Signal Transduction In B Lymphocytes: Identification Of Key Signaling Molecules

CD 38是一种能够产生钙动员第二信使的细胞表面受体。它与宿主防御和癌症生物学有关，但CD 38下游的信号传导机制仍不清楚。LRRK 2（富含亮氨酸重复序列激酶2）的突变是帕金森病最常见的遗传原因;它也是克罗恩病，麻风病和某些类型癌症的风险因素。这些疾病的发病机制涉及炎症和巨自噬/自噬，CD 38和LRRK 2都参与其中。我们已经将CD 38和LRRK 2作为TFEB（转录因子EB）的上游激活剂在机械上和功能上连接，TFEB是宿主防御转录因子和自噬/溶酶体机制的主转录调节因子。在B淋巴细胞和巨噬细胞中，我们发现CD 38和LRRK 2存在于质膜上的复合物中。CD 38与单克隆抗体克隆90的连接导致CD 38-LRRK 2复合物的内化及其靶向内溶酶体系统。这产生NAADP依赖性钙信号，其需要LRRK 2激酶活性，并导致TFEB的下游激活。因此，lrrk 2 KO巨噬细胞在CD 38或LPS刺激后具有TFEB活化缺陷，并且在LPS处理后不能转换为糖酵解代谢。在过表达模型中，致病性LRRK 2G 2019 S突变体通过稳定TFEB和经由异常钙信号传导促进其核转位，即使在不存在CD 38的情况下也促进TFEB的超活化。总之，我们已经鉴定了免疫细胞中的生理性CD 38-LRRK 2-TFEB信号传导轴。常见的致病突变体LRRK 2G 2019 S似乎劫持了这一途径。 当通过促进B细胞粘附限制膜结合抗原时，B细胞整联蛋白LFA-1与ICAM-1的连接促进B细胞活化和免疫突触形成。在这项研究中，我们使用原代B细胞的超分辨率成像显示，LFA-1：ICAM-1相互作用也促进了支配B细胞免疫突触的肌动球蛋白网络的形成。该网络由肌球蛋白2A的双极细丝形成，并组织成同心的收缩弧。定量延时成像显示，该网络和B细胞受体：抗原簇以相同的速度向内流动。成像显示单个B细胞受体微簇被单个肌动球蛋白弧向内扫过。在整联蛋白共刺激促进突触形成的条件下，抑制肌球蛋白收缩性损害突触形成，如通过减少的抗原集中、减少的B细胞受体依赖性信号传导和突触处信号传导蛋白分布的缺陷所证明的。总之，这些结果表明，整合素连接下游产生的收缩性肌动球蛋白弧网络在LFA-1共刺激促进B细胞活化和免疫突触形成的机制中起重要作用（本研究与John Hammers实验室合作）。 配体结合趋化因子受体触发异源三聚体G蛋白i亚基核苷酸交换，刺激细胞骨架重组和细胞极性变化。为了更好地理解负责的信号传导事件，我们专注于小鼠脾B细胞CXCR 5参与后的早期F-肌动蛋白变化。F-肌动蛋白水平迅速增加，这取决于Gi-信号传导，PI-3激酶/AKT通路，ERK激活，磷脂酶C活性和Dock 2介导的Rac 1/2激活。AKT底物和pT 60 WNK 1免疫印迹鉴定了WNK 1（无赖氨酸激酶1）作为AKT活化下游的潜在早期效应子。由于其重要性，用特异性WNK抑制剂处理B细胞减少了pAKT和pERK活化，破坏了F-肌动蛋白动力学，并损害了B细胞的极性、运动性和趋化性。对小鼠一次性给予WNK抑制剂可短暂降低体内淋巴结B细胞运动性和极性。这些结果表明，WNK 1信号转导维持了B细胞对趋化因子的反应性。WNK 1在自然杀伤（NK）细胞中也有很好的表达。 WNK激酶抑制剂显著降低了IL-2激活的NK细胞的细胞体积、细胞溶解活性和迁移。 抑制WNK激酶可增加AKT、FOXO 1、PRAS 40、LKB 1、AMPK、p38和TSC 2的磷酸化。 相反，它降低了OXSR 1，mTOR，70 S6 K，S6和4 E-BP 1的磷酸化。此外，WNK激酶抑制剂增加NK细胞中的自噬通量。成像实验表明，NK细胞的体积和运动明显减少的抑制剂。最后，向小鼠施用WNK抑制剂WNK 463抑制了NK细胞在黑色素瘤模型中的抗转移作用。

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.