SYK and ZAP70 kinases in lymphocyte selection

摘要 虽然B细胞承受着消除自身反应和癌前克隆的巨大选择压力， 我们将SYK确定为中央激酶，以设定负选择的阈值。SYK及其高级化 同源亲缘ZAP70启动B细胞受体和T细胞受体信号转导 分别为T淋巴细胞。尽管这两个激酶几乎完全相同，并且服务类似 功能，它们在B细胞和T细胞中的表达在整个进化过程中是严格分离的。鉴于 分离两种蛋白的原因不明，Syk和ZAP70的异常共表达 以前报道过B细胞慢性淋巴细胞白血病(CLL)和外周T细胞淋巴瘤 (PTCL)。我们的研究小组最近证实，ZAP70的异常表达是Pre-Pre的常见特征。 生发中心(GC)B细胞恶性肿瘤(B-ALL、CLL、MCL；SADRAS等人，Mol Cell 2021)。在遗传方面 B-ALL和B-CLL小鼠模型，诱导共表达ZAP70加速疾病发病，而 基因缺失阻碍了恶性转化。同样，ZAP70的诱导表达在 早期B细胞发育颠覆自身反应性B细胞负选择促进弥漫性 自身抗体的产生。 从机制上讲，ZAP70与SYK竞争，对SYK依赖的SYK产生显性负向作用 钙离子--信号转导。通过占用而不是磷酸化BLNK、BTK和PLC2底物 通过钙信号转导，ZAP70显著降低了自主钙振荡的频率。快地 在SYK(4.5 MHz)单独存在的情况下，振荡由NFATC1解码，并导致无能和 细胞死亡。相反，在ZAP70(0.25 MHz)存在下，缓慢的钙振荡促进选择性 激活核因子-B和B细胞的存活和增殖。 我们在这里测试了一个中心假设，即B细胞感知A细胞下游的病理性信号 自身反应性bcr或通过SYK依赖的高频钙离子-转化癌基因- 震荡。在高频下，钙离子振荡激活NFATC1启动B细胞无能和克隆性 删除。相反，ZAP70可减缓钙离子振荡以激活核因子-B，而不是NFATC1. 使自身反应性和癌前B细胞克隆持续存在，并最终导致自身免疫 疾病或GC前B细胞恶性肿瘤。 我们提出三个目标：(1)阐明ZAP70延缓SYK- 依赖的高频钙振荡，以(2)确定ZAP70的共表达如何颠覆 自身免疫性疾病中自身反应性B细胞的阴性选择和(3)揭示ZAP70是如何 使癌前B细胞发生致癌信号和明显的恶变。

ABSTRACT While B-cells are under intense selective pressure to eliminate autoreactive and pre-malignant clones, we identified SYK as central kinase to set the thresholds for negative selection. SYK and its highly homologous relative ZAP70 initiate B-cell receptor (BCR) and T-cell receptor (TCR) signaling in B- and T-lymphocytes, respectively. Even though the two kinases are almost identical and serve analogous functions, their expression in B- and T-cells is strictly segregated throughout evolution. Whereas the reason for separation of the two kinases is not known, aberrant coexpression of Syk and Zap70 was previously reported in B-cell chronic lymphocytic leukemia (CLL) and in peripheral T-cell lymphoma (PTCL). Our group recently demonstrated that aberrant ZAP70 expression is a common feature in pre- germinal center (GC) B-cell malignancies (B-ALL, CLL, MCL; Sadras et al., Mol Cell 2021). In genetic mouse models for B-ALL and B-CLL, inducible coexpression of Zap70 accelerated disease onset, while genetic deletion impaired malignant transformation. Likewise, inducible expression of Zap70 during early B-cell development subverted negative selection of autoreactive B-cells to promote pervasive autoantibody production. Mechanistically, ZAP70 competes with SYK and exerts a dominant-negative effect on SYK-dependent Ca2+-signaling. By occupying but not phosphorylating BLNK, BTK and PLC2 substrates upstream of calcium signaling, ZAP70 dramatically reduces the frequency of autonomous Ca2+-oscillations. Fast oscillations in the sole presence of SYK (4.5 mHz) are decoded by NFATC1 and result in anergy and cell death. In contrast, slow Ca2+-oscillations in the presence of ZAP70 (0.25 mHz) promote selective activation of NF-B and B-cell survival and proliferation. We test here the central hypothesis that B-cells sense pathological signaling downstream of an autoreactive BCR or a transforming oncogene through SYK-dependent high-frequency Ca2+- oscillations. At high frequencies, Ca2+-oscillations activate NFATC1 to initiate B-cell anergy and clonal deletion. Conversely, ZAP70 slows down Ca2+-oscillations to activate NF-B instead of NFATC1 and enables autoreactive and premalignant B-cell clones to persist and eventually give rise to autoimmune disease or pre-GC B-cell malignancies. We are proposing three Aims to (1) elucidate the mechanisms by which ZAP70 slows down SYK- dependent high-frequency Ca2+-oscillations, to (2) determine how coexpression of ZAP70 subverts negative selection of autoreactive B-cells in autoimmune disease and (3) to uncover how ZAP70 enables oncogenic signaling and overt malignant transformation of pre-malignant B-cells.