Phosphorylation of ORF1p is required for L1 retrotransposition
Phosphorylation of ORF1p is required for L1 retrotransposition
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DOI：
10.1073/pnas.1416869112
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发表时间：
2015-04-07
影响因子：
11.1
通讯作者：
Furano, Anthony V.
中科院分区：
文献类型：
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作者：
Cook, Pamela R.;Jones, Charles E.;Furano, Anthony V.
尽管L1(line-1)非LTR反转录转座子L1分支的成员可能是有害的，但L1分支在大多数哺乳动物中保持活跃已有近1亿年的时间，并产生了近40%的人类基因组。然而，L1与宿主相互作用的细节在很大程度上是未知的。在这里，我们报告L1的活性需要L1 ORF1(ORF1p)编码的蛋白质的磷酸化。关键的磷酸受体残基(两个丝氨酸和两个苏氨酸)位于四个保守的脯氨酸导向蛋白激酶(PDPK)靶点。PDPK家族包括丝裂原活化蛋白激酶和细胞周期蛋白依赖性激酶。任何PDPK磷酸受体的突变都会抑制L1逆转录转座。拟磷天冬氨酸可以恢复两个丝氨酸位置的活性，但不能恢复苏氨酸位置的活性，因为它在这两个位置上具有强烈的抑制作用。ORF1p还包含保守的PDPK对接位点，促进PDPK与其靶标的特异性相互作用。正如预期的那样，这些位点的突变也抑制了L1的活性。ORF1p上使L1失活的PDPK突变对ORF1p在体外退火RNA的能力没有显著影响，这是蛋白质的一种重要生化性质。我们发现，ORF1p中的磷酸化PDPK位点是与肽基脯氨酰异构酶1(Pin1)相互作用所必需的，PIN1是PDPK介导的调节的关键成分。Pin1通过磷酸化的PDPK基序上的Pro侧链的异构化作用，从而影响底物的构象和活性。我们的证据表明，L1的活性依赖于细胞磷酸化调节级联，并与之整合在一起，这大大增加了我们对L1与其宿主之间相互作用的理解。
Although members of the L1 (LINE-1) clade of non-LTR retrotransposons can be deleterious, the L1 clade has remained active in most mammals for similar to 100 million years and generated almost 40% of the human genome. The details of L1-host interaction are largely unknown, however. Here we report that L1 activity requires phosphorylation of the protein encoded by the L1 ORF1 (ORF1p). Critical phospho-acceptor residues (two serines and two threonines) reside in four conserved proline-directed protein kinase (PDPK) target sites. The PDPK family includes mitogen-activated protein kinases and cyclin-dependent kinases. Mutation of any PDPK phospho-acceptor inhibits L1 retrotransposition. The phosphomimetic aspartic acid can restore activity at the two serine sites, but not at either threonine site, where it is strongly inhibitory. ORF1p also contains conserved PDPK docking sites, which promote specific interaction of PDPKs with their targets. As expected, mutations in these sites also inhibit L1 activity. PDPK mutations in ORF1p that inactivate L1 have no significant effect on the ability of ORF1p to anneal RNA in vitro, an important biochemical property of the protein. We show that phosphorylated PDPK sites in ORF1p are required for an interaction with the peptidyl prolyl isomerase 1 (Pin1), a critical component of PDPK-mediated regulation. Pin1 acts via isomerization of proline side chains at phosphorylated PDPK motifs, thereby affecting substrate conformation and activity. Our demonstration that L1 activity is dependent on and integrated with cellular phosphorylation regulatory cascades significantly increases our understanding of interactions between L1 and its host.