Chunyan Tian

KRAB-type zinc-finger protein Apak specifically regulates p53-dependent apoptosis

Only a few p53 regulators have been shown to participate in the selective control of p53-mediated cell cycle arrest or apoptosis. How p53-mediated apoptosis is negatively regulated remains largely unclear. Here we report that Apak (ATM and p53-associated KZNF protein), a Krüppel-associated box (KRAB)-type zinc-finger protein, binds directly to p53 in unstressed cells, specifically downregulates pro-apoptotic genes, and suppresses p53-mediated apoptosis by recruiting KRAB-box-associated protein (KAP)-1 and histone deacetylase 1 (HDAC1) to attenuate the acetylation of p53. Apak inhibits p53 activity by interacting with ATM, a previously identified p53 activator. In response to stress, Apak is phosphorylated by ATM and dissociates from p53, resulting in activation of p53 and induction of apoptosis. These findings revealed Apak to be a negative regulator of p53-mediated apoptosis and showed the dual role of ATM in p53 regulation.

PACT is a negative regulator of p53 and essential for cell growth and embryonic development

The tumor suppressor p53 regulates cell cycle progression and apoptosis in response to various types of stress, whereas excess p53 activity creates unwanted effects. Tight regulation of p53 is essential for maintaining normal cell growth. p53-associated cellular protein-testes derived (PACT, also known as P2P-R, RBBP6) is a 250-kDa Ring finger-containing protein that can directly bind to p53. PACT is highly up-regulated in esophageal cancer and may be a promising target for immunotherapy. However, the physiological role of the PACT–p53 interaction remains largely unclear. Here, we demonstrate that the disruption of PACT in mice leads to early embryonic lethality before embryonic day 7.5 (E7.5), accompanied by an accumulation of p53 and widespread apoptosis. p53-null mutation partially rescues the lethality phenotype and prolonged survival to E11.5. Endogenous PACT can interact with Hdm2 and enhance Hdm2-mediated ubiquitination and degradation of p53 as a result of the increase of the p53–Hdm2 affinity. Consequently, PACT represses p53-dependent gene transcription. Knockdown of PACT significantly attenuates the p53–Hdm2 interaction, reduces p53 polyubiquitination, and enhances p53 accumulation, leading to both apoptosis and cell growth retardation. Taken together, our data demonstrate that the PACT–p53 interaction plays a critical role in embryonic development and tumorigenesis and identify PACT as a member of negative regulators of p53.

Role for the pleckstrin homology domain- containing protein CKIP-1 in AP-1 regulation and apoptosis

The oncogenic transcription factor c‐Jun plays an important role in cell proliferation, transformation and differentiation. All identified c‐Jun‐interacting proteins are localized to the nucleus or cytoplasm and function in their intact forms. Here we show that the pleckstrin homology domain‐containing protein CKIP‐1 (casein kinase 2‐interacting protein‐1) functions as a plasma membrane‐bound AP‐1 regulator. During apoptosis, CKIP‐1 is cleaved by caspase‐3 and translocated to the cytoplasm and then to the nucleus. C‐terminal fragments of cleaved CKIP‐1 strongly repress AP‐1 activity. Importantly, CKIP‐1 overexpression promotes apoptosis by forming a positive feedback loop between CKIP‐1 and caspase‐3. RNA interference of CKIP‐1 or overexpression of c‐Jun attenuates the sensitivity to apoptosis, indicating a novel role of CKIP‐1 in apoptosis. CKIP‐1 is the first case of a c‐Jun‐interacting protein that regulates AP‐1 activity via caspase‐3‐dependent cleavage and translocation.

Smad Ubiquitylation Regulatory Factor 1/2 (Smurf1/2) Promotes p53 Degradation by Stabilizing the E3 Ligase MDM2

The tumor suppressor p53 protein is tightly regulated by a ubiquitin-proteasomal degradation mechanism. Several E3 ubiquitin ligases, including MDM2 (mouse double minute 2), have been reported to play an essential role in the regulation of p53 stability. However, it remains unclear how the activity of these E3 ligases is regulated. Here, we show that the HECT-type E3 ligase Smurf1/2 (Smad ubiquitylation regulatory factor 1/2) promotes p53 degradation by enhancing the activity of the E3 ligase MDM2. We provide evidence that the role of Smurf1/2 on the p53 stability is not dependent on the E3 activity of Smurf1/2 but rather is dependent on the activity of MDM2. We find that Smurf1/2 stabilizes MDM2 by enhancing the heterodimerization of MDM2 with MDMX, during which Smurf1/2 interacts with MDM2 and MDMX. We finally provide evidence that Smurf1/2 regulates apoptosis through p53. To our knowledge, this is the first report to demonstrate that Smurf1/2 functions as a factor to stabilize MDM2 protein rather than as a direct E3 ligase in regulation of p53 degradation.

Histone methyltransferase protein SETD2 interacts with p53 and selectively regulates its downstream genes.

SETD2 (SET domain containing protein 2) is a histone H3K36 trimethyltransferase protein that associates with hyperphosphorylated RNA polymerase II and involves in transcriptional elongation. However, whether and how SETD2 is implicated in the specific regulation of gene transcription remains unknown. Here we show that SETD2 could interact with p53 and selectively regulate the transcription factor activity of p53. The interaction was dependent of C-terminal region of SETD2, which contains the SET and WW domains, and the N-terminal transactivation domain (residues 1-45) of p53. Overexpression of SETD2 upregulated the expression levels of a subset of p53 targets including puma, noxa, p53AIP1, fas, p21, tsp1, huntingtin, but downregulated that of hdm2. In contrast, it had no significant effect on those of 14-3-3sigma, gadd45 and pig3. Consistently, knockdown of endogenous SETD2 expression by RNA interference resulted in converse effects as expected. In p53-deficient H1299 cells, SETD2 lost the ability to regulate these gene expression except hdm2, indicating the dependence of p53. Furthermore, we demonstrated that SETD2 downregulated hdm2 expression by targeting its P2 promoter and then enhanced p53 protein stability. Collectively, these findings suggest that the histone methyltransferase SETD2 could selectively regulate the transcription of subset genes via cooperation with the transcription factor p53.

CKIP-1 Inhibits Cardiac Hypertrophy by Regulating Class II Histone Deacetylase Phosphorylation Through Recruiting PP2A

Background— Sustained cardiac pressure overload–induced hypertrophy and pathological remodeling frequently leads to heart failure. Casein kinase-2 interacting protein-1 (CKIP-1) has been identified to be an important regulator of cell proliferation, differentiation, and apoptosis. However, the physiological role of CKIP-1 in the heart is unknown. Methods and Results— The results of echocardiography and histology demonstrate that CKIP-1–deficient mice exhibit spontaneous cardiac hypertrophy with aging and hypersensitivity to pressure overload–induced pathological cardiac hypertrophy, as well. Transgenic mice with cardiac-specific overexpression of CKIP-1 showed resistance to cardiac hypertrophy in response to pressure overload. The results of GST pull-down and coimmunoprecipitation assays showed the interaction between CKIP-1 and histone deacetylase 4 (HDAC4), through which they synergistically inhibited transcriptional activity of myocyte-specific enhancer factor 2C. By directly interacting with the catalytic subunit of phosphatase 2A, CKIP-1 overexpression enhanced the binding of catalytic subunit of phosphatase-2A to HDAC4 and promoted HDAC4 dephosphorylation. Conclusions— CKIP-1 was found to be an inhibitor of cardiac hypertrophy by upregulating the dephosphorylation of HDAC4 through the recruitment of protein phosphatase 2A. These results demonstrated a unique function of CKIP-1, by which it suppresses cardiac hypertrophy through its capacity to regulate HDAC4 dephosphorylation and fetal cardiac genes expression.

HECT ubiquitin ligase Smurf1 targets the tumor suppressor ING2 for ubiquitination and degradation

MINT‐7894249: ING2 (uniprotkb:Q9H160) physically interacts (MI:0915) with ubiquitin (uniprotkb:P62988) by anti tag co‐immunoprecipitation (MI:0007)

Apak competes with p53 for direct binding to intron 1 of p53AIP1 to regulate apoptosis

The KRAB‐type zinc‐finger protein Apak was recently identified as a negative regulator of p53‐mediated apoptosis. However, the mechanism of this selective regulation is not fully understood. Here, we show that Apak recognizes the TCTTN2−30TTGT consensus sequence through its zinc‐fingers. This sequence is specifically found in intron 1 of the proapoptotic p53 target gene p53AIP1 and largely overlaps with the p53‐binding sequence. Apak competes with p53 for binding to this site to inhibit p53AIP1 expression. Upon DNA damage, Apak dissociates from the DNA, which abolishes its inhibitory effect on p53‐mediated apoptosis.

p53 Degradation by a Coronavirus Papain-like Protease Suppresses Type I Interferon Signaling

Background: The molecular mechanism of coronavirus PLPs suppressing the innate immune response remains unclear. Results: PLP2 induces the degradation of p53 through stabilizing MDM2, and IRF7 is a novel target gene of p53. Conclusion: PLP2 inhibits the p53-mediated production of type I IFN and apoptosis to ensure viral growth. Significance: We identify the mechanism with which coronavirus induces the low dosage IFN production. Infection by human coronaviruses is usually characterized by rampant viral replication and severe immunopathology in host cells. Recently, the coronavirus papain-like proteases (PLPs) have been identified as suppressors of the innate immune response. However, the molecular mechanism of this inhibition remains unclear. Here, we provide evidence that PLP2, a catalytic domain of the nonstructural protein 3 of human coronavirus NL63 (HCoV-NL63), deubiquitinates and stabilizes the cellular oncoprotein MDM2 and induces the proteasomal degradation of p53. Meanwhile, we identify IRF7 (interferon regulatory factor 7) as a bona fide target gene of p53 to mediate the p53-directed production of type I interferon and the innate immune response. By promoting p53 degradation, PLP2 inhibits the p53-mediated antiviral response and apoptosis to ensure viral growth in infected cells. Thus, our study reveals that coronavirus engages PLPs to escape from the innate antiviral response of the host by inhibiting p53-IRF7-IFNβ signaling.

ATM-mediated NuSAP phosphorylation induces mitotic arrest

NuSAP is a microtubule-associated protein that plays an important role in spindle assembly. NuSAP deficiency in mice leads to early embryonic lethality. Spindle assembly in NuSAP-deficient cells is highly inefficient and chromosomes remain dispersed in the mitotic cytoplasm. ATM is a key kinase that phosphorylates a series of substrates to mediate G1/S control. However, the role of ATM at the G2/M phase is not well understood. Here we demonstrate that ectopic expression of NuSAP lead to mitotic arrest observably dependent on the kinase activity of ATM. When endogenous ATM was depleted or its kinase activity was inhibited, NuSAP could not cause mitotic arrest. We further show ATM interacts with NuSAP and phosphorylates NuSAP on Ser124. The phosphorylation and interaction occur specifically at G2/M-phase. Collectively, our work has uncovered an ATM-dependent checkpoint pathway that prevents mitotic progression by targeting a microtubule-associated protein, NuSAP.