Regulation and Function of Cyclin D3 in B Cell Subsets

腹膜B-1细胞构成了独特的B细胞亚群，以许多表型和功能特征区分。B-1细胞是研究的重要亚群，因为它们负责大部分非免疫血清免疫球蛋白（IG），其在获得性免疫之前提供针对一系列微生物的血清学保护。B-1细胞与自身免疫和恶性转化的发病机制有关。值得注意的是，B-1细胞代表人慢性淋巴细胞白血病（CLL）的起源细胞。在成年动物中，B-1细胞是自我更新的，而传统的脾B（B-2）细胞不是。这表明G 0/GI-S期进展的调节在B-1和B-2细胞之间不同。我们已经报道了在细胞周期中B-1和B-2细胞之间存在显著的功能差异。 进入S阶段所需的信号。B-1细胞不能对抗Ig刺激增殖，这驱使B-2细胞进入S期。相反，B-1细胞响应佛波酯（PMA）增殖，并且增殖速度非常快，而B-2细胞增殖需要PMA加钙离子载体。我们已经研究了细胞周期对PMA的反应，作为理解B-1和更普遍地所有B细胞中G 0/G1-S期进展的调节的手段。我们的工作已经导致了几个关键的发现：（i）细胞周期蛋白D3是唯一的定位，以介导PMA刺激的B-1细胞的S期进入;（ii）PMA诱导细胞周期蛋白D3-cdk 4复合物组装在B-1和B-2细胞，但后者中的复合物是无活性的。类似地，IL-4诱导B-2细胞中无活性的细胞周期蛋白D3-cdk 4复合物的组装;和（iii）cdk 4表现出与其磷酸化抑制相一致的独特磷酸化特征。 PMA刺激的B-2细胞中的激酶活性。本项目的长期目标是了解细胞周期蛋白D3（和细胞周期蛋白D3-cdk 4复合物）在B-1和B-2细胞中的调节和功能，作为解剖所有B细胞限制点进展和进入S期的重要因素的切入点。这将通过三个具体目标来实现。1.阐明细胞周期蛋白D3在腹膜B-1细胞中的生物学作用; 2.绘制B-2和B-1细胞中cdk 4上的磷酸化位点;和3.阐明cdk 4中个别磷酸受体位点的生物学作用及调控。信息来源于 该项目将阐明控制B细胞中G 0/G1-到-S进程的调节的新原理，并将普遍适用于所有哺乳动物细胞类型中细胞周期进程的调节。

Peritoneal B-1 cells constitute a unique B cell subset, distinguished by numerous phenotypic and functional characteristics. B-1 cells are an important subset to study because they are responsible for the majority of non-immune serum immunoglobulin (Ig), which provides serological protection against a range of microorganisms prior to adaptive immunity. B-1 cells have been implicated in the pathogenesis of autoimmunity and malignant transformation. Notably, B-1 cells represent the cell of origin for human chronic lymphocytic leukemia (CLL). In adult animals B-1 cells are self-renewing, whereas conventional splenic B (B-2) cells are not. This suggests that the regulation of G0/GI-S phase progression differs between B-1 and B-2 cells. We have reported that dramatic functional differences exist between B-1 and B-2 cells in the signals required for S-phase entry. B-1 cells fail to proliferate to anti-lg stimulation, which drives B-2 cells into S phase. Conversely, B-1 cells proliferate in response to phorbol ester (PMA) and do so unusually rapidly, whereas B-2 cell proliferation requires PMA plus calcium ionophore. We have studied the cell cycle response to PMA as a means to understand the regulation of G0/G1-S phase progression in B-1 and, more generally, all B cells. Our work has led to several key findings: (i) cyclin D3 is uniquely positioned to mediate S-phase entry in PMA stimulated B-1 cells; (ii) PMA induces cyclin D3-cdk4 complex assembly in B-1 and B-2 cells, but complexes in the latter are inactive. Similarly, IL-4 induces the assembly of inactive cyclin D3-cdk4 complexes in B-2 cells; and (iii) cdk4 exhibits a distinct phosphorylation profile coincident with inhibition of its kinase activity in PMA-stimulated B-2 cells. The long term objective of this project is to understand the regulation and function of cyclin D3 (and cyclinD3-cdk4 complexes) in B-1 and B-2 cells as an entry point for dissecting important elements that govern restriction point progression and commitment to S-phase entry of all B cells. This will be accomplished through three specific aims. 1. Elucidate the biological role of cyclin D3 in peritoneal B-1 cells; 2. Map the phosphorylation sites on cdk4 in B-2 and B-1 cells; and 3. Elucidate the biological role and regulation of individual phosphoacceptor sites in cdk4. The information developed from this project will elucidate novel principles that govern the regulation of G0/G1-to-S progression in B cells and will be of general applicability to the regulation of cell cycle progression in all mammalian cell types.