Reprogramming kinase substrate specificity

这一建议源于我最近发现的一个值得注意的现象：由一个小的非酶细菌(沙门氏菌)毒力蛋白Stee重新编程到不同的氨基酸和底物上。丝氨酸(S)、苏氨酸(T)和酪氨酸(Y)残基的磷酸化是细胞中广泛存在的一个调节系统，它为蛋白质组功能提供了一个巨大的可逆扩展。我发现STEE与特征很好的真核生物S/T激酶GSK3相互作用，这导致GSK3在两个非规范底物上磷酸化Y残基：STEE和宿主转录因子STAT3。GSK3需要STE磷酸化STAT3，这驱动抗炎巨噬细胞极化和沙门氏菌毒力。因此，S/T激酶磷酸化受体位点重编程在机制上是可行的，在生物学上也是相关的，但只描述了一个例子。新的初步数据表明，存在以前未研究过的、假定的、由不同细菌编码的激酶重新编程蛋白。我将对这些进行研究，以检验一种假说，即激酶重编程代表了一种更一般的机制，可以改变不同真核细胞激酶的磷酸受体位点特异性，并破译跨不同激酶重编程的分子基础。我还将研究真核蛋白是否具有激酶重编程活性。通过这种方式，我将挑战将激酶指定为S/T-导向或双特异性(磷酸化S/T和Y)的教条，并期待找到一组只有在被调节蛋白结合时才能磷酸化Y残基的S/T激酶。最后，通过定向进化，我的目标是开始设计合成的激酶改变蛋白，因为这可能会彻底改变基于激酶的疗法的发展。总体而言，这项工作将改变我们对激酶在健康和疾病过程中调节的理解。

This proposal stems from a remarkable phenomenon I discovered recently: the reprogramming of canonical kinase specificity to a different amino acid and substrate by a small non-enzymatic bacterial (Salmonella) virulence protein, SteE. Phosphorylation of serine (S), threonine (T) and tyrosine (Y) residues isa widespread regulatory system in cells that provides a vast and reversible expansion to proteome function. I found that SteE interacts with the well-characterised eukaryotic S/T kinase, GSK3, and that this causesGSK3 to phosphorylates Y residues on two non-canonical substrates: SteE and the host transcription factor STAT3.SteE phosphorylation is required for STAT3 phosphorylation by GSK3, and this drives anti-inflammatory macrophage polarisation and Salmonella virulence. Therefore, S/T kinase phospho-acceptor site reprogramming is both mechanistically feasible and biologically relevant yet only described for one example. New preliminary data suggests the existence of previously unstudied, putative, kinase reprogramming proteins that are encoded by diverse bacteria. I will study these to test the hypothesis that kinase reprogramming represents a more general mechanism that can change the phospho-acceptor site specificity of diverse eukaryotic kinases and decipher the molecular basis of reprogramming across different kinases. I will also investigate whether eukaryotic proteins have kinase reprogramming activity. In this way, I will challenge the dogma that assigns kinases as either S/T-directed or dual specificity (phosphorylatesS/T and Y) and anticipate identifying a group of S/T kinases that can phosphorylate Y residues only when bound by a regulatory protein.Finally, through directed evolution I aim to begin the design of synthetic kinase-altering proteins as this could revolutionise the development of kinase-based therapeutics. Overall, this work will change our understanding of kinase regulation during health and disease.