Department of Chemistry and Biochemistry, University of Arizona , 1306 East University Boulevard, Tucson, Arizona 85721, United States.
J Am Chem Soc. 2014 Dec 10;136(49):17078-86. doi: 10.1021/ja5080745. Epub 2014 Nov 26.
Protein kinases phosphorylate client proteins, while protein phosphatases catalyze their dephosphorylation and thereby in concert exert reversible control over numerous signal transduction pathways. We have recently reported the design and validation of split-protein kinases that can be conditionally activated by an added small molecule chemical inducer of dimerization (CID), rapamycin. Herein, we provide the rational design and validation of three split-tyrosine phosphatases (PTPs) attached to FKBP and FRB, where catalytic activity can be modulated with rapamycin. We further demonstrate that the orthogonal CIDs, abscisic acid and gibberellic acid, can be used to impart control over the activity of split-tyrosine kinases (PTKs). Finally, we demonstrate that designed split-phosphatases and split-kinases can be activated by orthogonal CIDs in mammalian cells. In sum, we provide a methodology that allows for post-translational orthogonal small molecule control over the activity of user defined split-PTKs and split-PTPs. This methodology has the long-term potential for both interrogating and redesigning phosphorylation dependent signaling pathways.
蛋白激酶使客户蛋白磷酸化，而蛋白磷酸酶则催化其去磷酸化，从而协同对众多信号转导通路进行可逆控制。我们最近报道了设计和验证了可通过外加小分子二聚化化学诱导剂（rapamycin）条件激活的分裂蛋白激酶。在此，我们提供了附着在 FKBP 和 FRB 上的三个分裂酪氨酸磷酸酶（PTP）的合理设计和验证，其中通过 rapamycin 可以调节催化活性。我们进一步证明，正交 CIDs（脱落酸和赤霉素）可用于控制分裂酪氨酸激酶（PTK）的活性。最后，我们证明设计的分裂磷酸酶和分裂激酶可以在哺乳动物细胞中被正交 CIDs 激活。总之，我们提供了一种方法，允许对用户定义的分裂 PTK 和分裂 PTP 的活性进行翻译后正交小分子控制。该方法具有长期潜力，可用于研究和重新设计磷酸化依赖的信号通路。
