Mahmoudi Tokameh, Li Vivian S W, Ng Ser Sue, Taouatas Nadia, Vries Robert G J, Mohammed Shabaz, Heck Albert J, Clevers Hans
Hubrecht Institute-KNAW and University Medical Centre Utrecht, Utrecht, The Netherlands.
EMBO J. 2009 Nov 4;28(21):3329-40. doi: 10.1038/emboj.2009.285. Epub 2009 Oct 8.
Wnt signalling maintains the undifferentiated state of intestinal crypt/progenitor cells through the TCF4/beta-catenin-activating transcriptional complex. In colorectal cancer, activating mutations in Wnt pathway components lead to inappropriate activation of the TCF4/beta-catenin transcriptional programme and tumourigenesis. The mechanisms by which TCF4/beta-catenin activate key target genes are not well understood. Using a proteomics approach, we identified Tnik, a member of the germinal centre kinase family as a Tcf4 interactor in the proliferative crypts of mouse small intestine. Tnik is recruited to promoters of Wnt target genes in mouse crypts and in Ls174T colorectal cancer cells in a beta-catenin-dependent manner. Depletion of TNIK and expression of TNIK kinase mutants abrogated TCF-LEF transcription, highlighting the essential function of the kinase activity in Wnt target gene activation. In vitro binding and kinase assays show that TNIK directly binds both TCF4 and beta-catenin and phosphorylates TCF4. siRNA depletion of TNIK followed by expression array analysis showed that TNIK is an essential, specific activator of Wnt transcriptional programme. This kinase may present an attractive candidate for drug targeting in colorectal cancer.
Wnt信号通路通过TCF4/β-连环蛋白激活转录复合物维持肠道隐窝/祖细胞的未分化状态。在结直肠癌中，Wnt通路成分的激活突变导致TCF4/β-连环蛋白转录程序的不适当激活和肿瘤发生。目前尚不清楚TCF4/β-连环蛋白激活关键靶基因的机制。我们采用蛋白质组学方法，在小鼠小肠增殖隐窝中鉴定出原生发中心激酶家族成员Tnik是Tcf4的相互作用蛋白。Tnik以β-连环蛋白依赖的方式被招募到小鼠隐窝和Ls174T结直肠癌细胞中Wnt靶基因的启动子上。TNIK的缺失和TNIK激酶突变体的表达消除了TCF-LEF转录，突出了激酶活性在Wnt靶基因激活中的重要作用。体外结合和激酶分析表明，TNIK直接结合TCF4和β-连环蛋白并使TCF4磷酸化。用小干扰RNA（siRNA）敲低TNIK后进行表达阵列分析表明，TNIK是Wnt转录程序的必需特异性激活剂。这种激酶可能是结直肠癌药物靶向治疗的一个有吸引力的候选靶点。
