E41K mutation activates Bruton’s tyrosine kinase by stabilizing an inositol hexakisphosphate-dependent invisible dimer

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.


Figure S1 .
Figure S1.BCR-mediated activation of BTK (A) Schematic representation of PIP3 mediated membrane recruitment of BTK upon BCR activation.(B) Structure alignment of PH-TH WT in complex with IP6 (PDB ID: 4Y94) (1) and PH-TH E41K domain in complex with IP4 (PDB ID: 1BWN) (2).(C)Rrepresentative immunoblot showing the IP6-dependent activation of purified full-length wild-type (WT) BTK and BTK E41K mutant.In the top panel, the level of autophosphorylation is determined with a total anti-phosphotyrosine antibody, and the bottom panel shows the loading control.(D)Densitometric analysis of immunoblots of the IP6-dependent activation of BTK shown in panel (C).Data are presented as mean values ± SD from three independent experiments.Data analyses were performed using GraphPad Prism version 9.5.1.An unpaired two-tailed t-test was used to calculate significance.

Figure S2 .
Figure S2.Characterization of the IP6-dependent PH-TH dimer in solution A) Representative gel-filtration elution profiles of indicated PH-TH constructs measured in the presence or absence of IP6.The dotted line represents the elution profile of a standard protein mixture comprised of BSA (66 kDa), Ovalbumin (44 kDa), and ULP1 (26 kDa).The plots were generated by XMGRACE Ver 5.1.25.

Figure S3 .
Figure S3.IP6-mediated activation of BTK in CHO cells (A) The left panel is the representative immunoblot of Y551 phosphorylation level in the indicated construct of BTK transiently expressed in the CHO cell line in the presence and absence of IP6.The densitometric analysis of the immunoblot is on the right.Data are presented as mean values ± SD from five independent experiments.Data analyses were performed using GraphPad Prism version 9.5.1.

Figure S4 .
Figure S4.(A-E) Intensity plots of indicated BTK constructs transiently expressed in CHO cell line, in the presence or absence of IP6.Figures S4 A-D are reused from Figure 4, and Figure S4E is reused from Figure S3B.The BTK expression level is shown in red, and the phosphorylation level of Y551 is shown in green.The blue represents the plasma membrane stained with Wheat Germ Agglutinin (WGA) fused to Alexa 633.Image analysis was done using Fiji Ver 1.54f.The plots were generated using GraphPad Prism version 9.5.1.Scale bar = 30 µm.

Figure S5 .
Figure S5.Measurement of resident time of BTK E41K mutant at the plasma membrane.(A) Representative total internal reflection fluorescence (TIRF) microscopy images of live CHO cell line transiently transfected with mCherry tagged BTK E41K before (t = 0 min) and after (t = 10 min) treating with the indicated concentration of IP6.Scale bar = 10 µm.(B)Plot of membrane fluorescence intensity of BTK E41K -mCherry versus time following IP6 treatment measured in the transiently transfected CHO cells.The table below shows the halflife of resident time (t1/2) of BTK E41K mutant at the plasma membrane, measured at the indicated IP6 concentration.Data are presented as mean values ± SD from three independent experiments.Data analyses were performed using GraphPad Prism version 9.5.1.The t1/2 is determined by fitting the decay of the fluorescence intensity to exponential decay.Image analysis was done using Fiji Ver 1.54f.

Figure S6 .
Figure S6.Cartoon representation of PH-TH molecule in the asymmetric unit of the PH-TH: IP6 crystal structure (PDB: 4Y94) (1).The Saraste dimer and the alternative dimers are shown in the box.

Table S2 : ∆Gunfolding and ∆∆Gunfolding of Apo and IP6 bound PH-TH domain of BTK
** The ∆Gunfolding was calculated at the Tm of the apo state of the respective PH-TH domain

Table S3 : Details of antibodies
B) Confocal images of BTK E41K/R28H/R49S mutant in the presence (bottom) or absence (up) of IP6.The BTK expression level is shown in red (lex= 552 nm, lem= 586-651 nm), and the phosphorylation status is shown in green (lex= 488 nm, lem= 505-531 nm).The blue represents the plasma membrane stained with Wheat Germ Agglutinin (WGA) fused to Alexa 633 (lex= 633 nm, lem= 647-692 nm).Scale bar = 30 µm.C) Quantification of colocalization of BTK E41K/R28H/R49S mutant and WGA by Pearson's correlation coefficient.n=22-25 over three independent experiments.Boxplots represent quartiles.The data points outside the whisker range are set as outliers.The black line inside the box represents the median value.boxplotswere generated using Origin Pro 2020b.Image analysis was done using Fiji Ver 1.54f (4).D) The plot of fraction phosphorylated for the BTK E41K/R28H/R49S mutant transiently expressed in CHO cell lines.n= 22-25 over five independent experiments.Boxplots represent quartiles.The data points outside the whisker range are set as outliers.The black line inside the box represents the median value.boxplots were generated using Origin Pro 2020b.An unpaired two-tailed t-test was used to calculate significance.E) One-dimensional 1 H NMR spectra of IP6 (top) and IP6 extracted from CHO cell line in untreated (middle) and treated (bottom) with IP6.The chemical structure of IP6 is presented on the right.