E41K mutation activates Bruton's tyrosine kinase by stabilizing an inositol hexakisphosphate-dependent invisible...

Bruton's tyrosine kinase (BTK) regulates diverse cellular signaling of the innate and adaptive immune system in response to microbial pathogens. Downregulation or constitutive activation of BTK is reported in patients with autoimmune diseases or various B-cell leukemias. BTK is a multidomain protein tyrosine kinase that adopts an Src-like autoinhibited conformation maintained by the interaction between the kinase and PH-TH domains. The PH-TH domain plays a central role in regulating BTK function. BTK is activated by binding to PIP₃ at the plasma membrane upon stimulation by the B-cell receptor (BCR). The PIP₃ binding allows dimerization of the PH-TH domain and subsequent transphosphorylation of the activation loop. Alternatively, a recent study shows that the multivalent T-cell-independent (TI) antigen induces BCR response by activating BTK independent of PIP₃ binding. It was proposed that a transiently stable IP₆-dependent PH-TH dimer may activate BTK during BCR activation by the TI antigens. However, no IP₆-dependent PH-TH dimer has been identified yet. Here, we investigated a constitutively active PH-TH mutant (E41K) to determine if the elusive IP₆-dependent PH-TH dimer exists. We showed that the constitutively active E41K mutation activates BTK by stabilizing the IP₆-dependent PH-TH dimer. We observed that a downregulating mutation in the PH-TH domain (R28H) linked to X-linked agammaglobulinemia impairs BTK activation at the membrane and in the cytosol by preventing PH-TH dimerization. We conclude that the IP₆ dynamically remodels the BTK active fraction between the membrane and the cytoplasm. Stimulating with IP₆ increases the cytosolic fraction of the activated BTK.