Signal Transduction In B Lymphocytes: Identification Of Key Signaling Molecules

National Institute Of Allergy And Infectious Diseases

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Abstract

CD38 is a cell surface receptor capable of generating calcium-mobilizing second messengers. It has been implicated in host defense and cancer biology, but signaling mechanisms downstream of CD38 remain unclear. Mutations in LRRK2 (leucine-rich repeat kinase 2) are the most common genetic cause of Parkinson disease; it is also a risk factor for Crohn disease, leprosy, and certain types of cancers. The pathogenesis of these diseases involves inflammation and macroautophagy/autophagy, processes both CD38 and LRRK2 are implicated in. We have mechanistically and functionally linked CD38 and LRRK2 as upstream activators of TFEB (transcription factor EB), a host defense transcription factor and the master transcriptional regulator of the autophagy/lysosome machinery. In B-lymphocytes and macrophages, we show that CD38 and LRRK2 exist in a complex on the plasma membrane. Ligation of CD38 with the monoclonal antibody clone 90 results in internalization of the CD38-LRRK2 complex and its targeting to the endolysosomal system. This generates an NAADP-dependent calcium signal, which requires LRRK2 kinase activity, and results in the downstream activation of TFEB. lrrk2 KO macrophages accordingly have TFEB activation defects following CD38 or LPS stimulation and fail to switch to glycolytic metabolism after LPS treatment. In overexpression models, the pathogenic LRRK2G2019S mutant promotes hyperactivation of TFEB even in the absence of CD38, both by stabilizing TFEB and promoting its nuclear translocation via aberrant calcium signaling. In sum, we have identified a physiological CD38-LRRK2-TFEB signaling axis in immune cells. The common pathogenic mutant, LRRK2G2019S, appears to hijack this pathway. Ligation of the B cell integrin LFA-1 with ICAM-1 promotes B cell activation and immune synapse formation when membrane-bound antigen is limiting by promoting B cell adhesion. In this study we showed using super-resolution imaging of primary B cells that LFA-1: ICAM-1 interaction also promotes the formation of an actomyosin network that dominates the B cell immune synapse. This network is created by the formin mDia1 and organized into concentric, contractile arcs by bipolar filaments of myosin 2A. Quantitative time-lapse imaging showed that this network and the B cell receptor: antigen clusters present within it flow inward at the same rate. The imaging revealed individual B cell receptor microclusters being swept inward by individual actomyosin arcs. Under conditions where integrin co-stimulation promotes synapse formation, inhibiting myosin contractility impaired synapse formation, as evidenced by reduced antigen centralization, diminished B cell receptor-dependent signaling, and defects in signaling protein distribution at the synapse. Together, these results argue that a contractile actomyosin arc network created downstream of integrin ligation plays an important role in the mechanism by which LFA-1 co-stimulation promotes B cell activation and immune synapse formation (This study was a collaboration with John Hammers laboratory). Ligand-engaged chemokine receptors trigger heterotrimeric G-protein i subunit nucleotide exchange that stimulates cytoskeletal reorganization and cell polarity changes. To better understand the responsible signaling events, we focused on early F-actin changes following murine splenic B cell CXCR5 engagement. F-actin levels rapidly increased, which depended upon Gi-signaling, the PI-3 kinase/AKT pathway, ERK activation, phospholipase C activity, and Dock2 mediated Rac1/2 activation. AKT substrate and pT60 WNK1 immunoblotting identified WNK1 (with no lysine kinase 1) as a potential early effector downstream of AKT activation. Verifying its importance, treating B cells with specific WNK inhibitors reduced pAKT and pERK activation, disrupted F-actin dynamics, and impaired B cell polarity, motility, and chemotaxis. A one-time administration of a WNK inhibitor to mice transiently reduced lymph node B cell motility and polarity in vivo. These results indicate that WNK1 signaling maintains B cell responsiveness to chemokines. WNK1 is also well expressed in natural killer (NK) cells. WNK kinase inhibitors dramatically decreased the cell volume, cytolytic activity, and the migration of IL-2 activated NK cells. Inhibition of WNK kinase increased the phosphorylation of AKT, FOXO1, PRAS40, LKB1, AMPK, p38, and TSC2. In contrast, it decreased phosphorylation of OXSR1, mTOR, 70S6K, S6, and 4E-BP1. Moreover, WNK kinase inhibitors increased autophagic flux in NK cells. Imaging experiments revealed that NK-cell volume and movement were clearly decreased by the inhibitors. Finally, administration of the WNK inhibitor WNK463 to mice inhibited anti-metastatic effects of NK cells in a melanoma model.