Lipid-targeting pleckstrin homology domain turns its autoinhibitory face toward the TEC kinases

Abstract

The pleckstrin homology (PH) domain is well known for its phospholipid targeting function. The PH-TEC homology (PHTH) domain within the TEC family of tyrosine kinases is also a crucial component of the autoinhibitory apparatus. The autoinhibitory surface on the PHTH domain has been previously defined, and biochemical investigations have shown that PHTH-mediated inhibition is mutually exclusive with phosphatidylinositol binding. Here we use hydrogen/deuterium exchange mass spectrometry, nuclear magnetic resonance (NMR), and evolutionary sequence comparisons to map where and how the PHTH domain affects the Bruton’s tyrosine kinase (BTK) domain. The data map a PHTH-binding site on the activation loop face of the kinase C lobe, suggesting that the PHTH domain masks the activation loop and the substrate-docking site. Moreover, localized NMR spectral changes are observed for non–surface-exposed residues in the active site and on the distal side of the kinase domain. These data suggest that the association of PHTH induces allosteric conformational shifts in regions of the kinase domain that are critical for catalysis. Through statistical comparisons of diverse tyrosine kinase sequences, we identify residues unique to BTK that coincide with the experimentally determined PHTH-binding surface on the kinase domain. Our data provide a more complete picture of the autoinhibitory conformation adopted by full-length TEC kinases, creating opportunities to target the regulatory domains to control the function of these kinases in a biological setting.

Differential impact of BTK active site inhibitors on the conformational state of full-length BTK

“…Previous work has established that the regulatory SH3 and SH2 domains of BTK assemble into a compact autoinhibitory conformation on the ‘distal’ surface of the kinase domain, similar to the autoinhibited SRC kinases ( Figure 1b ; Joseph et al, 2017 ; Wang et al, 2015 ). Solution data support an additional autoinhibitory interface on the activation loop face of the kinase domain occupied by the PHTH domain ( Figure 1b ; Amatya et al, 2019 ; Devkota et al, 2017 ). Upon receptor-mediated activation, the regulatory domains of BTK bind their activating ligands, which releases the catalytic domain from its intramolecular interactions, thereby permitting activation loop phosphorylation and a conformational shift of the catalytic machinery from the inactive to active state.…”

“…Solution data support an additional autoinhibitory interface on the activation loop face of the kinase domain occupied by the PHTH domain ( Fig. 1b) (12,13). Upon receptor mediated activation, the regulatory domains of BTK bind their activating ligands, which releases the catalytic domain from its intramolecular interactions, thereby permitting activation loop phosphorylation and a conformational shift of the catalytic machinery from the inactive to active state.…”

“…PHTH, Pleckstrin homology-Tec homology domain; PRR, proline-rich region; SH3, Src homology three domain; SH2, Src homology two domain; L, SH2-kinase linker; FL, full-length; 32LKD, SH3-SH2-linker-kinase domain; and LKD, linker-kinase domain. ( b ) Domain arrangement of BTK full-length (FL) in the autoinhibited conformation (left) based on the crystal structure of the BTK SH3-SH2-kinase (32LKD) fragment (PDB ID: 4XI2) and solution data supporting an autoinhibitory interaction between the PHTH domain and the activation loop face of the kinase domain ( Amatya et al, 2019 ; Devkota et al, 2017 ). The location of T316 in the SH2 domain is indicated with a black circle.…”

Reining in BTK: Interdomain Interactions and Their Importance in the Regulatory Control of BTK

“…The BTK PHTH domain in Pose 2 (Figure 4) blocks access of substrate to the kinase domain active site (Amatya et al, 2019) and the residues of the PHTH domain that mediate this autoinhibitory interaction (Y42 and D43) (Devkota et al, 2017;Joseph et al, 2017) are the same residues that mediate the Saraste dimer interface suggesting that this autoinhibitory pose is likely mutually exclusive with the active BTK dimer. The (D) Once PIP 3 levels in the membrane surpass the required threshold, BTK engages PIP 3 via the canonical and peripheral binding sites in PHTH and BTK dimerization at the membrane is stabilized.…”

Inositol Polyphosphate-Based Compounds as Inhibitors of Phosphoinositide 3-Kinase-Dependent Signaling

“…Bruton ‘s tyrosine kinase (Btk) and Interleukin-2-inducible T cell kinase (Itk) are known to play a key role in B cell and T cell receptor signaling, respectively [ 77 ]. Both Btk and Itk are localized in the cytoplasm in resting cells and translocate to the plasma membrane upon receptor activation through binding of their PH domains to PtdIns(3,4,5) P 3 [ 78 ]. Small compounds targeting the interaction between Btk PH domain and PtdIns(3,4,5) P 3 have been developed [ 79 ].…”