Signal Transduction In B Lymphocyte--Signaling Molecules

该项目的重点是两个丝氨酸苏氨酸蛋白激酶，GCK和GCKR和酪氨酸激酶PYK 2。GCK和GCKR属于蛋白激酶的亚家族，其还包括GLK、HPK 1和NIK。它们的特征在于与酵母STE 20蛋白激酶相关的N-末端激酶结构域、大的C-末端调节结构域和激活应激活化蛋白激酶（SAPK，也称为Jun激酶或JNK）途径的能力。该途径被许多细胞应激激活，炎性细胞因子肿瘤坏死因子（TNF）和CD 40配体（CD 40 L）。CD 40 L存在于活化的T淋巴细胞上，并与其反受体CD 40相互作用，后者由免疫系统中的许多重要细胞表达，包括树突状细胞、单核细胞和B淋巴细胞。以前，我们发现GCK和GCKR是CD 40和TNF介导的SAPK活化的主要中间体，并且通过这些受体的信号传导导致GCKR，TRAF 2和TANK之间形成三分子复合物。CD 40和TNF α诱导的生发中心激酶的活化和随后的应激活化蛋白激酶的活化需要TRAF 2。我们已经发现GCKR以依赖于TRAF 2和Ubc 13的方式激活SAPK通路，Ubc 13是二聚体泛素缀合酶复合物中与Uev 1A一起沿着的成员。干扰Ubc 13功能或表达可抑制TNF α和TRAF 2介导的GCKR和SAPK活化。TNF α信号传导导致TRAF 2多聚泛素化、TRAF 2寡聚化以及GCKR的募集、泛素化和活化，所有这些都对Ubc 13功能的破坏敏感。这些结果表明，由Ubc 13/Uev 1A组装TRAF 2赖氨酸-63连接的多聚泛素链是TNFa介导的GCKR和SAPK激活所必需的。为了更好地理解GCK和GCKR在体内的作用，已经分离了小鼠GCK和GCKR基因。GCK-/-和GCKR -/-小鼠均已创建。这两种突变都没有显著改变小鼠的发育，因为GCK和GCKR缺陷小鼠出生时具有正常的孟德尔频率。对GCKR小鼠免疫组织的评价显示了异常免疫反应性的证据。这些小鼠具有扩增的生殖中心B细胞群，并且它们的B细胞对包括CD 40的接合在内的几种增殖信号具有高应答性。来自这些小鼠的B细胞异常地激活通常由TNF α、CD 40和Wnt配体刺激的信号传导途径。GCK-/- GCKR-/-小鼠已在双杂合子小鼠杂交中鉴定。这些小鼠表现出与GCKR-/-小鼠相似的免疫表型，但除此之外，雄性不育，雌性与野生型雄性杂交时，产仔次数少且小。为了更好地理解这些小鼠中的缺陷，我们从GCK和GCKR敲除小鼠以及GCKR/GCK双敲除小鼠中分离胚胎成纤维细胞系。我们目前正在测试它们对已知激活Jun和p38 MAPK通路的几种不同配体的反应。这些数据将与用这些小鼠制备的淋巴细胞进行的信号传导试验获得的数据相结合。

This project focuses on two serine threonine protein kinases, GCK and GCKR and a tyrosine kinase PYK2. GCK and GCKR belong to a subfamily of protein kinases, which also includes GLK, HPK1, and NIK. They are characterized by an N-terminal kinase domain related to the yeast STE20 protein kinase, a large C-terminal regulatory domain, and the ability to activate the stress- activated protein kinase (SAPK, also referred to as Jun kinase or JNK) pathway. This pathway is activated by many cellular stresses, the inflammatory cytokine tumor necrosis factor (TNF) and CD40 ligand (CD40L). CD40L is present on activated T lymphocytes and interacts with its counter-receptor CD40, which is expressed by many of the important cells in the immune system, including dendritic cells, monocytes, and B- lymphocytes. Previously, we showed that GCK and GCKR are major intermediaries in CD40 and TNF-mediated SAPK activation, and that signaling through these receptors leads to the formation of a trimolecular complex between GCKR, TRAF2, and TANK. CD40 and TNFa-induced activation of germinal center kinases and the subsequent activation of stress activated protein kinases requires TRAF2. We have found that GCKR activates the SAPK pathway in a manner that depends upon TRAF2 and Ubc13, a member along with Uev1A of a dimeric ubiquitin-conjugating enzyme complex. Interference with Ubc13 function or expression inhibits both TNFa- and TRAF2-mediated GCKR and SAPK activation. TNFa signaling leads to TRAF2 polyubiquitination, TRAF2 oligomerization, and the recruitment, ubiquitination, and activation of GCKR, all sensitive to disruption of Ubc13 function. These results indicate that the assembly of a TRAF2 lysine-63 linked polyubiquitin chain by Ubc13/Uev1A is required for TNFa-mediated GCKR and SAPK activation. To better understand the role of GCK and GCKR in vivo, the murine GCK and GCKR gene have been isolated. Both GCK-/- and GCKR -/- mice have been created. Neither mutation dramatically alters mouse development as GCK and GCKR deficient mice are born with a normal Mendelian frequency. Evaluation of the immune tissues from the GCKR mice revealed evidence of abnormal immune reactivity. These mice possess an expanded population of germinal center B cells and their B cells are hyper-responsive to several proliferative signals including engagement of CD40. The B cells from these mice aberrantly activate signaling pathways normally stimulated by TNFalpha, CD40, and Wnt ligands. GCK-/- GCKR-/- mice have identified in crosses from interbreeding of double heterozygotic mice. These mice exhibit a similar immune phenotype to that of the GCKR-/- mice, but in addition the males are sterile and the females have infrequent and small litters when crossed with wild type males. To better understand the defects in these mice we have isolated embryonic fibroblast cell lines from GCK and GCKR knock-out mice as well as from the GCKR/GCK double knock-out mice. We are currently testing their response to several different ligands known to activate the Jun and p38 MAPK pathways. These data will be combined with data obtained from signaling assays performed with lymphocytes prepared from these mice.