Tec family kinases : transcriptional and posttranslational regulation
Author: Yu, Liang
Date: 2009-04-17
Location: C1:87, Karolinska Universitetssjukhuset Huddinge
Time: 09.00
Department: Institutionen för laboratoriemedicin / Department of Laboratory Medicine
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thesis.pdf (2.106Mb)
Abstract
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.
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.
List of papers:
I. Yu L, Mohamed AJ, Vargas L, Berglöf A, Finn G, Lu KP, Smith CI (2006). "Regulation of Bruton tyrosine kinase by the peptidylprolyl isomerase Pin1." J Biol Chem 281(26): 18201-7. Epub 2006 Apr 27
Pubmed
II. Yu L, Mohamed AJ, Simonson OE, Vargas L, Blomberg KE, Björkstrand B, Arteaga HJ, Nore BF, Smith CI (2008). "Proteasome-dependent autoregulation of Bruton tyrosine kinase (Btk) promoter via NF-kappaB." Blood 111(9): 4617-26. Epub 2008 Feb 21
Pubmed
III. Yu L, Mohanram V, Simonson OE, Smith CIE, Spetz AL, Mohamed AJ (2009). "Proteasome inhibitors block HIV-1 replication by affecting both cellular and viral targets." (Submitted)
IV. Yu L, Simonson OE, Mohamed AJ, Smith CIE (2009). "NF-kappaB regulates the transcription of protein tyrosine kinase Tec." (Manuscript)
I. Yu L, Mohamed AJ, Vargas L, Berglöf A, Finn G, Lu KP, Smith CI (2006). "Regulation of Bruton tyrosine kinase by the peptidylprolyl isomerase Pin1." J Biol Chem 281(26): 18201-7. Epub 2006 Apr 27
Pubmed
II. Yu L, Mohamed AJ, Simonson OE, Vargas L, Blomberg KE, Björkstrand B, Arteaga HJ, Nore BF, Smith CI (2008). "Proteasome-dependent autoregulation of Bruton tyrosine kinase (Btk) promoter via NF-kappaB." Blood 111(9): 4617-26. Epub 2008 Feb 21
Pubmed
III. Yu L, Mohanram V, Simonson OE, Smith CIE, Spetz AL, Mohamed AJ (2009). "Proteasome inhibitors block HIV-1 replication by affecting both cellular and viral targets." (Submitted)
IV. Yu L, Simonson OE, Mohamed AJ, Smith CIE (2009). "NF-kappaB regulates the transcription of protein tyrosine kinase Tec." (Manuscript)
Issue date: 2009-03-27
Rights:
Publication year: 2009
ISBN: 978-91-7409-397-1
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