Borar Prateeka, Biswas Tapan, Chaudhuri Ankur, Rao T Pallavi, Raychaudhuri Swasti, Huxford Tom, Chakrabarti Saikat, Ghosh Gourisankar, Polley Smarajit
Department of Biological Sciences, Bose Institute, Kolkata, India.
Department of Chemistry and Biochemistry, University of California San Diego, La Jolla, USA.
bioRxiv. 2024 Dec 31:2023.06.27.546692. doi: 10.1101/2023.06.27.546692.
Rapid and high-fidelity phosphorylation of two serines (S32 and S36) of IκBα by a prototype Ser/Thr kinase IKK2 is critical for fruitful canonical NF-κB activation. Here, we report that IKK2 is a dual specificity Ser/Thr kinase that autophosphorylates itself at tyrosine residues in addition to its activation loop serines. Mutation of one such tyrosine, Y169, located in proximity to the active site, to phenylalanine, renders IKK2 inactive for phosphorylation of S32 of IκBα. Surprisingly, auto-phosphorylated IKK2 relayed phosphate group(s) to IκBα without ATP when ADP is present. We also observed that mutation of K44, an ATP-binding lysine conserved in all protein kinases, to methionine renders IKK2 inactive towards specific phosphorylation of S32 or S36 of IκBα, but not non-specific substrates. These observations highlight an unusual evolution of IKK2, in which autophosphorylation of tyrosine(s) in the activation loop and the invariant ATP-binding K44 residue define its signal-responsive substrate specificity ensuring the fidelity of NF-κB activation.
原型丝氨酸/苏氨酸激酶IKK2对IκBα的两个丝氨酸（S32和S36）进行快速且高保真的磷酸化，这对于有效的经典NF-κB激活至关重要。在此，我们报告IKK2是一种双特异性丝氨酸/苏氨酸激酶，除了在其激活环丝氨酸处自身磷酸化外，还在酪氨酸残基处进行自身磷酸化。其中一个位于活性位点附近的酪氨酸Y169突变为苯丙氨酸后，IKK2对IκBα的S32磷酸化失去活性。令人惊讶的是，当存在ADP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，所有蛋白激酶中保守的ATP结合赖氨酸K44突变为甲硫氨酸后，IKK2对IκBα的S32或S36的特异性磷酸化失去活性，但对非特异性底物仍有活性。这些观察结果突出了IKK2不同寻常的进化过程，其中激活环中酪氨酸的自身磷酸化以及不变的ATP结合K44残基决定了其信号响应底物特异性，确保了NF-κB激活的保真度。
