Identification of molecular switch regulating tyrosine phosphorylation of Intestinal breast cancer resistance protein (BCRP) mediated mucosal wound repair and barrier functions in obesity

Abstract

Breast cancer resistance protein (BCRP) is a member of ATP-binding cassette (ABC) transporter proteins whose primary function is to efflux substrates bound to the plasma membrane. In our previous study, we reported that obese individuals have compromised intestinal BCRP functions and that diet-induced obese mice recapitulate these outcomes. Furthermore we demonstrated that the compromised BCRP functions during obesity are because of loss of Janus kinase 3 (JAK3)-mediated tyrosine phosphorylation of BCRP. Janus kinase 3 (Jak3) is a non receptor tyrosine kinase expressed in both hematopoietic and nonhematopoietic cells. Although mutations that abrogate Jak3 functions cause different immunological disorders, its constitutive activation leads to various types of inflammatory diseases. Mechanistically, JAK3-mediated phosphorylation of BCRP promoted its interactions with membrane-localized β-catenin essential not only for BCRP expression and surface localization, but also for the maintenance of BCRP-mediated intestinal drug efflux and barrier functions. In the present study, we demonstrate that, BCRP interacted with Jak3, thereby facilitating intestinal barrier function and drug efflux function. Furthermore, we characterized the structural determinants that regulate the interactions between BCRP and Jak3 and pBCRP and β-catenin. Functional reconstitution of kinase activity by recombinant full-length (wt) Jak3 using Jak3-wt or BCRP-wt as substrate showed that Jak3 autophosphorylation was the rate-limiting step during interactions between Jak3 and drug transporter protein. Determination of kinetic parameters showed that phosphorylated (P) Jak3-wt binds to p-BCRP-wt with a dissociation constant K(d)) of 15 nM and a Hill's coefficient of 2.5. Pairwise binding between Jak3 mutants and P-BCRP-wt showed that the FERM domain of Jak3 was sufficient for binding to p-BCRP with a K(d) of 20.0 nM whereas NBD domain of BCRP was contributing more towards it's binding to pJak3 with a K(d) value of 30 nM. Furthermore, Phosphorylated form of F2-subdomain of FERM was responsible for binding of FERM with BCRP. On the other hand NBD domain of BCRP was sufficient to bind to pβ-catenin with K(d)of 22nM. Physiologically, Jak3-mediated phosphorylation of BCRP increased IL2-mediated mucosal wound repair and also facilitated epithelial barrier functions. Our results also show that both BCRP and β-catenin bind to FERM domain of Jak3 non-competitively. We demonstrate that mutation (Y-F) in the tyrosine residue at 123 position of NBD domain of BCRP prevented Jak3 and β-catenin significantly from binding to BCRP. Results from cell culture: Thus, we demonstrate the molecular mechanism of interactions between Jak3 and BCRP and pBCRP and further we are investigating to determine the molecular mechanism involved in BCRP phosphorylation mediated mucosal wound repair.