Janus kinase 2 activation loop as a regulator of catalysis and trans-activation.

Protein kinases regulate essential cellular processes such as apoptosis, proliferation, and growth. Precise regulation of kinase activity is critical for proper signal transduction and is often mediated by activation-loop phosphorylation. Janus kinases (JAKs) employ a unique regulatory mechanism: they remain autoinhibited by their pseudokinase domains until cytokine stimulation triggers dimerization and trans-phosphorylation of two activation-loop tyrosines. Despite its relevance for JAK inhibitor design, the molecular mechanism of JAK activation by activation-loop phosphorylation is not fully understood. Here, we show that phosphorylation of the JAK2 activation loop is essential for stabilizing substrate binding and in the absence of phosphorylation, the activation loop fails to bind substrates. Our findings reveal a unique regulatory role for the activation loop in JAK2, where phosphorylation enhances loop stability rather than inducing a major conformational change as in most protein kinases. In addition, the flexibility of the unphosphorylated activation loop allows conformational changes required for trans-phosphorylation during JAK activation. These insights provide a mechanistic basis for JAK activation and offer new insights for the development of JAK inhibitors.