Phosphorylation-driven effector switching of Rab7 and Rab12 by the leucine-rich repeat kinase 1 in mast cells.

INTRODUCTION: Mast cells (MCs) mediate immune, allergic, and neuroinflammatory responses by releasing inflammatory mediators upon activation through the immunoglobulin E (IgE) receptor FcεRI or innate stimuli acting through Mas-related G protein coupled receptors (Mrgprs). We previously showed that Rab12 negatively regulates mediator release by recruiting the Rab-interacting lysosomal protein (RILP)-dynein complex to the secretory granules (SGs). Because Rab12 also interacts with the RILP-like proteins RILP-L1 and RILP-L2, we examined whether phosphorylation controls Rab12 distribution among its RILP family effectors. METHODS: Pulldown assays were used to compare the effects of MC activation on Rab12 interactions with its effectors. RESULTS: Here we show that activation by either IgE/antigen or the neuropeptide substance P, which binds to MRGPRX2, induces phosphorylation of the Rab GTPases Rab7 and Rab12. Phosphorylation of both GTPases was sensitive to protein kinase C (PKC) inhibition but resistant to inhibition of the leucine-rich repeat kinase 2 (LRRK2), a known Rab12 phosphorylating kinase. Furthermore, knockdown of the Leucine-Rich Repeat kinase 1 (LRRK1) suppressed phosphorylation of both Rab7 and Rab12, implicating LRRK1 in their phosphorylation by a PKC-dependent mechanism. Like phosphorylation by LRRK2, LRRK1-mediated phosphorylation of Rab12 increased its affinity for RILP-L1 and RILP-L2 while reducing binding to RILP. In contrast, LRRK1 phosphorylation of Rab7 enhanced its affinity for RILP.