Dual-specific autophosphorylation of kinase IKK2 enables phosphorylation of substrate IκBα through a phosphoenzyme intermediate.

Borar Prateeka, Biswas Tapan, Chaudhuri Ankur, T Pallavi Rao, Raychaudhuri Swasti, Huxford Tom, Chakrabarti Saikat, Ghosh Gourisankar, Polley Smarajit
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Abstract

Rapid and high-fidelity phosphorylation of serine residues at positions 32 and 36 of IκBα by IKK2, a prototypical Ser/Thr kinase, is critical for canonical NF-κB activation. Here, we report that IKK2 not only phosphorylates substrate serine residues and autophosphorylates its own activation loop, but also autophosphorylates at a tyrosine residue proximal to the active site and is, therefore, a dual-specificity kinase. We observed that mutation of Y169, an autophosphorylatable tyrosine located at the DFG+1 (DLG in IKK1) position, to phenylalanine renders IKK2 incapable of catalyzing phosphorylation at S32 within its IκBα substrate. We also observed that mutation of the phylogenetically conserved ATP-contacting residue K44 in IKK2 to methionine converts IKK2 to an enzyme that no longer catalyzes specific phosphorylation of IκBα at S32 or S36, but rather directs phosphorylation of IκBα at other residues. Lastly, we report evidence of a phospho-relay from autophosphorylated IKK2 to IκBα in the presence of ADP. These observations suggest an unusual evolution of IKK2, in which autophosphorylation of tyrosine(s) in the activation loop, and the conserved ATP-contacting K44 residue provide its signal-responsive substrate specificity and ensure fidelity during NF-κB activation.

激酶IKK2的双特异性自磷酸化使底物IκBα磷酸化而不需要ATP。
原型S/T激酶IKK2对IκBα的两个丝氨酸(S32和S36)的快速高保真磷酸化对于富有成效的典型NF-κB活化至关重要。在这里,我们报道了IKK2是一种双特异性激酶,除了其激活环丝氨酸外,它还在顺式的酪氨酸残基上自我磷酸化。位于活性位点附近的一种酪氨酸Y169突变为苯丙氨酸,使IKK2对IκBα的S32磷酸化没有活性。令人惊讶的是,当ADP存在时,自身磷酸化的IKK2在没有ATP的情况下将磷酸基团传递给IκBα。我们还观察到,K44(一种在所有蛋白激酶中保守的ATP结合赖氨酸)突变为甲硫氨酸,使IKK2对IκBα的S32或S36的特异性磷酸化没有活性,但对非特异性底物没有活性。这些观察结果突出了IKK2的一种不同寻常的进化,其中激活环中酪氨酸的自磷酸化和不变的ATP结合K44残基定义了其信号响应性底物特异性,确保了NF-κB激活的保真度。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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