Control of Oncogenic Signaling Through Spatial Organization of Kinases and mRNAs

通过激酶和 mRNA 的空间组织控制致癌信号传导

基本信息

  • 批准号:
    10926503
  • 负责人:
  • 金额:
    $ 147.21万
  • 依托单位:
  • 依托单位国家:
    美国
  • 项目类别:
  • 财政年份:
  • 资助国家:
    美国
  • 起止时间:
  • 项目状态:
    未结题

项目摘要

Our research is organized into two inter-related subprojects. The first focuses on how 3'UTR sequences in mRNAs encoding substrates of oncogenic kinases such as ERK and CK2 regulate access of these proteins to their kinases in tumor cells. The 3'UTR mechanism involves subcellular mRNA localization, which can either inhibit or stimulate phosphorylation of the encoded protein by controlling access to kinases. The kinases in turn display different localization in normal and transformed cells. The second subproject involves investigating how oncogenic kinases partition to the perinuclear cytoplasm in tumor cells to form perinuclear signaling centers, or PSCs. PSCs serve as critical signaling engines that drive malignant transformation and cancer. 1. 3'UTR regulation of protein activity (UPA) and its underlying mechanisms. 3'UTR regulation of protein activity was discovered through our earlier observation that the CEBPB 3'UTR inhibits RAS-induced post-translational activation of the C/EBPb protein in tumor cells. Thus, the 3'UTR uncouples C/EBPb from RAS signaling, thereby constraining its pro-senescence activity. UPA also represses the ability of C/EBPb to activate transcription of pro-inflammatory senescence-associated secretory phenotype (SASP) genes. UPA requires a long G/U-rich element (GRE) motif in the 3'UTR and its cognate binding protein, HuR/ELAV1. These components exclude CEBPB mRNAs from a perinuclear cytoplasmic compartment where ERK1/2 and CK2 are present on signaling endosomes embedded within the ER network. Hence, newly translated C/EBPb is prevented from accessing its kinases. We later found that the RNA decay proteins UPF1 and Staufen (STAU1/2) are also essential UPA factors and are enriched within the perinuclear cytoplasm. Together with HuR, these proteins promote perinuclear mRNA decay (PMD) of CEBPB transcripts. Depletion of UPF1 or STAU in tumor cells increased the nuclear-proximal population of CEBPB transcripts, leading to C/EBPb phosphorylation on its CK2 site and senescence. High resolution imaging showed that the perinuclear CEBPB mRNAs frequently colocalize with CK2 foci. These observations suggest that when UPA is disabled, C/EBPb undergoes phosphorylation by CK2 which is contingent on close proximity of CEBPB transcripts (and thus newly translated CEBPb) with activating kinases. We identified a STAU binding site (SBS) adjacent to the GRE which, when deleted, activates the pro-senescence functions of C/EBPb and phosphorylation by CK2 but not its ability to induce SASP genes. Furthermore, deletion of the GRE alone also leads to C/EBPb-induced senescence but not phosphorylation by CK2. These and other observations imply that distinct 3'UTR sequences repress different C/EBPb functions, likely by differential inhibition of PTMs. We propose that various mRNA decay factors (e.g., STAU) which recognize discrete 3'UTR sequences are tethered to different types of signaling endosomes; e.g., those carrying ERK, CK2 or other kinases. Hence, individual 3'UTR elements may promote mRNA decay in the vicinity of particular kinases and thereby inhibit protein phosphorylation on specific sites. Future work will expand upon this novel relationship between modular 3'UTR motifs, localized mRNA decay and inhibition of specific PTMs on the encoded protein. To examine the in vivo relevance of UPA, we generated mice with a deletion that removes the Cebpb GRE and part of the adjacent SBS. This mutant strain was tested in a Kras model of lung tumorigenesis. Although overall lung tumor burdens in deltaGRE mice were similar to WT animals, regions of malignant adenocarcinoma were significantly reduced. Benign lesions such as adenomas were unaffected. These findings provide the first in vivo evidence that UPA constrains C/EBPb activity to facilitate tumor progression to carcinomatous lesions. We are currently performing senescent cell analysis and RNA-seq studies on tumors of different stages in the two genotypes to assess whether the delGRE mutation increases senescence and how this allele alters the C/EBPb transcriptome. A key goal of our work is to determine whether UPA is a general mechanism that regulates many proteins. We are using CRISPR-mediated deletion of 3'UTRs and 3'UTR swap strategies to identify other UPA-regulated genes. p53 is one such candidate. Its3'UTR suppresses the cytostatic activity of a p53 in RAS tumor cells, without affecting p53 protein levels, and excludes TP53 mRNAs from the kinase-rich perinuclear region, inhibiting phosphorylation on its CK2 site, Ser392. TP53 transcripts partition away from the nuclear-proximal region in tumor cells. However, TP53 mRNAs undergo perinuclear translocation in exposed to chemotherapeutic DNA damaging agents that induce p53-dependent senescence or apoptosis, coinciding with increased Ser392 phosphorylation. In summary, our findings demonstrate that 3'UTR-dependent changes in mRNA localization control the activity of p53, C/EBPb, and probably many other proteins. 2. Mechanisms and function of perinuclear signaling centers (PSCs) as key signaling engines in cancer cells. Oncogenic RAS induces perinuclear translocation of p-ERK and CK2 and the signaling scaffold KSR1. These proteins form signaling hubs on endosomes tethered to the ER network. These PSCs are critical to the 3'UTR (UPA) mechanism and are observed in all cancer cell lines and tumor tissues tested. PSCs are key signaling engines that drive cancer, allowing oncogenic kinases to access targets that are important for neoplastic transformation. We found that the endosomal adaptor TOLLIP is required for perinuclear localization of RAB11A+ endosomes harboring CK2 and KSR1, but not ERK. ERK resides on a different class of signaling endosomes. TOLLIP is perinuclear in human cancer cells and KRasG12D-driven mouse tumors but is pan-cytoplasmic in non-transformed cells and thus coincides with the presence of PSCs. A conserved "linker" region in TOLLIP mediates interactions with KSR1 pseudo-kinase domain. This association recruits CK2 signaling complexes to endosomes. A series of phosphoproteomic experiments showed that perinuclear CK2 phosphorylates selective substrates, including proteins involved in ribosome biogenesis and translation. One such target is the atypical kinase RIOK1, which regulates 18S rRNA processing and 40S subunit maturation. Mutant analysis suggests that phosphorylation on RIOK1 Ser22 by perinuclear CK2 is essential for RIOK1 function in tumor cells. KRasG12D-driven lung tumors in Tollip-/- mice progress less efficiently to the malignant adenocarcinoma stage. Furthermore, tumor cell lines carrying mutant KRAS or NRAS, but not HRAS or BRAF mutations, require TOLLIP for proliferation/survival. TOLLIP is therefore a key signaling adaptor in K/NRAS tumor cells whose inhibition is a potential vulnerability of these aggressive, treatment-resistant cancers. in collaboration with Dr. Nadya Tarasova (CIL), we are identifying small molecules that are projected to dock in TOLLIP pockets. One such candidate shows potent inhibition of proliferation/survival in KRAS mutant cancer cells. This and other compounds that that block PSC formation in tumor cells will be considered for further development as anti-cancer agents. As cancer cells driven by mutant HRAS, BRAF and other oncogenes display reduced dependence on TOLLIP but nevertheless exhibit perinuclear CK2, we believe that an alternative adaptor protein(s) provides a redundant perinuclear tethering function in these cells. Furthermore, ERK PSCs remain perinuclear in the absence of TOLLIP. Therefore, our future research will include identifying and characterizing additional endosomal adaptors involved in PSC formation by ERK, CK2 and other components of the RAS pathway.
我们的研究被组织成两个相关的亚参数。第一个侧重于在编码诸如ERK和CK2等致癌激酶底物的mRNA中的3'UTR序列如何调节这些蛋白在肿瘤细胞中的激酶的访问。 3'UTR机制涉及亚细胞mRNA定位,可以通过控制对激酶的访问来抑制或刺激编码蛋白的磷酸化。激酶依次在正常和转化的细胞中显示出不同的定位。第二次次要注射涉及研究肿瘤细胞中的致癌激酶如何与肿瘤细胞中核周细胞质分配,以形成核周信号传导中心或PSC。 PSC是驱动恶性转化和癌症的关键信号发动机。 1。3'UTR调节蛋白质活性(UPA)及其基本机制。 3'UTR调节蛋白活性是通过我们较早的观察结果发现的,即CEBPB 3'UTR抑制RAS诱导的肿瘤细胞中C/EBPB蛋白的翻译后激活。因此,3'UTR从RAS信号传导中c/eBPB c/eBPB,从而限制了其渐染活性。 UPA还抑制了C/EBPB激活促炎性衰老相关分泌表型(SASP)基因转录的能力。 UPA需要3'UTR中的长G/U元素(GRE)基序及其同源结合蛋白HUR/ELAV1。这些成分将CEBPB mRNA从核周细胞室中排除,其中ERK1/2和CK2存在于嵌入ER网络中的信号内体上。因此,新翻译的C/EBPB被阻止了其激酶。后来,我们发现RNA衰减蛋白UPF1和Staufen(STAU1/2)也是必不可少的UPA因子,并且在核周细胞质内富集。这些蛋白与HUR一起促进CEBPB转录本的核周核mRNA衰变(PMD)。肿瘤细胞中UPF1或Stau的耗竭增加了CEBPB转录本的核氧化群,导致其CK2位点和衰老的C/EBPB磷酸化。高分辨率成像表明,核周CEBPB mRNA经常与CK2焦点共定位。这些观察结果表明,当UPA被禁用时,C/EBPB会通过CK2进行磷酸化,这取决于CEBPB转录本的紧密近端(因此是与激活的激酶新翻译的CEBPB)。我们确定了与GRE相邻的Stau结合位点(SBS),该位点(SBS)被删除后激活C/EBPB和CK2磷酸化的促染色功能,而不是其诱导SASP基因的能力。此外,仅GRE的缺失也会导致C/EBPB诱导的衰老,但不会导致CK2磷酸化。这些和其他观察结果表明,不同的3'UTR序列抑制了不同的C/EBPB函数,这可能是由于PTM的差异抑制。我们提出,识别离散3'UTR序列的各种mRNA衰减因子(例如,stau)被束缚在不同类型的信号内体上。例如,那些携带ERK,CK2或其他激酶的人。因此,单个3'UTR元素可能会促进特定激酶附近的mRNA衰变,从而抑制特定部位的蛋白质磷酸化。未来的工作将扩展到模块化3'UTR基序,局部mRNA衰变和对编码蛋白上特定PTM的抑制之间的新型关系。为了检查UPA的体内相关性,我们用缺失产生了删除CEBPB GRE和相邻SB的一部分的小鼠。该突变菌株在肺肿瘤发生的KRAS模型中进行了测试。尽管三角洲小鼠的总体肺肿瘤负担与WT动物相似,但恶性腺癌区域显着降低。良性病变(例如腺瘤)不受影响。这些发现提供了第一个体内证据,即UPA限制了C/EBPB活性以促进肿瘤发展为癌变。目前,我们正在对两种基因型不同阶段肿瘤的衰老细胞分析和RNA-seq研究进行评估,以评估Delgre突变是否会增加衰老以及该等位基因如何改变C/EBPB转录组。我们工作的关键目标是确定UPA是否是调节许多蛋白质的一般机制。我们正在使用CRISPR介导的3'UTR和3'UTR交换策略的缺失来识别其他受调的基因。 p53就是这样的候选人。 ITS3'UTR抑制了RAS肿瘤细胞中p53的胞抑制活性,而不会影响p53蛋白水平,并排除了来自富含激酶的核周核区域的TP53 mRNA,抑制了其CK2位点上的磷酸化,SER392,SER392。 TP53转录本从肿瘤细胞中的核能区域分开。然而,TP53 mRNA在暴露于化学治疗DNA损伤剂的暴露于p53依赖性衰老或细胞凋亡的情况下经历核周易位,与Ser392磷酸化的增加相吻合。总而言之,我们的发现表明,mRNA定位的3'UTR依赖性变化控制p53,c/eBPB以及许多其他蛋白质的活性。 2。核周信号传导中心(PSC)作为癌细胞中的关键信号发动机的机制和功能。致癌性RAS诱导P-ERK和CK2的核周易位以及信号支架KSR1。这些蛋白质在内体上形成信号枢纽,这些蛋白会束缚在ER网络上。这些PSC对于3'UTR(UPA)机制至关重要,并且在所有癌细胞系和肿瘤组织中都观察到。 PSC是驱动癌症的关键信号发动机,使致癌激酶能够访问对肿瘤转化很重要的靶标。我们发现,内体适配器Tollip是携带CK2和KSR1的Rab11a+内体的核糖核定位置所必需的,但不是ERK。 ERK位于不同类别的信号内体上。 Tollip在人类癌细胞和KRASG12D驱动的小鼠肿瘤中是核周的,但在非转化细胞中是泛胞质的,因此与PSC的存在相吻合。 Tollip中保守的“接头”区域介导了与KSR1伪激酶结构域的相互作用。该关联将CK2信号复合物募集到内体。一系列磷酸蛋白质组学实验表明,核周CK2磷酸化选择性底物,包括参与核糖体生物发生和翻译的蛋白质。这样的靶标是非典型激酶RIOK1,它调节18S rRNA加工和40S亚基成熟。突变分析表明,核周CK2对RIOK1 SER22的磷酸化对于肿瘤细胞中的RIOK1功能至关重要。 Tollip中的KRASG12D驱动的肺部肿瘤 - / - 小鼠的进展较小,效率较小。此外,携带突变体KRAS或NRA的肿瘤细胞系,但不需要HRA或BRAF突变,需要Tollip才能增殖/存活。因此,Tollip是K/NRAS肿瘤细胞中的一个关键信号适配器,其抑制作用是这些侵略性,耐药性癌症的潜在脆弱性。与Nadya Tarasova博士(CIL)合作,我们正在确定预计将在Tollip口袋里停靠的小分子。这样的候选者显示出对KRAS突变癌细胞增殖/存活的有效抑制。这种阻断肿瘤细胞中PSC形成的这种化合物将被视为抗癌剂的进一步发展。由于由突变HRA驱动的癌细胞,BRAF和其他癌基因对Tollip的依赖性降低,但仍表现出核周核CK2,我们相信替代的衔接蛋白(S)在这些细胞中提供了冗余的核周链球菌功能。此外,在没有Tollip的情况下,ERK PSC仍然保持核糖周核。因此,我们未来的研究将包括识别和表征ERK,CK2和RAS途径其他组件参与PSC形成的其他内体适配器。

项目成果

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peter f johnson其他文献

peter f johnson的其他文献

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{{ truncateString('peter f johnson', 18)}}的其他基金

Role of CEBP transcription factors in regulating cell growth and tumorigenesis
CEBP转录因子在调节细胞生长和肿瘤发生中的作用
  • 批准号:
    8763075
  • 财政年份:
  • 资助金额:
    $ 147.21万
  • 项目类别:
Role of CEBP transcription factors in regulating cell growth and tumorigenesis
CEBP转录因子在调节细胞生长和肿瘤发生中的作用
  • 批准号:
    9153546
  • 财政年份:
  • 资助金额:
    $ 147.21万
  • 项目类别:
Role of CEBP transcription factors in cell growth and tumorigenesis
CEBP转录因子在细胞生长和肿瘤发生中的作用
  • 批准号:
    9556264
  • 财政年份:
  • 资助金额:
    $ 147.21万
  • 项目类别:
Role of CEBP transcription factors in cell growth and tumorigenesis
CEBP转录因子在细胞生长和肿瘤发生中的作用
  • 批准号:
    10262069
  • 财政年份:
  • 资助金额:
    $ 147.21万
  • 项目类别:
Role of CEBP transcription factors in regulating cell growth and tumorigenesis
CEBP转录因子在调节细胞生长和肿瘤发生中的作用
  • 批准号:
    8552666
  • 财政年份:
  • 资助金额:
    $ 147.21万
  • 项目类别:
Role of CEBP transcription factors in regulating cell growth and tumorigenesis
CEBP转录因子在调节细胞生长和肿瘤发生中的作用
  • 批准号:
    7965252
  • 财政年份:
  • 资助金额:
    $ 147.21万
  • 项目类别:
Role of CEBP transcription factors in regulating cell growth and tumorigenesis
CEBP转录因子在调节细胞生长和肿瘤发生中的作用
  • 批准号:
    8348976
  • 财政年份:
  • 资助金额:
    $ 147.21万
  • 项目类别:
Role of CEBP transcription factors in regulating cell gr
CEBP转录因子在调节细胞GR中的作用
  • 批准号:
    7291710
  • 财政年份:
  • 资助金额:
    $ 147.21万
  • 项目类别:
Role of CEBP transcription factors in regulating cell growth and tumorigenesis
CEBP转录因子在调节细胞生长和肿瘤发生中的作用
  • 批准号:
    8937710
  • 财政年份:
  • 资助金额:
    $ 147.21万
  • 项目类别:
Role of CEBP transcription factors in regulating cell growth and tumorigenesis
CEBP转录因子在调节细胞生长和肿瘤发生中的作用
  • 批准号:
    8175307
  • 财政年份:
  • 资助金额:
    $ 147.21万
  • 项目类别:

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Regulation of Human Tumorigensis by Cancer Specific NXF1 Adaptor Proteins
癌症特异性 NXF1 接头蛋白对人类肿瘤发生的调节
  • 批准号:
    10411472
  • 财政年份:
    2022
  • 资助金额:
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  • 项目类别:
Regulation of Human Tumorigensis by Cancer Specific NXF1 Adaptor Proteins
癌症特异性 NXF1 接头蛋白对人类肿瘤发生的调节
  • 批准号:
    10596156
  • 财政年份:
    2022
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METTL3-NUP93 interaction facilitates the nuclear export of m6A-modified mRNAs
METTL3-NUP93 相互作用促进 m6A 修饰 mRNA 的核输出
  • 批准号:
    10461179
  • 财政年份:
    2021
  • 资助金额:
    $ 147.21万
  • 项目类别:
METTL3-NUP93 interaction facilitates the nuclear export of m6A-modified mRNAs
METTL3-NUP93 相互作用促进 m6A 修饰 mRNA 的核输出
  • 批准号:
    10299046
  • 财政年份:
    2021
  • 资助金额:
    $ 147.21万
  • 项目类别:
Transcript-selective translational control of Th17 cell development and function
Th17 细胞发育和功能的转录选择性翻译控制
  • 批准号:
    10373280
  • 财政年份:
    2021
  • 资助金额:
    $ 147.21万
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