FHL2 interacts with iASPP and impacts the biological functions of leukemia cells.
ABSTRACT: iASPP is an inhibitory member of apoptosis-stimulating proteins of p53 (ASPP) family, which inhibits p53-dependent apoptosis. iASPP was highly expressed in acute leukemia, inhibited leukemia cells apoptosis and promoted leukemogenesis. In order to clarify its mechanism, a yeast two-hybrid screen was performed and FHL2 was identified for the first time as one of the binding proteins of iASPP. FHL2 was highly expressed in K562 and Kasumi-1 cells. FHL2 and iASPP interacted with each other and co-localized in both nucleus and cytoplasm. Either FHL2 or iASPP silenced could reduce cell proliferation, induce cell cycle arrest at G0/G1 phase, and increase cell apoptosis. Western blot analysis showed that the level of p21 and p27 increased, CDK4, E2F1, Cyclin E and anti-apoptotic proteins Bcl-2 and Bcl-xL reduced. Interestingly, when FHL2 was knocked down, the protein expression level of iASPP also decreased. Similarly, the expression of FHL2 would reduce when iASPP was silenced. These results indicated that FHL2 might be a novel potential target for acute myelocytic leukemia treatment.
Project description:iASPP is an inhibitory member of the apoptosis-stimulating proteins of P53 (ASPP) family. iASPP is over expressed in several malignant tumors and potentially affects cancer progression. However, the expression and potential role of iASPP in oral tongue squamous cell carcinoma (OTSCC) have not been addressed. In our study, we detected iASPP expression in OTSCC by immunohistochemistry. iASPP expression is up-regulated in OTSCC tissues. Moreover, in clinical pathology specimens, we found that increased iASPP expression correlates with poor differentiation and lymph node metastasis. Using multicellular tumor spheroids (MTS) and flow cytometry, we demonstrated that iASPP down-regulation arrests OTSCC cells at the G0/G1 phase, induces OTSCC cell apoptosis and inhibits OTSCC cell proliferation. These results indicate that iASPP plays a significant role in the progression of OTSCC and may serve as a biomarker or therapeutic target for OTSCC patients.
Project description:BACKGROUND:Gestational trophoblastic disease (GTD) is a heterogeneous group of diseases developed from trophoblasts. ASPP (Ankyrin-repeat, SH3-domain and proline-rich region containing protein) family proteins, ASPP1 and ASPP2, have been reported to be dysregulated in GTD. They modulate p53 activities and are responsible for multiple cellular processes. Nevertheless, the functional role of the ASPP family inhibitory member, iASPP, is not well characterized in GTD. METHODS:To study the functional role of iASPP in GTD, trophoblastic tissues from normal placentas, hydatidiform mole (HM) and choriocarcinoma were used for immunohistochemistry, whereas siRNAs were used to manipulate iASPP expression in choriocarcinoma cell lines and study the subsequent molecular changes. RESULTS:We demonstrated that iASPP was overexpressed in both HM and choriocarcinoma when compared to normal placenta. Progressive increase in iASPP expression from HM to choriocarcinoma suggests that iASPP may be related to the development of trophoblastic malignancy. High iASPP expression in HM was also significantly associated with a high expression of autophagy-related protein LC3. Interestingly, iASPP silencing retarded the growth of choriocarcinoma through senescence instead of induction of apoptosis. LC3 expression decreased once iASPP was knocked down, suggesting a downregulation on autophagy. This may be due to iASPP downregulation rendered decrease in Atg5 expression and concomitantly hindered autophagy in choriocarcinoma cells. Autophagy inhibition per se had no effect on the growth of choriocarcinoma cells but increased the susceptibility of choriocarcinoma cells to oxidative stress, implying a protective role of iASPP against oxidative stress through autophagy in choriocarcinoma. CONCLUSIONS:iASPP regulates growth and the cellular responses towards oxidative stress in choriocarcinoma cells. Its overexpression is advantageous to the pathogenesis of GTD. (266 words).
Project description:iASPP, an inhibitory member of the ASPP (apoptosis stimulating protein of p53) family, is an evolutionarily conserved inhibitor of p53 which is frequently upregulated in human cancers. However, little is known about the role of iASPP under physiological conditions. Here, we report that iASPP is a critical regulator of epithelial development. We demonstrate a novel autoregulatory feedback loop which controls crucial physiological activities by linking iASPP to p63, via two previously unreported microRNAs, miR-574-3p and miR-720. By investigating its function in stratified epithelia, we show that iASPP participates in the p63-mediated epithelial integrity program by regulating the expression of genes essential for cell adhesion. Silencing of iASPP in keratinocytes by RNA interference promotes and accelerates a differentiation pathway, which also affects and slowdown cellular proliferation. Taken together, these data reveal iASPP as a key regulator of epithelial homeostasis.
Project description:Cytokinesis is the last step of cell division and is concluded by the abscission of the intercellular bridge that connects two daughter cells. The tight regulation of cytokinesis completion is essential because cytokinesis failure is associated with various human diseases. Here, we report that iASPP, a member of the apoptosis-stimulating proteins of p53 (ASPP) family, is required for proper cell division. iASPP depletion results in abnormal midbody structure and failed cytokinesis. We used protein affinity purification methods to identify the functional partners of iASPP. We found that iASPP associates with centrosomal protein of 55?kDa (CEP55), an important cytokinetic abscission regulator. Mechanically, iASPP acts as a PP1-targeting subunit to facilitate the interaction between PP1 and CEP55 and to remove PLK1-mediated Ser436 phosphorylation in CEP55 during late mitosis. The latter step is critical for the timely recruitment of CEP55 to the midbody. The present observations revealed a previously unrecognized function of iASPP in cytokinesis. This function, in turn, likely contributes to the roles of iASPP in tumor development and genetic diseases.
Project description:Suppression of apoptosis is a key Hallmark of cancer cells, and reactivation of apoptosis is a major avenue for cancer therapy. We reveal an interaction between the two anti-apoptotic proteins iASPP and NAF-1, which are overexpressed in many types of cancer cells and tumors. iASPP is an inhibitory member of the ASPP protein family, whereas NAF-1 belongs to the NEET 2Fe-2S protein family. We show that the two proteins are stimulated to interact in cells during apoptosis. Using peptide array screening and computational methods we mapped the interaction interfaces of both proteins to residues 764-778 of iASPP that bind to a surface groove of NAF-1. A peptide corresponding to the iASPP 764-780 sequence stabilized the NAF-1 cluster, inhibited NAF-1 interaction with iASPP, and inhibited staurosporine-induced apoptosis activation in human breast cancer, as well as in PC-3 prostate cancer cells in which p53 is inactive. The iASPP 764-780 IC50 value for inhibition of cell death in breast cancer cells was 13 ± 1 ?M. The level of cell death inhibition by iASPP 764-780 was altered in breast cancer cells expressing different levels and/or variants of NAF-1, indicating that the peptide activity is associated with NAF-1 function. We propose that the interaction between iASPP and NAF-1 is required for apoptosis activation in cancer cells. This interaction uncovers a new layer in the highly complex regulation of cell death in cancer cells and opens new avenues of exploration into the development of novel anticancer drugs that reactivate apoptosis in malignant tumors.
Project description:Inhibitor of apoptosis-stimulating protein of p53 (iASPP) is the most ancient member of the ASPP family of proteins and an evolutionarily conserved inhibitor of p53. iASPP is also a binding partner and negative regulator of p65RelA. Because p65RelA and the p53 family members often have opposite effects in controlling cell fate, it is important to understand the cellular context in which iASPP can regulate their activities. To address this question and to study the biological importance of iASPP in vivo, we generated a transgenic mouse in which iASPP expression is controlled by the Cre/loxP recombination system. We observed that iASPP is able to prevent premature cellular senescence in mouse embryonic fibroblasts. iASPP loss resulted in increased differentiation of primary keratinocytes both in vitro and in vivo. In stratified epithelia, nuclear iASPP often colocalized with p63 in the nuclei of basal keratinocytes. Consistent with this, iASPP bound p63 and inhibited the transcriptional activity of both TAp63? and ?Np63? in vitro and influenced the expression level of p63-regulated genes such as loricrin and involucrin in vivo. In contrast, under the same conditions, p65RelA was frequently expressed as a cytoplasmic protein in the suprabasal layers of stratified epithelia and rarely colocalized with nuclear iASPP. Thus, iASPP is likely to control epithelial stratification by regulating p63's transcriptional activity, rather than p65RelA's. This study identifies iASPP as an inhibitor of senescence and a key player in controlling epithelial stratification.
Project description:p53-dependent apoptosis is modulated by the ASPP family of proteins (apoptosis-stimulating proteins of p53; also called ankyrin repeat-, Src homology 3 domain-, and Pro-rich region-containing proteins). Its three known members, ASPP1, ASPP2, and iASPP, were previously found to interact with p53, influencing the apoptotic response of cells without affecting p53-induced cell cycle arrest. More specifically, the bona fide tumor suppressors, ASPP1 and ASPP2, bind to the core domain of p53 and stimulate transcription of apoptotic genes, whereas oncogenic iASPP also binds to the p53 core domain but inhibits p53-dependent apoptosis. Although the general interaction regions are known, details of the interfaces for each p53-ASPP complex have not been evaluated. We undertook a comprehensive biophysical characterization of ASPP-p53 complex formation and mapped the binding interfaces by NMR. We found that the interaction interface on p53 for the proapoptotic protein ASPP2 is distinct from that for the antiapoptotic iASPP. ASPP2 primarily binds to the core domain of p53, whereas iASPP predominantly interacts with a linker region adjacent to the core domain. Our detailed structural analyses of the ASPP-p53 interactions provide insight into the structural basis of the differential behavior of pro- and antiapoptotic ASPP family members.
Project description:ASPP (apoptosis-stimulating proteins of p53) proteins bind PP-1c (protein phosphatase 1) and regulate p53 impacting cancer cell growth and apoptosis. Here we determine the crystal structure of the oncogenic ASPP protein, iASPP, bound to PP-1c. The structure reveals a 1:1 complex that relies on interactions of the iASPP SILK and RVxF motifs with PP-1c, plus interactions of the PP-1c PxxPxR motif with the iASPP SH3 domain. Small-angle X-ray scattering analyses suggest that the crystal structure undergoes slow interconversion with more extended conformations in solution. We show that iASPP, and the tumor suppressor ASPP2, enhance the catalytic activity of PP-1c against the small-molecule substrate, pNPP as well as p53. The combined results suggest that PxxPxR binding to iASPP SH3 domain is critical for complex formation, and that the modular ASPP-PP-1c interface provides dynamic flexibility that enables functional binding and dephosphorylation of p53 and other diverse protein substrates.
Project description:We present a possible molecular basis for the opposite activity of two homologues proteins that bind similar ligands and show that this is achieved by fine-tuning of the interaction interface. The highly homologous ASPP proteins have opposite roles in regulating apoptosis: ASPP2 induces apoptosis while iASPP inhibits it. The ASPP proteins are regulated by an autoinhibitory interaction between their Ank-SH3 and Pro domains. We performed a detailed biophysical and molecular study of the Pro - Ank-SH3 interaction in iASPP and compared it to the interaction in ASPP2. We found that iASPP Pro is disordered and that the interaction sites are entirely different: iASPP Ank-SH3 binds iASPP Pro via its fourth Ank repeat and RT loop while ASPP2 Ank-SH3 binds ASPP2 Pro via its first Ank repeat and the n-src loop. It is possible that by using different moieties in the same interface, the proteins can have distinct and specific interactions resulting in differential regulation and ultimately different biological activities.
Project description:The t(10;11)(p13;q14) translocation results in the fusion of the CALM (clathrin assembly lymphoid myeloid leukemia protein) and AF10 genes. This translocation is observed in acute myeloblastic leukemia (AML M6), acute lymphoblastic leukemia (ALL) and malignant lymphoma. Using a yeast two-hybrid screen, the four and a half LIM domain protein 2 (FHL2) was identified as a CALM interacting protein. Recently, high expression of FHL2 in breast, gastric, colon, lung as well as in prostate cancer was shown to be associated with an adverse prognosis. The interaction between CALM and FHL2 was confirmed by glutathione S-transferase-pulldown assay and co-immunoprecipitation experiments. The FHL2 interaction domain of CALM was mapped to amino acids 294-335 of CALM. The transcriptional activation capacity of FHL2 was reduced by CALM, but not by CALM/AF10, which suggests that regulation of FHL2 by CALM might be disturbed in CALM/AF10-positive leukemia. Extremely high expression of FHL2 was seen in acute erythroid leukemia (AML M6). FHL2 was also highly expressed in chronic myeloid leukemia and in AML with complex aberrant karyotype. These results suggest that FHL2 may play an important role in leukemogenesis, especially in the case of AML M6.