Abdalla J. Mohamed

A peptide nucleic acid–nuclear localization signal fusion that mediates nuclear transport of DNA

We have combined a peptide nucleic acid (PNA) with the SV40 core nuclear localization signal (NLS), to create a bifunctional PNA–NLS peptide. The PNA–NLS peptide increased the nuclear uptake of oligonucleotides and enhanced the transfection efficacy of plasmids. Gene expression from an enhanced green fluorescent protein plasmid and a lacZ plasmid was preserved when hybridized to PNA–NLS. In combination with the transfection agent polyethyleneimine, we have improved both the nuclear translocation of fluorescence-marked oligonucleotides, and the efficacy of plasmid transfection, up to eightfold. The technique obviates the use of cumbersome coupling procedures of the vector due to DNA–PNA duplex formation or displacement of the antisense plasmid DNA strand by a PNA molecule.

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

Summary:  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‐κB (NF‐κB) and nuclear factor of activated T cells (NFAT). In B cells, NF‐κB was shown to bind to the Btk promoter and induce transcription, whereas the B‐cell receptor‐dependent NF‐κB 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.

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

Summary:  Brutons 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.

Redistribution of Bruton's tyrosine kinase by activation of phosphatidylinositol 3-kinase and Rho-family GTPases.

Brutons tyrosine kinase (Btk) is a member of the Tec family of protein tyrosine kinases (PTK) characterized by an N‐terminal pleckstrin homology domain (PH) thought to directly interact with phosphoinositides. We report here that wild‐type (wt) and also a gain‐of‐function mutant of Btk are redistributed following a wide range of receptor‐mediated stimuli through phosphatidylinositol 3‐kinase (PI 3‐K) activation. Employing chimeric Btk with green fluorescent protein in transient transfections resulted in Btk translocation to the cytoplasmic membrane of live cells through various forms of upstream PI 3‐K activation. The redistribution was blocked by pharmacological and biological inhibitors of PI 3‐K. A gain‐of‐function mutant of Btk was found to be a potent inducer of lamellipodia and / or membrane ruffle formation. In the presence of constitutively active forms of Rac1 and Cdc42, Btk is co‐localized with actin in these regions. Formation of the membrane structures was blocked by the dominant negative form of N17‐Rac1. Therefore, Btk forms a link between a vast number of cell surface receptors activating PI 3‐K and certain members of the Rho‐family of small GTPases. In the chicken B cell line, DT40, cells lacking Btk differed from wt cells in the actin pattern and showed decreased capacity to form aggregates, further suggesting that cytoskeletal regulation mediated by Btk may be of physiological relevance.

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.

Signalling of Bruton's tyrosine kinase, Btk.

Brutons tyrosine kinase, which is encoded by the BTK gene, is a cytoplasmic protein tyrosine kinase (PTK) crucial for B‐cell development and differentiation. It belongs to the Tec family of PTKs containing several domains that are characteristic of signalling molecules. In humans, mutations that disrupt the function of this gene lead to the classical XLA syndrome (X‐linked agammaglobulinaemia), a primary immunodeficiency mainly characterized by lack of mature B cells as well as low levels of immunoglobulins. In contrast, animal models of this disease such as the xid mice display profoundly milder XLA phenotype. BTK phosphorylation and activation in response to engagement of the B‐cell receptor (BCR) by antigen is a dynamic process whereby a variety of proteins interact with each other and recruit signalling molecules resulting in a physiological response such as B‐cell proliferation and antibody production. The main players, however, that participate in the intracellular downstream cascade have not yet been identified and are therefore under intense scrutiny in several laboratories. This review discusses certain aspects of BTK activation following receptor stimulation by agonists and how this event is translated into the biochemical signals within the cell that eventually lead to nuclear responses.

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.

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 (pep‐tide 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.—Ge R., Heinonen, J. E., Svahn, M. G., Mohamed, A. J., Lundin, K. E., Smith C. I. E. Zorro locked nucleic acid induces sequence‐specific gene silencing. FASEB J. 21, 1902–1914 (2007)

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

ABSTRACT Brutons 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.

Regulation of Nucleocytoplasmic Shuttling of Bruton's Tyrosine Kinase (Btk) through a Novel SH3-Dependent Interaction with Ankyrin Repeat Domain 54 (ANKRD54)

ABSTRACT Brutons 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 Btks SH3 domain. Liar also showed reduced binding to an aspartic acid phosphomimetic SH3 mutant. Three other investigated nucleus-located proteins, Abl, estrogen receptor β (ERβ), 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.