PKD2 involvement in the Btk-PKCBeta-NFkappaBeta pathway*

描述(由申请人提供)： B细胞受体复合体(BCR)的激活导致核因子-KB(NF-KB)/Rel转录因子家族的激活，这是正常B淋巴细胞生存和激活所必需的。异常激活的核因子-KB参与了B细胞淋巴瘤和自身免疫性疾病的发生和发展。BCR-nF-kB级联反应是通过不同的信号小体介导的，包括BCR、CARMA1、IKK和NFkB等多分子复合体。多个事件可以调节这些信号体的特定定位、相互作用和激活或抑制。虽然具有接头功能的蛋白质构成了这些复合体的核心，但多项研究表明，蛋白质和肌醇激酶在这一过程中发挥着至关重要的作用。虽然BTK和PKCbeta在免疫受体诱导的NF-kB激活中起着重要作用，但控制这一过程的分子事件仍未完全确定。BCR-NF-kB级联的潜在成员包括蛋白激酶D(PKD)家族的成员，这是最近发现的第二信使刺激的丝氨酸/苏氨酸(Ser/Thr)蛋白激酶家族。 有四条证据表明PKD/2可能参与了NFKB通路：在B细胞中，PKD与BTK密切相关；在经典的PKCs中，PKD被反式磷酸化；在上皮细胞中，PKD1通过对IKKβ的磷酸化而参与NFKB通路；CARMA1的调节连接区含有共同的PKD2磷酸化位点。考虑到大多数淋巴细胞表达PKD2而不表达PKD1，本研究的总体目标是确定PKD2在调节BCR-BTK-PKCbeta介导的B细胞NFKB活化中的作用。在目前的提案中，我们将定义介导BTK依赖的PKD2激活的分子事件。进一步，我们将检验PKCbeta直接参与BCR依赖的PKD2激活的假设。我们还将解决CARMA1是否可以作为PKD2的直接底物的问题。最后，我们将确定在弥漫性大B细胞淋巴瘤(DLBCL)细胞中观察到的PKCβ表达和激活升高是否与PKD2表达和活性的变化有关。这将需要在人类DLBCL细胞系和从预后良好(生发中心表型)与预后较差(活化B细胞表型)疾病患者的样本中平行研究PKCB和PKD的表达和活性。这项基于Fogarty计划的研究工作将主要在乌克兰的R.E.Kavetsky国家科学院实验病理学、肿瘤学和放射生物学研究所与Svetlana Sidorenko合作进行，作为NIH补助金#4 R01 HD037091-07的延伸。

DESCRIPTION (provided by applicant): The triggering of B cell receptor complex (BCR) leads to activation of nuclear factor-KB(NF-KB)/REL family of transcription factors that is essential for survival and activation of normal B lymphocytes. Dysregulated NF-KB activation contributes to development and progression of B cell lymphoma and autoimmune diseases. The BCR NF-kB cascade is mediated via distinct "signalosomes" separated in cells in space and time and includes the BCR, CARMA1, IKK and NFkB multimolecular complexes. Multiple events can modulate specific localization, interaction and activation, or inhibition, of these signalosomes. While proteins with adaptor functions form a core for these complexes, multiple lines of investigation demonstrate crucial roles for protein and inositol kinases in this process. While it is now clear that Btk and PKCbeta play an essential role in immunoreceptor-induced NF-kB activation, the molecular events controlling this process remain to be fully defined. Among potential components of BCR-NF-kB cascade are members of the Protein Kinase D (PKD) family, a relatively recently recognized family of second-messenger-stimulated serine/threonine (Ser/Thr) protein kinases. Four lines of evidence suggest that PKD1/2 may participate in NFKB pathway: PKD is constitutively associated with Btk in B cells; PKDs are trans-phosphorylated by classic PKCs; PKD1 participates in NFKB pathway in epithelial cells via phosphorylation of IKKbeta; and the regulatory linker region of CARMA1 contains consensus PKD2 phosphorylation sites. Taking into account that majority of lymphocytes express PKD2 but not PKD1, the general objective of this proposal is to determine the role for PKD2 in modulation of BCR-Btk- PKCbeta mediated NFKB activation in B cells. In the current proposal, we will define molecular events that mediate Btk-dependent PKD2 activation. Further, we will test the hypotheses that PKCbeta is directly involved in the BCR dependent activation of PKD2. We will also address the question whether CARMA1 can serve as a direct substrate for PKD2. Finally, we will determine if the elevated PKCbeta expression and activation observed in diffuse large B cell lymphoma (DLBCL) cells correlates with alterations in PKD2 expression and activity. This will entail parallel studies of PKCB and PKD expression and activity in human DLBCL cell lines and samples obtained from patients with good prognosis (germinal center phenotype) vs. poor prognosis (activated B cell phenotype) disease. This Fogarty program based research work will be carried out primarily in the Ukraine at the R.E. Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology National Academy of Sciences of Ukraine in collaboration with Svetlana Sidorenko as an extension of NIH Grant #4 R01 HD037091-07.