Specific properties of shRNA-mediated CCR5 downregulation that enhance the inhibition of HIV-1 infection in combination with shRNA targeting HIV-1 rev.

Treatment with RNAi against HIV-1 transcripts efficiently inhibits viral replication but induces selection of escape mutants; therefore, the CCR5 coreceptor was suggested as an additional target. Blocking viral and host transcripts improved the antiviral effect. We have used short hairpin RNA (shRNA) targeting the human CCR5 (shCCR5) or the HIV-1 rev (shRev) transcripts to demonstrate distinctive properties of anti-CCR5 shRNA: shCCR5 induced more sustained protection than shRev; partial reduction in CCR5 expression substantially decreased HIV-1 infection, and shCCR5 performed better than shRev in the mixed shRNA-treated and untreated cultures. These observations indicate that CCR5 inhibitors should be conveniently included in HIV-1 gene silencing treatment schedules when only a certain cell fraction is protected to further reduce endogenous virus in a properly ART-treated HIV-1 infected individual.

Protein kinase B (AKT) regulates SYK activity and shuttling through 14-3-3 and importin 7.

The Protein kinase B (AKT) regulates a plethora of intracellular signaling proteins to fine-tune signaling of multiple pathways. Here, we found that following B-cell receptor (BCR)-induced tyrosine phosphorylation of the cytoplasmic tyrosine kinase SYK and the adaptor BLNK, the AKT/PKB enzyme strongly induced BLNK (>100-fold) and SYK (>100-fold) serine/threonine phosphorylation (pS/pT). Increased phosphorylation promoted 14-3-3 binding to BLNK (37-fold) and SYK (2.5-fold) in a pS/pT-concentration dependent manner. We also demonstrated that the AKT inhibitor MK2206 reduced pS/pT of both BLNK (3-fold) and SYK (2.5-fold). Notably, the AKT phosphatase, PHLPP2 maintained the activating phosphorylation of BLNK at Y84 and increased protein stability (8.5-fold). In addition, 14-3-3 was required for the regulation SYK's interaction with BLNK and attenuated SYK binding to Importin 7 (5-fold), thereby perturbing shuttling to the nucleus. Moreover, 14-3-3 proteins also sustained tyrosine phosphorylation of SYK and BLNK. Furthermore, substitution of S295 or S297 for alanine abrogated SYK's binding to Importin 7. SYK with S295A or S297A replacements showed intense pY525/526 phosphorylation, and BLNK pY84 phosphorylation correlated with the SYK pY525/526 phosphorylation level. Conversely, the corresponding mutations to aspartic acid in SYK reduced pY525/526 phosphorylation. Collectively, these and previous results suggest that AKT and 14-3-3 proteins down-regulate the activity of several BCR-associated components, including BTK, BLNK and SYK and also inhibit SYK's interaction with Importin 7.

A murine model of acute myeloid leukemia with Evi1 overexpression and autocrine stimulation by an intracellular form of GM-CSF in DA-3 cells.

The poor treatment response of acute myeloid leukemia (AML) overexpressing high-risk oncogenes such as EVI1, demands specific animal models for new treatment evaluations. Evi1 is a common site of activating integrations in murine leukemia virus (MLV)-induced AML and in retroviral and lentiviral gene-modified HCS. Still, a model of overt AML induced by Evi1 has not been generated. Cell lines from MLV-induced AML are growth factor-dependent and non-transplantable. Hence, for the leukemia maintenance in the infected animals, a growth factor source such as chronic immune response has been suggested. We have investigated whether these leukemias are transplantable if provided with growth factors. We show that the Evi1(+)DA-3 cells modified to express an intracellular form of GM-CSF, acquired growth factor independence and transplantability and caused an overt leukemia in syngeneic hosts, without increasing serum GM-CSF levels. We propose this as a general approach for modeling different forms of high-risk human AML using similar cell lines.

Dual phosphorylation of Btk by Akt/protein kinase b provides docking for 14-3-3ζ, regulates shuttling, and attenuates both tonic and induced signaling in B cells.

Bruton's tyrosine kinase (Btk) is crucial for B-lymphocyte activation and development. Mutations in the Btk gene cause X-linked agammaglobulinemia (XLA) in humans and X-linked immunodeficiency (Xid) in mice. Using tandem mass spectrometry, 14-3-3ζ was identified as a new binding partner and negative regulator of Btk in both B-cell lines and primary B lymphocytes. The activated serine/threonine kinase Akt/protein kinase B (PKB) phosphorylated Btk on two sites prior to 14-3-3ζ binding. The interaction sites were mapped to phosphoserine pS51 in the pleckstrin homology domain and phosphothreonine pT495 in the kinase domain. The double-alanine, S51A/T495A, replacement mutant failed to bind 14-3-3ζ, while phosphomimetic aspartate substitutions, S51D/T495D, caused enhanced interaction. The phosphatidylinositol 3-kinase (PI3-kinase) inhibitor LY294002 abrogated S51/T495 phosphorylation and binding. A newly characterized 14-3-3 inhibitor, BV02, reduced binding, as did the Btk inhibitor PCI-32765 (ibrutinib). Interestingly, in the presence of BV02, phosphorylation of Btk, phospholipase Cγ2, and NF-κB increased strongly, suggesting that 14-3-3 also regulates B-cell receptor (BCR)-mediated tonic signaling. Furthermore, downregulation of 14-3-3ζ elevated nuclear translocation of Btk. The loss-of-function mutant S51A/T495A showed reduced tyrosine phosphorylation and ubiquitination. Conversely, the gain-of-function mutant S51D/T495D exhibited intense tyrosine phosphorylation, associated with Btk ubiquitination and degradation, likely contributing to the termination of BCR signaling. Collectively, this suggests that Btk could become an important new candidate for the general study of 14-3-3-mediated regulation.

Signaling of the ITK (interleukin 2-inducible T cell kinase)-SYK (spleen tyrosine kinase) fusion kinase is dependent on adapter SLP-76 and on the adapter function of the kinases SYK and ZAP70.

The inducible T cell kinase-spleen tyrosine kinase (ITK-SYK) oncogene consists of the Tec homology-pleckstrin homology domain of ITK and the kinase domain of SYK, and it is believed to be the cause of peripheral T cell lymphoma. We and others have recently demonstrated that this fusion protein is constitutively tyrosine-phosphorylated and is transforming both in vitro and in vivo. To gain a deeper insight into the molecular mechanism(s) underlying its activation and signaling, we mutated a total of eight tyrosines located in the SYK portion of the chimera into either phenylalanine or to the negatively charged glutamic acid. Although mutations in the interdomain-B region affected ITK-SYK kinase activity, they only modestly altered downstream signaling events. In contrast, mutations that were introduced in the kinase domain triggered severe impairment of downstream signaling. Moreover, we show here that SLP-76 is critical for ITK-SYK activation and is particularly required for the ITK-SYK-dependent phosphorylation of SYK activation loop tyrosines. In Jurkat cell lines, we demonstrate that expression of ITK-SYK fusion requires an intact SLP-76 function and significantly induces IL-2 secretion and CD69 expression. Furthermore, the SLP-76-mediated induction of IL-2 and CD69 could be further enhanced by SYK or ZAP-70, but it was independent of their kinase activity. Notably, ITK-SYK expression in SYF cells phosphorylates SLP-76 in the absence of SRC family kinases. Altogether, our data suggest that ITK-SYK exists in the active conformation state and is therefore capable of signaling without SRC family kinases or stimulation of the T cell receptor.

Regulation of nucleocytoplasmic shuttling of Bruton's tyrosine kinase (Btk) through a novel SH3-dependent interaction with ankyrin repeat domain 54 (ANKRD54).

Bruton's tyrosine kinase (Btk), belonging to the Tec family of tyrosine kinases (TFKs), is essential for B-lymphocyte development. Abrogation of Btk signaling causes human X-linked agammaglobulinemia (XLA) and murine X-linked immunodeficiency (Xid). We employed affinity purification of Flag-tagged Btk, combined with tandem mass spectrometry, to capture and identify novel interacting proteins. We here characterize the interaction with ankryin repeat domain 54 protein (ANKRD54), also known as Lyn-interacting ankyrin repeat protein (Liar). While Btk is a nucleocytoplasmic protein, the Liar pool was found to shuttle at a higher rate than Btk. Importantly, our results suggest that Liar mediates nuclear export of both Btk and another TFK, Txk/Rlk. Liar-mediated Btk shuttling was enriched for activation loop, nonphosphorylated Btk and entirely dependent on Btk's SH3 domain. Liar also showed reduced binding to an aspartic acid phosphomimetic SH3 mutant. Three other investigated nucleus-located proteins, Abl, estrogen receptor ß (ERß), and transcription factor T-bet, were all unaffected by Liar. We mapped the interaction site to the C terminus of the Btk SH3 domain. A biotinylated, synthetic Btk peptide, ARDKNGQEGYIPSNYVTEAEDS, was sufficient for this interaction. Liar is the first protein identified that specifically influences the nucleocytoplasmic shuttling of Btk and Txk and belongs to a rare group of known proteins carrying out this activity in a Crm1-dependent manner.

TEC family kinases in health and disease--loss-of-function of BTK and ITK and the gain-of-function fusions ITK-SYK and BTK-SYK.

The TEC family is ancient and constitutes the second largest family of cytoplasmic tyrosine kinases. In 1993, loss-of-function mutations in the BTK gene were reported as the cause of X-linked agammaglobulinemia. Of all the existing 90 tyrosine kinases in humans, Bruton's tyrosine kinase (BTK) is the kinase for which most mutations have been identified. These experiments of nature collectively provide a form of mutation scanning with direct implications for the several hundred endogenous signaling proteins carrying domains also found in BTK. In 2009, an inactivating mutation in the ITK gene was shown to cause susceptibility to lethal Epstein-Barr virus infection. Both kinases represent interesting targets for inhibition: in the case of BTK, as an immunosuppressant, whereas there is evidence that the inhibition of inducible T-cell kinase (ITK) could influence the infectivity of HIV and also have anti-inflammatory activity. Since 2006, several patients carrying a fusion protein, originating from a translocation joining genes encoding the kinases ITK and spleen tyrosine kinase (SYK), have been shown to develop T-cell lymphoma. We review these disease processes and also describe the role of the N-terminal pleckstrin homology-Tec homology (PH-TH) domain doublet of BTK and ITK in the downstream intracellular signaling of such fusion proteins.

NF-kappaB regulates the transcription of protein tyrosine kinase Tec.

The tyrosine kinase expressed in hepatocellular carcinoma (Tec) is a non-receptor protein tyrosine kinase (PTK) that is expressed in hematopoietic cells, such as B and T lymphocytes, myeloid lineage cells and neutrophils. Mutations in the human Btk gene cause X-linked agammaglobulinemia (XLA), but the corresponding mutation in mice results in a much milder defect. However, the combined inactivation of Btk and Tec genes in mice cause a severe phenotype resembling XLA. Tec is involved in the regulation of both B and T lymphocytes, fine-tuning of TCR/BCR signaling, and also activation of the nuclear factor of activated T cells. Previous work has shown that the transcription factors Sp1 and PU.1 can bind and regulate the Tec promoter. In this study, we demonstrate that NF-kappaB is an essential transcription factor for optimal expression of the Tec gene, and identify a unique functionally active NF-kappaB binding site in its promoter. The NF-kappaB subunit p65/RelA directly induced transcriptional activity of the Tec promoter. Moreover, we also found that proteasome inhibitors, including Bortezomib, repress Tec transcription through inactivation of the NF-kappaB signaling pathway. This study, together with our previous findings on the transcriptional regulation of Btk (Bruton's tyrosine kinase) by proteasome inhibitors, provides important insight into the molecular mechanism(s) underlying the role of NF-kappaB in Tec family kinase signaling and lymphocyte development.

Phosphatidylinositol-3-kinase-dependent phosphorylation of SLP-76 by the lymphoma-associated ITK-SYK fusion-protein.

Recurrent chromosomal translocations have long been implicated in various types of lymphomas and other malignancies. Novel recurrent t(5;9)(q33;q22) has been recently discovered in un-specified peripheral T-cell lymphoma. To elucidate the role of this translocation, the corresponding fusion construct encoding the N-terminal portion of the ITK kinase and the C-terminal catalytic region of the SYK kinase was generated. We herein show that the ITK-SYK fusion-protein is constitutively active. Moreover, we demonstrate that ITK-SYK is phosphorylated on key tyrosine residues and is capable of potently phosphorylating the related adapter proteins BLNK and SLP-76. In transiently transfected cells, SYK was phosphorylated at Y352 but not detectably at the activation-loop tyrosines Y525/Y526. In contrast, ITK-SYK was phosphorylated both at Y212 and the activation-loop tyrosines Y385/Y386, corresponding to Y352 and Y525/Y526 in SYK, respectively. In resting primary lymphocytes, ITK-SYK predominantly localizes to the cell surface. In addition, we demonstrate that following stimulation, the ITK-SYK fusion-protein in cell lines translocates to the cell membrane and, moreover, that this phenomenon as well as SLP-76 phosphorylation are blocked upon phosphatidylinositol-3-kinase (PI3-kinase) inhibition.

Proteasome inhibitors block HIV-1 replication by affecting both cellular and viral targets.

HIV-1 has proved to be notoriously difficult to tackle despite the availability of more than 20 clinically approved drugs. The majority of these drugs, however, target viral genes and their continued use will select for drug-resistant strains. Since NF-kappaB signaling is critical for viral replication, we wanted to investigate the effect of proteasome inhibitors on viral gene expression. We herein demonstrate that proteasome and NF-kappaB inhibitors effectively shut down transcription from the HIV-1 LTR-promoter. We further show that replication of HIV-1 in PBMC was severely compromised following treatment with proteasome inhibitors alone or in combination with other antiretroviral drugs. Finally, incubation of PBMC with these drugs reduced expression of IL-2 inducible T cell kinase (Itk), a Tec-family kinase, recently shown to be required for HIV-1 replication. These results suggest that proteasome inhibitors suppress LTR-promoter activity by interfering with cellular targets required for viral replication.

Bruton's tyrosine kinase (Btk): function, regulation, and transformation with special emphasis on the PH domain.

Bruton's agammaglobulinemia tyrosine kinase (Btk) is a cytoplasmic tyrosine kinase important in B-lymphocyte development, differentiation, and signaling. Btk is a member of the Tec family of kinases. Mutations in the Btk gene lead to X-linked agammaglobulinemia (XLA) in humans and X-linked immunodeficiency (Xid) in mice. Activation of Btk triggers a cascade of signaling events that culminates in the generation of calcium mobilization and fluxes, cytoskeletal rearrangements, and transcriptional regulation involving nuclear factor-kappaB (NF-kappaB) and nuclear factor of activated T cells (NFAT). In B cells, NF-kappaB was shown to bind to the Btk promoter and induce transcription, whereas the B-cell receptor-dependent NF-kappaB signaling pathway requires functional Btk. Moreover, Btk activation is tightly regulated by a plethora of other signaling proteins including protein kinase C (PKC), Sab/SH3BP5, and caveolin-1. For example, the prolyl isomerase Pin1 negatively regulates Btk by decreasing tyrosine phosphorylation and steady state levels of Btk. It is intriguing that PKC and Pin1, both of which are negative regulators, bind to the pleckstrin homology domain of Btk. To this end, we describe here novel mutations in the pleckstrin homology domain investigated for their transforming capacity. In particular, we show that the mutant D43R behaves similar to E41K, already known to possess such activity.

Proteasome-dependent autoregulation of Bruton tyrosine kinase (Btk) promoter via NF-kappaB.

Bruton tyrosine kinase (Btk) is critical for B-cell development. Btk regulates a plethora of signaling proteins, among them nuclear factor-[kappa]B (NF-kappaB). Activation of NF-kappaB is a hallmark of B cells, and NF-kappaB signaling is severely compromised in Btk deficiency. We here present strong evidence indicating that NF-kappaB is required for efficient transcription of the Btk gene. First, we found that proteasome blockers and inhibitors of NF-kappaB signaling suppress Btk transcription and intracellular expression. Similar to Btk, proteasome inhibitors also reduced the expression of other members of this family of kinases, Itk, Bmx, and Tec. Second, 2 functional NF-kappaB-binding sites were found in the Btk promoter. Moreover, in live mice, by hydrodynamic transfection, we show that bortezomib (a blocker of proteasomes and NF-kappaB signaling), as well as NF-kappaB binding sequence-oligonucleotide decoys block Btk transcription. We also demonstrate that Btk induces NF-kappaB activity in mice. Collectively, we show that Btk uses a positive autoregulatory feedback mechanism to stimulate transcription from its own promoter via NF-kappaB.

Sequence-specific inhibition of RNA polymerase III-dependent transcription using Zorro locked nucleic acid (LNA).

BACKGROUND: RNA polymerase III (pol III)-dependent transcripts are involved in many fundamental activities in a cell, such as splicing and protein synthesis. They also regulate cell growth and influence tumor formation. During recent years vector-based systems for expression of short hairpin (sh) RNA under the control of a pol III promoter have been developed as gene-based medicines. Therefore, there is an increasing interest in means to regulate pol III-dependent transcription. Recently, we have developed a novel anti-gene molecule 'Zorro LNA (Locked Nucleic Acid)', which simultaneously hybridizes to both strands of super-coiled DNA and potently inhibits RNA polymerase II-derived transcription. We have now applied Zorro LNA in an attempt to also control U6 promoter-driven expression of shRNA. METHODS: In this study, we constructed pshluc and pshluc2BS plasmids, in which U6 promoter-driven small hairpin RNA specific for luciferase gene (shluc) was without or with Zorro LNA binding sites, respectively. After hybridization of Zorro LNA to pshluc2BS, the LNA-bound plasmid was cotransfected with pEGFPluc into mammalian cells and into a mouse model. In cellular experiments, cotransfection of unhybridized pshluc2BS, Zorro LNA and pEGFPluc was also performed. RESULTS: The results showed that the Zorro LNA construct efficiently inhibited pol III-dependent transcription as an anti-gene reagent in a cellular context, including in vivo in a mouse model. CONCLUSIONS: Thus, this new form of gene silencer 'Zorro LNA' could potentially serve as a versatile regulator of pol III-dependent transcription, including various forms of shRNAs.

Zorro locked nucleic acid induces sequence-specific gene silencing.

Locked nucleic acids (LNAs) are synthetic analogs of nucleic acids that contain a bridging methylene carbon between the 2' and 4' positions of the ribose ring. In this study, we generated a novel sequence-specific antigene molecule "Zorro LNA", which simultaneously binds to both strands, and that induced effective and specific strand invasion into DNA duplexes and potent inhibition of gene transcription, also in a cellular context. By comparing the Zorro LNA with linear LNA as well as an optimized bisPNA (peptide nucleic acid) oligonucleotide directed against the same target sites, respectively, we found that the Zorro LNA construct was unique in its ability to arrest gene transcription in mammalian cells. To our knowledge, this is the first time that in mammalian cells, gene transcription was blocked by a nucleic acid analog in a sequence-specific way using low but saturated binding of a blocking agent. This offers a novel type of antigene drug that is easy to synthesize.

Regulation of Bruton tyrosine kinase by the peptidylprolyl isomerase Pin1.

Bruton tyrosine kinase (Btk) is expressed in B-lymphocytes. Mutations in Btk cause X-linked agammaglobulinemia in humans. However, the mechanism of activation and signaling of this enzyme has not been fully investigated. We have here shown that the peptidylprolyl cis/trans isomerase (PPIase) Pin1 is a negative regulator of Btk, controlling its expression level by reducing its half-life, whereas the catalytic activity of Btk was unaffected. The negative regulatory effect of Pin1 was observed both in cell lines and in Pin(-/-) mice and was found to be dependent on a functionally intact Btk. This may constitute a feedback loop for the regulation of Btk. The target region in Btk was localized to the pleckstrin homology domain suggesting that interphase phosphorylation of serine 115 (Ser-115) in Btk is required, whereas mitosis phosphorylation of serine 21 (Ser-21) is critical. Accordingly, Pin 1 was shown to associate with Btk through binding to Ser-21 and -115, respectively, both of which lie in a classical Pin1-binding pocket. Using a phosphomitotic antibody, it was found that Btk harbors a bona fide MPM2 epitope corresponding to a phosphorylated serine or threonine residue followed by a proline. Our results indicate that the peptidylprolyl isomerase Pin1 interacts with Btk in a cell cycle-dependent manner, regulating the Btk expression level.

Inducible H1 promoter-driven lentiviral siRNA expression by Stuffer reporter deletion.

Introduction of 19-23-bp small interfering RNA (siRNA) into mammalian cells has become a standard procedure to downregulate mRNA with high efficacy. siRNAs can be introduced into cells either as synthetic duplexes or as hairpin structures produced by Pol III promoter-driven vectors. Pol III promoter-expressed small hairpin RNAs (shRNAs) offer a great possibility for the production of endogenous siRNA, which can be used for stable siRNA production in vivo. A major drawback of this strategy is the incapability of detecting rapidly occurring cellular responses. Here, we present a lentiviral shRNA-producing vector system, which can be induced by CRE recombinase enzyme to overcome these limitations. Following the addition of CRE, the pLIND (LentiINDucible) will activate siRNA production by deleting EGFP and a stop cassette between the promoter and siRNA oligo. Target gene downregulation capacity was comparable to that of a noninducible siRNA system.

Bruton's tyrosine kinase: cell biology, sequence conservation, mutation spectrum, siRNA modifications, and expression profiling.

Bruton's tyrosine kinase (Btk) is encoded by the gene that when mutated causes the primary immunodeficiency disease X-linked agammaglobulinemia (XLA) in humans and X-linked immunodeficiency (Xid) in mice. Btk is a member of the Tec family of protein tyrosine kinases (PTKs) and plays a vital, but diverse, modulatory role in many cellular processes. Mutations affecting Btk block B-lymphocyte development. Btk is conserved among species, and in this review, we present the sequence of the full-length rat Btk and find it to be analogous to the mouse Btk sequence. We have also analyzed the wealth of information compiled in the mutation database for XLA (BTKbase), representing 554 unique molecular events in 823 families and demonstrate that only selected amino acids are sensitive to replacement (P < 0.001). Although genotype-phenotype correlations have not been established in XLA, based on these findings, we hypothesize that this relationship indeed exists. Using short interfering-RNA technology, we have previously generated active constructs downregulating Btk expression. However, application of recently established guidelines to enhance or decrease the activity was not successful, demonstrating the importance of the primary sequence. We also review the outcome of expression profiling, comparing B lymphocytes from XLA-, Xid-, and Btk-knockout (KO) donors to healthy controls. Finally, in spite of a few genes differing in expression between Xid- and Btk-KO mice, in vivo competition between cells expressing either mutation shows that there is no selective survival advantage of cells carrying one genetic defect over the other. We conclusively demonstrate that for the R28C-missense mutant (Xid), there is no biologically relevant residual activity or any dominant negative effect versus other proteins.

Functional interaction of caveolin-1 with Bruton's tyrosine kinase and Bmx.

Bruton's tyrosine kinase (Btk), a member of the Tec family of protein-tyrosine kinases, has been shown to be crucial for B cell development, differentiation, and signaling. Mutations in the Btk gene lead to X-linked agammaglobulinemia in humans and X-linked immunodeficiency in mice. Using a co-transfection approach, we present evidence here that Btk interacts physically with caveolin-1, a 22-kDa integral membrane protein, which is the principal structural and regulatory component of caveolae membranes. In addition, we found that native Bmx, another member of the Tec family kinases, is associated with endogenous caveolin-1 in primary human umbilical vein endothelial cells. Second, in transient transfection assays, expression of caveolin-1 leads to a substantial reduction in the in vivo tyrosine phosphorylation of both Btk and its constitutively active form, E41K. Furthermore, a caveolin-1 scaffolding peptide (amino acids 82--101) functionally suppressed the autokinase activity of purified recombinant Btk protein. Third, we demonstrate that mouse splenic B-lymphocytes express substantial amounts of caveolin-1. Interestingly, caveolin-1 was found to be constitutively phosphorylated on tyrosine 14 in these cells. The expression of caveolin-1 in B-lymphocytes and its interaction with Btk may have implications not only for B cell activation and signaling, but also for antigen presentation.