Understanding the role of Inositol Hexakisphosphate in the regulation of Bruton’s Tyrosine Kinase in B cell signaling

Chowdhury, Subhankar (2024) Understanding the role of Inositol Hexakisphosphate in the regulation of Bruton’s Tyrosine Kinase in B cell signaling. PhD thesis, Indian Institute of Science Education and Research Kolkata.

Text (PhD thesis of Subhankar Chowdhury (13IP011)) 13IP011.pdf - Submitted Version Restricted to Repository staff only Download (6MB)

Abstract

Bruton’s tyrosine kinase (BTK) is a non-receptor tyrosine kinase required for regulation of B lymphocyte development, differentiation and signalling. The activation of BTK is a crucial event in the B-Cell signaling pathway. BTK was known to be activated by its recruitment to the plasma membrane and subsequent phosphorylation by Src family of kinases. Recently, it was found that inositol hexakisphosphate (IP₆) activates BTK, even in the absence of a membrane through its interaction with the PH-TH domain. This interaction is hypothesized to stabilize a transient dimer of PH-TH domain which is yet to be detected in solution or in cellular environment. Interestingly, a point mutation (E41K) in the PH domain constitutively activates BTK, the mechanism of which is yet to be completely understood. This E41K mutation has higher affinity towards IP₆ than the wild type BTK, which raises the question that if there is any role of IP6 in the constitutive activation of E41K. In this study, we focus to characterize the IP6 mediated PH-TH dimer and try to investigate the role of IP₆ in constitutive activation of E41K mutant. Our data showed that IP₆ activates E41K by forming a stable PH-TH dimer in solution. We further investigated the IP₆ dependent activation of E41K in cellular environment. In the untreated cells, E41K showed mild concentration dependent activation at the membrane. Interestingly, in presence of IP6, the E41K protein showed significantly increased cytosolic activation compared to the untreated cells. Here, we speculate that the active E41K fraction peels off from membrane and moves to cytosol in presence of IP₆.

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