Molecular mechanism of B cell development governed by surrogate light chain.

前B细胞受体（preBCR）在B细胞发育的大前B细胞阶段瞬时表达，由μ重链、VpreB/β 5替代轻链和Igalpha/Igbeta异源二聚体组成。识别参与preBCR信号传导的分子对于阐明preBCR如何调控B细胞分化的机制是必不可少的。在本研究中，我们建立了一个新的系统，提供了一个上级的方式来分析前BCR的信号转导，通过使用骨髓proB细胞刺激在体内和exvivo.When RAG-2^<-1->小鼠与抗Ig β单克隆抗体处理，发育停滞的proB细胞诱导分化为小的前B细胞阶段。这表明前B细胞上Ig β的交联激发分化信号，类似于正常B细胞发育中前BCR所传递的信号。生化分析显示，Ig β的交联诱导了proB细胞中Igalpha、Sky、P13-激酶、Vav和SLP-76的快速和瞬时的酪氨酸磷酸化以及MAP激酶ERK的激活。有趣的是，35- 40 kD蛋白的酪氨酸磷酸化在用抗Ig β mAb刺激的前B细胞中是明显的，但在以相同方式刺激的B细胞中不是。我们已经从preB细胞中克隆了一个编码丝氨酸/苏氨酸激酶LOK的基因，LOK主要在淋巴细胞中表达。LOK具有激酶结构域，其显示出与酵母中的酵母MAPKKKK STE 20的激酶结构域的一些同源性，以及富含脯氨酸的区域和卷曲螺旋区域。为了阐明LOK在淋巴细胞中的功能，我们目前正在建立和分析LOK缺陷小鼠。

The preB cell receptor (preBCR), composed of muheavy chain, VpreB/lambda5 surrogate light chain and Igalpha/Igbeta heterodimer, is expressed transiently at the large preB cell stage of B cell development. Identification of molecules involved in the preBCR signaling is essential for elucidating the mechanism how preBCR governs B cell differentiation. In the present study, we have established a novel system that provides a superior way to analyze the signal transduction of preBCR by using bone marrow proB cells stimulated in vivo and ex vivo.When RAG-2^<-1-> mice were treated with an anti-Igbeta mAb, developmentally-arrested proB cells were induced to differentiate to the small preB cell stage. This indicates that the cross-linking of Igbeta on proB cells elicits differentiation signals analogouw to those delivered by preBCR in normal B cell development. The biochemical analyzes revealed that the cross-linking of Igbeta induced a rapid and transient tyrosine phosphorylation of Igalpha, Sky, P13-kinase, Vav and SLP-76 as well as the activation of MAP kinase ERK in proB cells. Intriguingly, the tyrosine phosphorylation of a protein (s) of 35-40kD was evident in proB cells stimulated with the anti-Igbeta mAb but not in B cells stimulated in the same way. We are in the process of identifying the nature of this molecule (s).We have cloned from preB cells a gene coding for a novel serine/threonine kinase, LOK,which is expressed predominantly in lymphocytes. LOK has a kinase domain, which shows some homology to that of a yeast MAPKKKK STE20 in yeast, as well as a proline-rich region and a coiled-coil region. In order to clarify the function of LOK in lymphocytes, we are currently establishing and analyzing mice deficient for LOK.