Functional phosphorylation sites in the C-terminal region of the multivalent multifunctional transcriptional factor CTCF
Functional phosphorylation sites in the C-terminal region of the multivalent multifunctional transcriptional factor CTCF
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DOI：
10.1128/mcb.21.6.2221-2234.2001
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发表时间：
2001-03-01
影响因子：
5.3
通讯作者：
Lobanenkov, VV
中科院分区：
文献类型：
--
作者：
Klenova, EM;Chernukhin, IV;Lobanenkov, VV
CTCF是一种广泛表达且高度保守的多锌指（ZF）核因子。与各种CTCF靶位点（CTS）的结合由不同ZF的组合贡献介导。不同的CTS介导不同的CTCF功能的转录调控，包括启动子抑制或激活和转录响应基因沉默。此外，必需的和足够的核心序列的不同增强子阻断（绝缘子）的元素，包括CpG甲基化敏感的，最近被精确定位到CTS。为了确定翻译后修饰是否可以调节CTCF功能，我们研究了CTCF磷酸化。我们证明，体内羧基末端发生的大多数修饰都可以在体外用酪蛋白激酶II（CKII）复制。主要修饰位点映射到在脊椎动物中高度保守的（SKKEDSSDSE）-K-604-S-609-D-610-E-612基序内的四个丝氨酸。这些丝氨酸的特定突变消除CTCF在体内的磷酸化和CKII诱导的体外磷酸化。此外，我们表明，完全防止磷酸化取代所有丝氨酸在这个网站导致显着增强抑制的CTS轴承脊椎动物c-myc启动子，但没有改变CTCF核定位或在体外DNA结合特性与c-myc CTS测定。此外，这些取代表现出对野生型CTCF的负细胞生长调节的深远影响。因此，CKII可能是CTCF活性减弱的原因，它要么自己起作用，要么通过提供其他激酶磷酸化信号和CTCF相互作用蛋白伴侣起作用。
CTCF is a widely expressed and highly conserved multi-Zn-finger (ZF) nuclear factor. Binding to various CTCF target sites (CTSs) is mediated by combinatorial contributions of different ZFs. Different CTSs mediate distinct CTCF functions in transcriptional regulation, including promoter repression or activation and hormone-responsive gene silencing. In addition, the necessary and sufficient core sequences of diverse enhancer-blocking (insulator) elements, including CpG methylation-sensitive ones, have recently been pinpointed to CTSs. To determine whether a posttranslational modification may modulate CTCF functions, we studied CTCF phosphorylation. We demonstrated that most of the modifications that occur at the carboxy terminus in vivo can be reproduced in vitro with casein kinase II (CKII). Major modification sites map to four serines within the (SKKEDSSDSE)-K-604-S-609-D-610-E-612 motif that is highly conserved in vertebrates. Specific mutations of these serines abrogate phosphorylation of CTCF in vivo and CKII-induced phosphorylation in vitro. In addition, we showed that completely preventing phosphorylation by substituting all serines within this site resulted in markedly enhanced repression of the CTS-bearing vertebrate c-myc promoters, but did not alter CTCF nuclear localization or in vitro DNA-binding characteristics assayed with c-myc CTSs. Moreover, these substitutions manifested a profound effect on negative cell growth regulation by wild-type CTCF. CKII may thus be responsible for attenuation of CTCF activity, either acting on its own or by providing the signal for phosphorylation by other kinases and for CTCF-interacting protein partners.