Tec family kinases : Transcriptional and posttranslational regulation

Detta är en avhandling från Stockholm : Karolinska Institutet, Department of Laboratory Medicine

Sammanfattning: Tec family tyrosine kinases consist of five members: Btk, Itk, Tec, Rlk/Txk and Bmx. These kinases are highly expressed in hematopoietic cells, including B cells, T cells and mast cells. Tec kinases execute important functions in signaling pathways downstream of the antigen receptors, and the activation of receptor and non-receptor protein tyrosine kinases is one of the first steps in the signal transduction cascades that lead to activation of hematopoietic cells. Following the identification of mutations in the Btk gene as the cause of human X-linked Agammaglobulinemia (XLA) and mice X-linked Immunodeficiency (xid) in 1993, Tec family kinases have received widespread research interest. Although much progress has been made in elucidating the signalling of Tec kinases, the molecular mechanisms that underlie the biology of these enzymes are largely unknown. This study was undertaken to address the transcriptional and posttranslational regulation of Tec kinases (Btk, Itk and Tec). In the first paper, we show that the peptidyl-prolyl cis/trans isomerase Pin1 is a negative regulator of Btk. The negative regulatory effect of Pin1 was observed both in B cell lines and in cells from Pin-/- mice and was found to be dependent on a functionally intact Btk. Our results indicate that Pin1 functionally interacts with Btk in a cell cycle dependent manner. Accordingly, Pin1 was found to interact with serine 21 (S21) during mitosis and serine 115 (S115) in interphase cells. In the second study, we found that NF-kappaB is required for the transcription of the Btk gene. Previously, it has been reported that NF-kappaB signals downstream of Btk. In this work, we found that proteasome and NF-kappaB inhibitors suppress Btk transcription. Second, two functionally active NF-kappaB binding sites were identified in the Btk promoter. Third, using hydrodynamic transfection technology, we show that Bortezomib can block Btk transcription in mice. Collectively, we show that Btk uses a positive auto-regulatory feedback mechanism to stimulate transcription from its own promoter via NF-kappaB. Paper III: We demonstrated that proteasome inhibitors effectively block HIV-1 replication by suppressing viral transcription and depleting a key cellular component Itk, a Tec-family kinase critical for HIV replication. First, we showed that proteasome inhibitors shut down transcription from the HIV-1 LTR-promoter. Second, replication of HIV-1 in PBMC was severely compromised following treatment with proteasome inhibitors. Finally, these drugs led to depletion of the steady state levels of Itk in PBMC. Altogether, these findings suggest that proteasome inhibitors not only function as bone fide antiretroviral drugs, but may also boost resistance of host cells by affecting a key endogenous component (Itk). Paper IV: We found that NF-kappaB is required for the optimal expression of the Tec gene, and identified a highly conserved and functionally active NF-kappaB binding site in the Tec promoter. Moreover, the NF-kappaB subunit p65/RelA was found to induce the Tec promoter. Finally, we showed that proteasome inhibitors repress Tec transcription by blocking the NF-kappaB signaling pathway. In conclusion, the present work shows that NF-kappaB is an important transcription factor for the expression of Tec family kinases (Btk, Tec and Itk), and proteasome inhibitors repress their transcription by disabling the NF-kappaB signaling pathway. This work also provides important insights into the molecular mechanisms underlying the functional regulation of Tec family kinases by NF-kappaB. Second, we demonstrate that Pin1 functionally interacts with Btk and regulates tyrosine phosphorylation and steady-state levels of Btk.

  HÄR KAN DU HÄMTA AVHANDLINGEN I FULLTEXT. (följ länken till nästa sida)