation of p53 in the presence of MDM2. KAP1 Veralipride has a RING domain and a PHD domain, which are features of ubiquitin E3 ligases (Joazeiro and Weissman, 2002). Figure 4A suggested that KAP1 alone does not function as E3 for p53. To determine which domain of KAP1 is important for cooperation with MDM2, KAP deletion mutants were tested in MDM2-null MEFs. The results showed that KAP1 239C835 mutant without the N-terminal RING domain showed reduced cooperation with MDM2 in p53 ubiquitination (Figure 4B). Interestingly, the N-terminal fragment 20C419 (RBCC) showed strong stimulation of p53 ubiquitination independent of MDM2 (Figure 4B). Since full-length KAP1 did not exhibit such activity, we introduced point mutations into the RBCC fragment to target conserved cysteine residues that may be important for ubiquitin E3 ligase function (C68S, C88S, Veralipride C156S, C209S) Veralipride or to cause disruption of the coiled-coil region (L306P). Surprisingly, these single point mutations failed to block the ability of RBCC to stimulate p53 ubiquitination level in MDM2-null cells (data not shown). It is noteworthy that another RBCC protein Efp has recently been shown to have RING domain-dependent ubiquitin E3 activity against 14-3-3 sigma (Urano population) in CD20-positive cells was quantified by FACS analysis after 48 h. Examples of FACS histograms are shown. Next, the effect of KAP1 on p53 apoptosis induction was examined. p53-deficient H1299 cells were transiently transfected with p53 and CD20 marker and the level of cell death was quantified by measuring the population of CD20-positive sub-2apoptotic cells in FACS. Expression of p53 induced efficient apoptosis in H1299 cells. Coexpression of MDM2 plasmid at 2:1 ratio only caused a small reduction in apoptosis. When KAP1 was cotransfected with this suboptimal amount of MDM2, significant inhibition of apoptosis was observed (Figure 5C). Therefore, KAP1 has the potential to regulate p53 transcription and apoptosis functions in cooperation with MDM2. ARF inhibits KAP1CMDM2 interaction The tumor suppressor ARF binds to the acidic region of MDM2 and inhibits p53 ubiquitination (Zhang and Xiong, 2001). ARF has also been shown to abrogate MDM2 inhibition of p53 acetylation (Ito suggests that this region may encode a cryptic ubiquitin E3 ligase activity that contributes to p53 ubiquitination when complexed with MDM2. Recent experiments also suggested that KAP1 RBCC can stimulate ubiquitination of MDM2 RING deletion mutants that normally cannot undergo self-ubiquitination (unpublished observations). Therefore, the biochemical activity of this fragment and its role in the context of full-length KAP1 protein remain to be further investigated. Several general transcription coactivators (p300, CBP, TAF250) have been shown to have ubiquitin E3 ligase function (Pham and Sauer, 2000; Grossman ubiquitination assay H1299 cells in 10 Veralipride cm plates were transfected with combinations of 5 g His6-ubiquitin expression NFIB plasmid, 1C5 g human MDM2, 5 g p53, and 5 g KAP1 expression plasmids. At 32 h after transfection, cells were lysed in buffer A (6 M guanidinium-HCl, 0.1 M Na2HPO4/NaH2PO4, 0.01 M TrisCHCl pH Veralipride 8.0, 5 mM imidazole, 10 mM -mercaptoethanol) and incubated.

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