IRAK4 autophosphorylation functions as a molecular switch to regulate death domain assembly and Myddosome maturation.

The organization of signaling proteins into multi-component complexes is essential for innate immune responses and is often achieved through the oligomerization of death domains (DDs). The Myddosome, composed of MyD88 and IRAK family kinases, exemplifies this mechanism. However, how DDs assemble in an ordered and regulated manner to form Myddosomes to control signaling output remains unclear. Here, we show that IRAK4 kinase activity and autophosphorylation are required for IRAK4:IRAK1 heterotypic DD interactions. We show that IRAK1 is autoinhibited by its kinase domain, and IRAK4 phosphorylation relieves this inhibition, enabling IRAK1 DD incorporation into Myddosomes. This phosphorylation-dependent switch similarly controls IRAK2/3 incorporation. Thus, IRAK4 autophosphorylation acts as an energy-dependent molecular switch that integrates phosphorylation with protein oligomerization to drive Myddosome maturation and signal transduction. This mechanism demonstrates how enzymatic activity can impose temporal and spatial control over macromolecular assembly to ensure directional and context-dependent inflammatory signaling.