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14-3-3? interacts with stat3 and regulates its constitutive activation in multiple myeloma cells.

ABSTRACT: The 14-3-3 proteins are a family of regulatory signaling molecules that interact with other proteins in a phosphorylation-dependent manner and function as adapter or scaffold proteins in signal transduction pathways. One family member, 14-3-3?, is believed to function in cell signaling, cycle control, and apoptotic death. A systematic proteomic analysis done in our laboratory has identified signal transducers and activators of transcription 3 (Stat3) as a novel 14-3-3? interacting protein. Following our initial finding, in this study, we provide evidence that 14-3-3? interacts physically with Stat3. We further demonstrate that phosphorylation of Stat3 at Ser727 is vital for 14-3-3? interaction and mutation of Ser727 to Alanine abolished 14-3-3?/Stat3 association. Inhibition of 14-3-3? protein expression in U266 cells inhibited Stat3 Ser727 phosphorylation and nuclear translocation, and decreased both Stat3 DNA binding and transcriptional activity. Moreover, 14-3-3? is involved in the regulation of protein kinase C (PKC) activity and 14-3-3? binding to Stat3 protects Ser727 dephosphorylation from protein phosphatase 2A (PP2A). Taken together, our findings support the model that multiple signaling events impinge on Stat3 and that 14-3-3? serves as an essential coordinator for different pathways to regulate Stat3 activation and function in MM cells.

SUBMITTER: Zhang J

PROVIDER: S-EPMC3261159 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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14-3-3ζ interacts with stat3 and regulates its constitutive activation in multiple myeloma cells.

Zhang Jia J Chen Fangjin F Li Wenliang W Xiong Qian Q Yang Mingkun M Zheng Peng P Li Chongyang C Pei Jianfeng J Ge Feng F

PloS one 20120118 1

The 14-3-3 proteins are a family of regulatory signaling molecules that interact with other proteins in a phosphorylation-dependent manner and function as adapter or scaffold proteins in signal transduction pathways. One family member, 14-3-3ζ, is believed to function in cell signaling, cycle control, and apoptotic death. A systematic proteomic analysis done in our laboratory has identified signal transducers and activators of transcription 3 (Stat3) as a novel 14-3-3ζ interacting protein. Follo ...[more]

PMID: 22279540

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Project description:BackgroundThe survival of INA-6 human multiple myeloma cells is strictly dependent upon the Interleukin-6-activated transcription factor STAT3. Although transcriptional analyses have revealed many genes regulated by STAT3, to date no protein-coding STAT3 target gene is known to mediate survival in INA-6 cells. Therefore, the aim here was to identify and analyze non-protein-coding STAT3 target genes. In addition to the oncogenic microRNA-21, we previously described five long noncoding RNAs (lncRNAs) induced by STAT3, named STAiRs. Here, we focus on STAT3-induced RNA 18 (STAiR18), an mRNA-like, long ncRNA that is duplicated in the human lineage. One STAiR18 locus is annotated as the already well described LINC00152/CYTOR, however, the other harbors the MIR4435-2HG gene and is, up to now, barely described.MethodsCAPTURE-RNA-sequencing was used to analyze STAiR18 transcript architecture. To identify the STAiR18 and STAT3 phenotype, siRNA-based knockdowns were performed and microarrays were applied to identify their target genes. RNA-binding partners of STAiR18 were determined by Chromatin-Isolation-by-RNA-Purification (ChIRP) and subsequent sequencing. STAT3 expression in dependence of STAiR18 was investigated by immunoblots, chromatin- and RNA-immunoprecipitations.ResultsAs identified by CAPTURE-RNA sequencing, a complex splice pattern originates from both STAiR18 loci, generating different transcripts. Knockdown of the most abundant STAiR18 isoforms dramatically decreased INA-6 cell vitality, suggesting a functional role in myeloma cells. Additionally, STAiR18 and STAT3 knockdowns yielded overlapping changes of transcription patterns in INA-6 cells, suggesting a close functional interplay between the two factors. Moreover, Chromatin isolation by RNA purification (ChIRP), followed by genome-wide RNA sequencing showed that STAiR18 associates specifically with the STAT3 primary transcript. Furthermore, the knockdown of STAiR18 reduced STAT3 levels on both the RNA and protein levels, suggesting a positive feedback between both molecules. Furthermore, STAiR18 knockdown changes the histone methylation status of the STAT3 locus, which explains the positive feedback and indicates that STAiR18 is an epigenetic modulator.ConclusionHence, STAiR18 is an important regulator of myeloma cell survival and is strongly associated with the oncogenic function of STAT3. The close functional interplay between STAT3 and STAiR18 suggests a novel principle of regulatory interactions between long ncRNAs and signaling pathways.

Project description:BackgroundHeme oxygenase 1 (HO-1) has been reported to be very important in the pathogenesis or progression of multiple types of cancer. Identification of novel hmox1 binding proteins may reveal undefined oncogenes, tumor suppressors, signaling pathways, and possible treatment targets.MethodsImmunoprecipitation and mass spectrometry analyses were used to identify novel regulators of HO-1. The association of the 14-3-3ζ protein with HO-1 and modulation of the stability of HO-1 were investigated by co-immunoprecipitation, immunofluorescence, western blotting, and quantitative RT-PCR. Degradation and in vivo ubiquitination assays were utilized to examine whether 14-3-3ζ stabilizes the HO-1 protein by inhibiting its ubiquitination. The effect of 14-3-3ζ on proliferation was investigated by function assays conducted in vitro using the CCK-8 and colony formation assays and in vivo in a xenograft mouse model. The biological functions of the 14-3-3ζ/HO-1 axis were demonstrated by western blotting and rescue experiments. Using gain-of-function and loss-of-function strategies, we further clarified the impact of 14-3-3ζ/HO-1 complex on the signal transducers and activators of transcription 3 (STAT3) signaling pathway in cancer cells.ResultsWe identified 14-3-3ζ as a novel HO-1 binding protein. The binding inhibited the ubiquitination and proteasome-mediated degradation of HO-1, thus facilitating its stabilization. Enforced expression of 14-3-3ζ significantly promoted cell proliferation in vitro, as well as tumorigenesis in vivo, while 14-3-3ζ knockdown had opposite effects. The data indicated that 14-3-3ζ can stabilize HO-1 expression and thus influence cancer cell proliferation. We further demonstrated the involvement of the STAT3 pathway in 14-3-3ζ/HO-1 regulation of hepatocellular carcinoma cell proliferation.ConclusionsCollectively, these data show that 14-3-3ζ regulates the stability of HO-1 to promote cancer cell proliferation and STAT3 signaling activation. The data establish the 14-3-3ζ-HO-1-STAT3 axis as an important regulatory mechanism of cancer cell growth and implicate HO-1 and 14-3-3ζ as potential therapeutic targets in hepatocellular carcinoma.

Dataset Information

14-3-3? interacts with stat3 and regulates its constitutive activation in multiple myeloma cells.

Publications

14-3-3ζ interacts with stat3 and regulates its constitutive activation in multiple myeloma cells.

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