Exposure to Therapeutic BTK Inhibitors Induces Phenocopying of Btk29A Mutants in the Fruit Fly Drosophila melanogaster.

BACKGROUND: Bruton's tyrosine kinase (BTK) is a non-receptor type tyrosine kinase originally identified as the genetic signature responsible for X-linked agammaglobulinemia (XLA) when mutated. Its functional form is required for B lymphocyte maturation in both humans and mice, whereas loss-of-function causes a different form of developmental defect in the fruit fly, Drosophila melanogaster. METHODS: Ibrutinib and other therapeutic inhibitors of BTK have been extensively used to successfully treat various leukemias and lymphomas. Btk29A type 2 is the ortholog of BTK in the fruit fly. We show that feeding wild-type flies an ibrutinib-containing diet induces phenocopying of Btk29A mutants, i.e., failure in the fusion of left and right halves of the dorsal cuticles, partial loss of wing tissues and dysregulation of germ cell production. RESULTS: We have previously reported that Btk29A phosphorylates Drosophila Arm (ß-catenin), and ibrutinib reduces phosphorylation at Tyrosine142 of endogenously expressed ß-catenin in Cos7 cells transfected with Btk29A type 2 cDNA. CONCLUSIONS: Thus, Drosophila is suitable for screens of novel BTK inhibitor candidates and offers a unique in vivo system in which the mode of action of BTK inhibitors can be examined at the molecular, cellular, and organismal levels.

Translocation-generated ITK-FER and ITK-SYK fusions induce STAT3 phosphorylation and CD69 expression.

Many cancer types carry mutations in protein tyrosine kinase (PTK) and such alterations frequently drive tumor progression. One category is gene translocation of PTKs yielding chimeric proteins with transforming capacity. In this study, we characterized the role of ITK-FER [Interleukin-2-inducible T-cell Kinase (ITK) gene fused with Feline Encephalitis Virus-Related kinase (FER) gene] and ITK-SYK [Interleukin-2-inducible T-cell Kinase (ITK) gene fused with the Spleen Tyrosine Kinase (SYK)] in Peripheral T Cell Lymphoma (PTCL) signaling. We observed an induction of tyrosine phosphorylation events in the presence of both ITK-FER and ITK-SYK. The downstream targets of ITK-FER and ITK-SYK were explored and STAT3 was found to be highly phosphorylated by these fusion kinases. In addition, the CD69 T-cell activation marker was significantly elevated. Apart from tyrosine kinase inhibitors acting directly on the fusions, we believe that drugs acting on downstream targets could serve as alternative cancer therapies for fusion PTKs.

ANKRD54 preferentially selects Bruton's Tyrosine Kinase (BTK) from a Human Src-Homology 3 (SH3) domain library.

Bruton's Tyrosine Kinase (BTK) is a cytoplasmic protein tyrosine kinase with a fundamental role in B-lymphocyte development and activation. The nucleocytoplasmic shuttling of BTK is specifically modulated by the Ankyrin Repeat Domain 54 (ANKRD54) protein and the interaction is known to be exclusively SH3-dependent. To identify the spectrum of the ANKRD54 SH3-interactome, we applied phage-display screening of a library containing all the 296 human SH3 domains. The BTK-SH3 domain was the prime interactor. Quantitative western blotting analysis demonstrated the accuracy of the screening procedure. Revealing the spectrum and specificity of ANKRD54-interactome is a critical step toward functional analysis in cells and tissues.

B Cell Receptor Activation Predominantly Regulates AKT-mTORC1/2 Substrates Functionally Related to RNA Processing.

Protein kinase B (AKT) phosphorylates numerous substrates on the consensus motif RXRXXpS/T, a docking site for 14-3-3 interactions. To identify novel AKT-induced phosphorylation events following B cell receptor (BCR) activation, we performed proteomics, biochemical and bioinformatics analyses. Phosphorylated consensus motif-specific antibody enrichment, followed by tandem mass spectrometry, identified 446 proteins, containing 186 novel phosphorylation events. Moreover, we found 85 proteins with up regulated phosphorylation, while in 277 it was down regulated following stimulation. Up regulation was mainly in proteins involved in ribosomal and translational regulation, DNA binding and transcription regulation. Conversely, down regulation was preferentially in RNA binding, mRNA splicing and mRNP export proteins. Immunoblotting of two identified RNA regulatory proteins, RBM25 and MEF-2D, confirmed the proteomics data. Consistent with these findings, the AKT-inhibitor (MK-2206) dramatically reduced, while the mTORC-inhibitor PP242 totally blocked phosphorylation on the RXRXXpS/T motif. This demonstrates that this motif, previously suggested as an AKT target sequence, also is a substrate for mTORC1/2. Proteins with PDZ, PH and/or SH3 domains contained the consensus motif, whereas in those with an HMG-box, H15 domains and/or NF-X1-zinc-fingers, the motif was absent. Proteins carrying the consensus motif were found in all eukaryotic clades indicating that they regulate a phylogenetically conserved set of proteins.

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.

Anti-leukaemic effects induced by APR-246 are dependent on induction of oxidative stress and the NFE2L2/HMOX1 axis that can be targeted by PI3K and mTOR inhibitors in acute myeloid leukaemia cells.

The small molecule APR-246 (PRIMA-1(MET) ) is a novel drug that restores the activity of mutated and unfolded TP53 protein. However, the mechanisms of action and potential off-target effects are not fully understood. Gene expression profiling in TP53 mutant KMB3 acute myeloid leukaemia (AML) cells showed that genes which protected cells from oxidative stress to be the most up-regulated. APR-246 exposure also induced reactive oxygen species (ROS) formation and depleted glutathione in AML cells. The genes most up-regulated by APR-246, confirmed by quantitative real time polymerase chain reaction, were heme oxygenase-1 (HMOX1, also termed HO-1), SLC7A11 and RIT1. Up-regulation of HMOX1, a key regulator of cellular response to ROS, was independent of TP53 mutational status. NFE2L2 (also termed Nrf2), a master regulator of HMOX1 expression, showed transcriptional up-regulation and nuclear translocation by APR-246. Down-regulation of NFE2L2 by siRNA in AML cells significantly increased the antitumoural effects of APR-246. The PI3K inhibitor wortmannin and the mTOR inhibitor rapamycin inhibited APR-246-induced nuclear translocation of NFE2L2 and counteracted the protective cellular responses to APR-246, resulting in synergistic cell killing together with APR-246. In conclusion, ROS induction is important for antileukaemic activities of APR-246 and inhibiting the protective response of the Nrf-2/HMOX1 axis using PI3K inhibitors, enhances the antileukaemic effects.

Longitudinal plication--a surgical strategy for complete rectal prolapse management.

BACKGROUND: Rectal prolapse is a known problem since antiquity and the cause is not fully understood. Despite the presence of more than 100 lines of treatment, none of them is ideal. METHODS: Between the years of (2005-2011), thirty patients with full-thickness rectal prolapse were operated upon. Age ranged between (2-65 years) with a mean of 21.5 year. Male to female ratio was (2:1). Each prolapsed rectum was repaired with longitudinal plication (LP) at two or three points accordingly using braded polyglycolic acid - absorbable 1.0 suture material. Plications started by inserting a stitch at the most proximal part of the prolapse, followed by successive similar transverse stiches continuing in a spiral fashion till the mucocutaneous junction. We used three LP in adults and two in children. All of the patients where operated upon as a day-case procedure and discharged 6 hours after the operation. RESULTS: In this series of patients, twenty-nine of them had complete recovery from the prolapse. Only one patient had recurrence 2 years after the operation, and the same procedure was applied successfully with uneventful post-operative period. Although twenty-three patients had fecal Incontinence, twenty-one of them regained continence after operation. CONCLUSIONS: This method is an easy perineal procedure, with fewer complications. It can be performed for all age groups, in an ordinary surgical unit, by an expert anorectal surgeon. We found that our procedure is simple, safe and less invasive.

Glutathione transferase mu 2 protects glioblastoma cells against aminochrome toxicity by preventing autophagy and lysosome dysfunction.

U373MG cells constitutively express glutathione S-transferase mu 2 (GSTM2) and exhibit (3)H-dopamine uptake, which is inhibited by 2 µM of nomifensine and 15 µM of estradiol. We generated a stable cell line (U373MGsiGST6) expressing an siRNA against GSTM2 that resulted in low GSTM2 expression (26% of wild-type U373MG cells). A significant increase in cell death was observed when U373MGsiGST6 cells were incubated with 50 µM purified aminochrome (18-fold increase) compared with wild-type cells. The incubation of U373MGsiGST6 cells with 75 µM aminochrome resulted in the formation of autophagic vacuoles containing undigested cellular components, as determined using transmission electron microscopy. A significant increase in autophagosomes was determined by measuring endogenous LC3-II, a significant decrease in cell death was observed in the presence of bafilomycin A 1, and a significant increase in cell death was observed in the presence of trehalose. A significant increase in LAMP2 immunostaining was observed, a significant decrease in bright red fluorescence of lysosomes with acridine orange was observed, and bafilomycin A 1 pretreatment reduced the loss of lysosome acidity. A significant increase in cell death was observed in the presence of lysosomal protease inhibitors. Aggregation of TUBA/α-tubulin (tubulin, α) and SQSTM1 protein accumulation were also observed. Moreover, a significant increase in the number of lipids droplets was observed compared with U373MG cells with normal expression of GSTM2. These results support the notion that GSTM2 is a protective enzyme against aminochrome toxicity in astrocytes and that aminochrome cell death in U373MGsiGST6 cells involves autophagic-lysosomal dysfunction.

Btk29A promotes Wnt4 signaling in the niche to terminate germ cell proliferation in Drosophila.

Btk29A is the Drosophila ortholog of the mammalian Bruton's tyrosine kinase (Btk), mutations of which in humans cause a heritable immunodeficiency disease. Btk29A mutations stabilized the proliferating cystoblast fate, leading to an ovarian tumor. This phenotype was rescued by overexpression of wild-type Btk29A and phenocopied by the interference of Wnt4-ß-catenin signaling or its putative downstream nuclear protein Piwi in somatic escort cells. Btk29A and mammalian Btk directly phosphorylated tyrosine residues of ß-catenin, leading to the up-regulation of its transcriptional activity. Thus, we identify a transcriptional switch involving the kinase Btk29A/Btk and its phosphorylation target, ß-catenin, which functions downstream of Wnt4 in escort cells to terminate Drosophila germ cell proliferation through up-regulation of piwi expression. This signaling mechanism likely represents a versatile developmental switch.

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.

A novel missense mutation in the CLCN7 gene linked to benign autosomal dominant osteopetrosis: a case series.

INTRODUCTION: Osteopetrosis is a rare inherited genetic disease characterized by sclerosis of the skeleton. The absence or malfunction of osteoclasts is found to be strongly associated with the disease evolution. Currently, four clinically distinct forms of the disease have been recognized: the infantile autosomal recessive osteopetrosis, the malignant and the intermediate forms, and autosomal dominant osteopetrosis, type I and type II forms. The autosomal recessive types are the most severe forms with symptoms in very early childhood, whereas the autosomal dominant classes exhibit a heterogeneous trait with milder symptoms, often at later childhood or adulthood. CASE PRESENTATION: Case 1 is the 12-year-old daughter (index patient) of an Iraqi-Kurdish family who, at the age of eight years, was diagnosed clinically to have mild autosomal dominant osteopetrosis. Presently, at 12-years old, she has severe complications due to the disease progression. In addition, the same family previously experienced the death of a female child in her late childhood. The deceased child had been misdiagnosed, at that time, with thalassemia major. In this report, we extended our investigation to identify the type of the inheritance patterns of osteopetrosis using molecular techniques, because consanguineous marriages exist within the family history. We have detected one heterozygous mutation in exon 15 of the Chloride Channel 7 gene in the index patient (Case 1), whereas other mutations were not detected in the associated genes TCIRG1, OSTM1, RANK, and RANKL. The missense mutation (CGG>TGG) located in exon 15 (c.1225C>T) of the Chloride Channel 7 gene changed the amino acid position 409 from arginine to tryptophan (p.R409W, c.1225C>T).Case 2 is the 16-year-old son (brother of the index patient) of the same family who was diagnosed clinically with mild autosomal dominant osteopetrosis. We have identified the same heterozygous mutation in exon 15 of the Chloride channel 7 gene in this patient (Case 2). The missense mutation (CGG>TGG) located in exon 15 (c.1225C>T) of the Chloride channel 7 gene changed the amino acid position 409 from arginine to tryptophan (p.R409W, c.1225C>T).In addition to the clinical diagnosis of both cases, the missense mutation we identified in one allele of the Chloride channel 7 gene could be linked to autosomal dominant osteopetrosis-II because the symptoms appear in late childhood or adolescence. CONCLUSION: In this family, the molecular diagnosis was confirmed after identification of the same mutation in the older son (sibling). Furthermore, we detected that the father and his brother (the uncle) are carriers of the same mutation, whereas the mother and her sister (the aunt) do not carry any mutation of the Chloride channel 7 gene. Thus, the disease penetrance is at least 60% in the family. The mother and father are cousins and a further consanguineous marriage between the aunt and the uncle is not recommended because the dominant allele of the Chloride channel 7 gene will be transferred to the progeny. However, a similar risk is also expected following a marriage between the uncle and an unrelated woman. The p.R409W mutation in the Chloride channel 7 gene has not yet been described in the literature and it possibly has a dominant-negative impact on the protein.

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.

Sporadic incidence of Fascioliasis detected during hepatobiliary procedures: a study of 18 patients from Sulaimaniyah governorate.

BACKGROUND: Fascioliasis is an often-neglected zoonotic disease and currently is an emerging infection in Iraq. Fascioliasis has two distinct phases, an acute phase, exhibiting the hepatic migratory stage of the fluke's life cycle, and a chronic biliary phase manifested with the presence of the parasite in the bile ducts through hepatic tissue. The incidence of Fascioliasis in Sulaimaniyah governorate was unexpected observation. We believe that shedding light on this disease in our locality will increase our physician awareness and experience in early detection, treatment in order to avoid unnecessary surgeries. FINDINGS: We retrospectively evaluated this disease in terms of the demographic features, clinical presentations, and managements by reviewing the medical records of 18 patients, who were admitted to the Sulaimani Teaching Hospital and Kurdistan Centre for Gastroenterology and Hepatology. Patients were complained from hepatobiliary and/or upper gastrointestinal symptoms and diagnosed accidentally with Fascioliasis during hepatobiliary surgeries and ERCP by direct visualization of the flukes and stone analysis. Elevated liver enzymes, white blood cells count and eosinophilia were notable laboratory indices. The dilated CBD, gallstones, liver cysts and abscess were found common in radiological images. Fascioliasis diagnosed during conventional surgical CBD exploration and choledochodoudenostomy, open cholecystectomy, surgical drainage of liver abscess, ERCP and during gallstone analysis. CONCLUSION: Fascioliasis is indeed an emerging disease in our locality, but it is often underestimated and ignored. We recommend the differential diagnosis of patients suffering from Rt. Hypochondrial pain, fever and eosinophilia. The watercress ingestion was a common factor in patient's history.

Precursor T-cell acute lymphoblastic leukemia presenting with bone marrow necrosis: a case report.

INTRODUCTION: Bone marrow necrosis is a clinicopathological condition diagnosed most often at postmortem examination, but it is also seen during the course of malignancy and is not always associated with a poor prognosis. The morphological features of bone marrow necrosis are disruption of the normal marrow architecture and necrosis of myeloid tissue and medullary stroma. Non-malignant conditions associated with bone marrow necrosis are sickle cell anemia, infections, drugs (sulfasalazine, interferon α, all-trans retinoic acid, granulocyte colony-stimulating factor and fludarabine), disseminated intravascular coagulation, antiphospholipid antibody syndrome and acute graft versus host diseases. The malignant causes are leukemia, lymphoma and metastatic carcinomas. Herein we report the case of a patient with precursor T-cell acute lymphoblastic leukemia and bone marrow necrosis at initial presentation. CASE PRESENTATION: A 10-year-old Kurdish boy was presented with generalized bone pain and fever of 1 month's duration which was associated with sweating, easy fatigability, nose bleeding, breathlessness and severe weight loss. On examination, we observed pallor, tachypnea, tachycardia, low blood pressure, fever, petechial hemorrhage, ecchymoses, tortuous dilated veins over the chest and upper part of abdomen, multiple small cervical lymph node enlargements, mildly enlarged spleen, palpable liver and gross abdominal distention. Blood analysis revealed pancytopenia and elevated lactate dehydrogenase and erythrocyte sedimentation rate. Imaging results showed mediastinal widening on a planar chest X-ray and diffuse focal infiltration of the axial bone marrow on magnetic resonance imaging of the lumbosacral vertebrae. Bone marrow aspiration and biopsy examination showed extensive bone marrow necrosis. Immunophenotyping analysis of the bone marrow biopsy confirmed T-cell acute lymphoblastic leukemia, as CD3 and terminal deoxynucleotidyl transferase markers were positive and CD10, CD20 and CD79a markers were negative. CONCLUSION: The aggressive initial clinical presentation of our patient with huge mediastinal widening, development of superior vein cava syndrome and extensive bone marrow necrosis as initial signs made the diagnosis of the case difficult. The necrotic hematopoietic cells gave inconclusive results on the initial immunohistochemistry tests. The prognosis of bone marrow necrosis is better secondary to acute lymphoblastic leukemia in the pediatric age group compared with adults and those with underlying solid tumors. Despite the aggressive behavior at initial presentation, the patient responded to chemotherapy and necrosis disappeared at day 28 after the start of the therapeutic regimen.

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.

Stable expression of short interfering RNA for DT-diaphorase induces neurotoxicity.

DT-Diaphorase has been proposed to play a neuroprotective role in dopaminergic neurons by preventing aminochrome neurotoxicity. There are several studies supporting this idea, but in all studies, we used dicoumarol, an inhibitor of DT-diaphorase. We have designed and developed two siRNA to silence the expression of DT-diaphorase to study its role in aminochrome metabolism. We transduced RCSN-3 cells with retroviral particles containing a pRetroSuper plasmid coding a siRNA for DT-diaphorase. The cells selected in the presence of puromycin generated a stable cell line RCSN-3Nq6 and RCSN-3Nq7 with low expression of DT-diaphorase (27% and 33% of wild type, respectively). A significant cell death was observed in RCSN-3 cells expressing siRNA Nq6 and Nq7 for DT-diaphorase when were incubated with 100 µM aminochrome during 48 (4- and 3.5-fold, respectively; P < 0.01). These results support the protective role of DT-diaphorase against aminochrome neurotoxicity in dopaminergic neurons containing neuromelanin and show that Nq6 and Nq7 siRNA are very useful tools to study the role of DT-diaphorase in aminochrome metabolism.

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.

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.