Leonardo Vargas

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.

Phosphorylation of Bruton's tyrosine kinase by c-Abl.

Brutons tyrosine kinase (Btk) is necessary for B-lymphocyte development. Mutation in the gene coding for Btk causes X-linked agammaglobulinemia (XLA) in humans. Similar to Btk, c-Abl is a tyrosine kinase shuttling between the cytoplasm and the nucleus where it is involved in different functions depending on the localization. In this report we describe for the first time that c-Abl and Btk physically interact and that c-Abl can phosphorylate tyrosine 223 in the SH3 domain of Btk. Interestingly, the Btk sequence matched a v-Abl substrate [correction] identified from a randomized peptide library and was also highly related to a number of previously found c-Abl substrates.