Comprehensive Analysis of G1 Cyclin Docking Motif Sequences that Control CDK Regulatory Potency In Vivo.
Comprehensive Analysis of G1 Cyclin Docking Motif Sequences that Control CDK Regulatory Potency In Vivo.
复制标题
DOI：
10.1016/j.cub.2020.08.099
复制
发表时间：
2020-11-16
期刊：
影响因子：
--
通讯作者：
Pryciak PM
中科院分区：
文献类型：
--
作者：
Bandyopadhyay S;Bhaduri S;Örd M;Davey NE;Loog M;Pryciak PM
许多蛋白质修饰酶通过对接基序来识别它们的底物，但是功能上允许的基序序列的范围通常是不明确的。在真核细胞分裂过程中，周期蛋白特异性对接基序帮助周期蛋白依赖性激酶（CDKs）在不同阶段磷酸化不同的底物，从而强制执行一系列时间顺序的事件。在出芽酵母中，具有Leu/Pro-rich （LP）对接基序的CDK底物在G1期后期被Cln1/2细胞周期蛋白识别，但这些基序的关键序列特征尚不清楚。在这里，我们通过结合竞争生长试验和深度突变扫描，全面分析了LP基序在体内的需求。我们使用来自不同真菌（包括医疗和农业病原体）的六种不同G1周期蛋白，量化了五种不同LP基序中所有单残基替换的影响。结果揭示了对共识序列偏差的实质性容忍，加上一些位置的要求取决于其他基序残基的有利性。它们还揭示了野生型基序之间功能效力变化的基础，并允许推导定量矩阵来预测其他候选基序序列的强度。最后，我们发现对接基序效力的变化可以提前或延迟CDK底物磷酸化发生的时间，从而控制细胞周期调节的时间顺序。总体结果为测量可行的对接基序序列和量化它们在体内的效力提供了一种通用方法，并揭示了对接强度的变化如何调节调控修饰的程度和时间。CDKs使用周期蛋白介导的对接相互作用来识别底物，但对接基序缺乏序列变化如何影响识别的定量描述。Bandyopadhyay等人将深度突变扫描与体内竞争生长分析相结合，量化了基序变异的功能强度，并定义了识别规则。
Many protein-modifying enzymes recognize their substrates via docking motifs, but the range of functionally permissible motif sequences is often poorly defined. During eukaryotic cell division, cyclin-specific docking motifs help cyclin-dependent kinases (CDKs) phosphorylate different substrates at different stages, thus enforcing a temporally-ordered series of events. In budding yeast, CDK substrates with Leu/Pro-rich (LP) docking motifs are recognized by Cln1/2 cyclins in late G1 phase, yet the key sequence features of these motifs were unknown. Here we comprehensively analyzed LP motif requirements in vivo by combining a competitive growth assay with deep mutational scanning. We quantified the impact of all single-residue replacements in five different LP motifs, using six distinct G1 cyclins from diverse fungi including medical and agricultural pathogens. The results uncover substantial tolerance for deviations from the consensus sequence, plus requirements at some positions that are contingent on the favorability of other motif residues. They also reveal the basis for variations in functional potency among wild-type motifs, and allow derivation of a quantitative matrix that predicts the strength of other candidate motif sequences. Finally, we find that variation in docking motif potency can advance or delay the time at which CDK substrate phosphorylation occurs, and thereby control the temporal ordering of cell cycle regulation. The overall results provide a general method for surveying viable docking motif sequences and quantifying their potency in vivo, and they reveal how variations in docking strength can tune the degree and timing of regulatory modifications. CDKs use cyclin-mediated docking interactions to recognize substrates, but the docking motifs lack a quantitative description of how sequence variations affect recognition. Bandyopadhyay et al combine deep mutational scanning with an in vivo competitive growth assay to quantify the functional strength of motif variants and define recognition rules.
登录
查看更多内容
影响因子：
14.8
作者：
Hietpas, Ryan;Roscoe, Benjamin;Jiang, Li;Bolon, Daniel N. A.
通讯作者：
Bolon, Daniel N. A.
DOI：
10.1186/s12964-015-0120-z
发表时间：
2015-11-21
期刊：
Cell communication and signaling : CCS
影响因子：
--
作者：
Davey NE;Cyert MS;Moses AM
通讯作者：
Moses AM
影响因子：
16.8
作者：
Koivomaegi, Mardo;Oerd, Mihkel;Iofik, Anna;Valk, Ervin;Venta, Rainis;Faustova, Ilona;Kivi, Rait;Balog, Eva Rose M.;Rubin, Seth M.;Loog, Mart
通讯作者：
Loog, Mart
影响因子：
--
作者：
Hungerbuehler, A. Katrin;Philippsen, Peter;Gladfelter, Amy S.
通讯作者：
Gladfelter, Amy S.
影响因子：
64.8
作者：
Nash, P;Tang, XJ;Tyers, M
通讯作者：
Tyers, M