PKD2 involvement in the Btk-PKCBeta-NFkappaBeta pathway*

描述（由申请人提供）： B细胞受体复合物（BCR）的触发导致核因子-κ B（NF-κ B）/REL家族转录因子的活化，其对于正常B淋巴细胞的存活和活化是必需的。NF-κ B活化失调有助于B细胞淋巴瘤和自身免疫性疾病的发生和发展。BCR NF-kB级联通过在空间和时间上在细胞中分离的不同“信号体”介导，包括BCR、CARMA 1、IKK和NFkB多分子复合物。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形成了这些复合物的核心，但多条研究线证明了蛋白质和肌醇激酶在此过程中的关键作用。虽然现在已经清楚Btk和PKC β在免疫受体诱导的NF-κ B活化中起重要作用，但控制这一过程的分子事件仍有待完全确定。BCR-NF-kB级联的潜在组分包括蛋白激酶D（PKD）家族的成员，PKD家族是一个相对较新认识的第二信使刺激丝氨酸/苏氨酸（Ser/Thr）蛋白激酶家族。 四条证据表明PKD 1/2可能参与NF κ B通路：PKD在B细胞中与Btk组成性相关; PKD被经典PKC反式磷酸化; PKD 1通过IKK β磷酸化参与上皮细胞中的NF κ B通路; CARMA 1的调节接头区含有共有PKD 2磷酸化位点。考虑到大多数淋巴细胞表达PKD 2而不表达PKD 1，本提案的总体目标是确定PKD 2在B细胞中BCR-Btk-PKC β介导的NF κ B活化的调节中的作用。 在目前的建议中，我们将定义介导Btk依赖性PKD 2激活的分子事件。此外，我们将测试PKC β直接参与PKD 2的BCR依赖性激活的假设。我们还将讨论CARMA 1是否可以作为PKD 2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PKC β表达和活化的升高是否与PKD 2表达和活性的改变相关。这将需要在人DLBCL细胞系和从具有良好预后（生发中心表型）与不良预后（活化B细胞表型）疾病的患者获得的样品中平行研究PKCB和PKD表达和活性。这项基于福格蒂计划的研究工作将主要在乌克兰进行。乌克兰国家科学院实验病理学、肿瘤学和放射生物学Kavetsky研究所与Svetlana Sidorenko合作，作为NIH Grant #4 R 01 HD 037091 -07的扩展。

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.