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

Preb细胞受体(PreBCR)由粘重链、VpreB/lambda5替代轻链和Igα/Igβ异源二聚体组成，在B细胞发育的大Preb细胞阶段瞬时表达。识别参与preBCR信号转导的分子对于阐明preBCR调控B细胞分化的机制至关重要。在本研究中，我们建立了一种新的系统，该系统利用体内和体外刺激的骨髓Prob细胞来分析PreBCR的信号转导。当RAG-2^-1-gt；小鼠被抗Igbeta单抗处理时，发育受阻的ProB细胞被诱导分化为小的PreB细胞阶段。这表明ProB细胞上的免疫球蛋白β的交联引起的分化信号类似于正常B细胞发育中前BCR所传递的信号。生化分析表明，在PROB细胞中，Igβ的交联物诱导了Igα、Sky、P13-K、Vav和SLP-76的快速和短暂的酪氨酸磷酸化，以及MAP激酶ERK的激活。有趣的是，一种35-40kD的蛋白(S)的酪氨酸磷酸化在抗IgB单抗刺激的PROB细胞中很明显，而在同样方式刺激的B细胞中则不明显。我们正在鉴定这种分子的性质(S)。我们已经从Preb细胞中克隆了编码一种新的丝氨酸/苏氨酸激酶LOK的基因，该基因主要在淋巴细胞中表达。LOK有一个与酵母中的酵母MAPKKKK STE20有同源性的激活区，以及一个富含脯氨酸的区域和一个卷曲的区域。为了阐明LOK在淋巴细胞中的功能，我们目前正在建立并分析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.