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Bruton's tyrosine kinase (BTK) is pivotal in B cell activation and development through its participation in the signaling pathways of multiple hematopoietic receptors. The mechanisms controlling BTK activation were studied here by examination of the biochemical consequences of an interaction between BTK and SRC family kinases. This interaction of BTK with SRC kinases transphosphorylated BTK on tyrosine at residue 551, which led to BTK activation. BTK then autophosphorylated at a second site. The same two sites were phosphorylated upon B cell antigen receptor cross-linking. The activated BTK was predominantly membrane-associated, which suggests that BTK integrates distinct receptor signals resulting in SRC kinase activation and BTK membrane targeting.

A hallmark of neurodegenerative proteinopathies is the formation of misfolded protein aggregates that cause cellular toxicity and contribute to cellular proteostatic collapse. Therapeutic options are currently being explored that target different steps in the production and processing of proteins implicated in neurodegenerative disease, including synthesis, chaperone-assisted folding and trafficking, and degradation via the proteasome and autophagy pathways. Other therapies, like mTOR inhibitors and activators of the heat shock response, can rebalance the entire proteostatic network. However, there are major challenges that impact the development of novel therapies, including incomplete knowledge of druggable disease targets and their mechanism of action as well as a lack of biomarkers to monitor disease progression and therapeutic response. A notable development is the creation of collaborative ecosystems that include patients, clinicians, basic and translational researchers, foundations and regulatory agencies to promote scientific rigor and clinical data to accelerate the development of therapies that prevent, reverse or delay the progression of neurodegenerative proteinopathies.

Bruton's tyrosine kinase (Btk) plays a crucial role in B cell development. Overexpression of Btk with a Src family kinase increases tyrosine phosphorylation and catalytic activity of Btk. This occurs by transphosphorylation at Y551 in the Btk catalytic domain and the enhancement of Btk autophosphorylation at a second site. A gain-of-function mutant called Btk* containing E41 to K change within the pleckstrin homology domain induces fibroblast transformation. Btk* enhances the transphosphorylation of Y551 by endogenous Src family tyrosine kinases and autophosphorylation at the second site. We mapped the major Btk autophosphorylation site to Y223 within the SH3 domain. Mutation of Y223 to F blocks Btk autophosphorylation and dramatically potentiates the transforming activity of Btk* in fibroblasts. The location of Y223 in a potential ligand-binding pocket suggests that autophosphorylation regulates SH3-mediated signaling by Btk.

Mutation of Bruton's tyrosine kinase (Btk) impairs B cell maturation and function and results in a clinical phenotype of X-linked agammaglobulinemia. Activation of Btk correlates with an increase in the phosphorylation of two regulatory Btk tyrosine residues. Y551 (site 1) within the Src homology type 1 (SH1) domain is transphosphorylated by the Src family tyrosine kinases. Y223 (site 2) is an autophosphorylation site within the Btk SH3 domain. Polyclonal, phosphopeptide-specific antibodies were developed to evaluate the phosphorylation of Btk sites 1 and 2. Crosslinking of the B cell antigen receptor (BCR) or the mast cell Fc receptor, or interleukin 5 receptor stimulation each induced rapid phosphorylation at Btk sites 1 and 2 in a tightly coupled manner. Btk molecules were singly and doubly tyrosinephosphorylated. Phosphorylated Btk comprised only a small fraction (<5%) of the total pool of Btk molecules in the BCR-activated B cells. Increased dosage of Lyn in B cells augmented BCR-induced phosphorylation at both sites. Kinetic analysis supports a sequential activation mechanism in which individual Btk molecules undergo serial transphosphorylation (site 1) then autophosphorylation (site 2), followed by successive dephosphorylation of site 1 then site 2. The phosphorylation of conserved tyrosine residues within structurally related Tec family kinases is likely to regulate their activation.Mutation of the Bruton's tyrosine kinase (Btk) gene produces X-linked (or Bruton's) agammaglobulinemia in humans and X-linked immunodeficiency in mice (1-4). At the cellular level, Btk mutation is manifested by abnormal B cell responses to multiple critical factors, such as interleukin 5 (IL-5) (5-7), IL-6 (8), IL-10 (9), anti-CD38 (10, 11), and the B cell antigen receptor (BCR) (12-17). A mechanism for activation of Btk has been derived from study of endogenous receptor signaling pathways as well as through heterologous expression of Btk in fibroblasts. Src family tyrosine kinases are rapidly activated after stimulation of the BCR (18, 19), then they phosphorylate Btk at Y551 (site 1) (17, 20), a consensus Src family phosphorylation site in the Src homology type 1 (SH1) domain. This phosphorylation event dramatically increases Btk protein tyrosine kinase activity and is required for promotion of fibroblast growth in soft agar by the activated Btk allele, Btk* (17, 20-22). A second major phosphorylated tyrosine residue (Y223) is located within the Btk SH3 domain (23). Phosphorylation of Y223 (site 2) occurs by a Btk kinase-dependent mechanism, i.e., autophosphorylation (17). In contrast to site 1, site 2 phosphorylation has little discernible influence on Btk catalytic activity in vitro or in vivo. The role of the SH3 domain, however, in protein-protein interactions is well established, and site 2 corresponds to a conserved residue important for binding to proline-rich peptide sequences (24-30). Y223 phosphorylation may be a mechanism to modify such interactions.A critical, but unresolved, feature of Btk activation is...

BackgroundIn Huntington's disease, expansion of a CAG triplet repeat occurs in exon 1 of the huntingtin gene (HTT), resulting in a protein bearing>35 polyglutamine residues whose N-terminal fragments display a high propensity to misfold and aggregate. Recent data demonstrate that polyglutamine expansion results in conformational changes in the huntingtin protein (HTT), which likely influence its biological and biophysical properties. Developing assays to characterize and measure these conformational changes in isolated proteins and biological samples would advance the testing of novel therapeutic approaches aimed at correcting mutant HTT misfolding. Time-resolved Förster energy transfer (TR-FRET)-based assays represent high-throughput, homogeneous, sensitive immunoassays widely employed for the quantification of proteins of interest. TR-FRET is extremely sensitive to small distances and can therefore provide conformational information based on detection of exposure and relative position of epitopes present on the target protein as recognized by selective antibodies. We have previously reported TR-FRET assays to quantify HTT proteins based on the use of antibodies specific for different amino-terminal HTT epitopes. Here, we investigate the possibility of interrogating HTT protein conformation using these assays.Methodology/Principal FindingsBy performing TR-FRET measurements on the same samples (purified recombinant proteins or lysates from cells expressing HTT fragments or full length protein) at different temperatures, we have discovered a temperature-dependent, reversible, polyglutamine-dependent conformational change of wild type and expanded mutant HTT proteins. Circular dichroism spectroscopy confirms the temperature and polyglutamine-dependent change in HTT structure, revealing an effect of polyglutamine length and of temperature on the alpha-helical content of the protein.Conclusions/SignificanceThe temperature- and polyglutamine-dependent effects observed with TR-FRET on HTT proteins represent a simple, scalable, quantitative and sensitive assay to identify genetic and pharmacological modulators of mutant HTT conformation, and potentially to assess the relevance of conformational changes during onset and progression of Huntington's disease.

Lupus erythematosus panniculitis (LEP) is an inflammatory disorder of the subcutaneous fat in patients with lupus erythematosus (LE). It is a rare variant of the disease, which occurs approximately in 1%-3% of patients with cutaneous LE. The purpose of this study was to investigate the clinical, histopathologic, immunophenotypical, and molecular profiles of LEP. We performed a retrospective study of 19 biopsy specimens from 17 patients with LEP. We reviewed their clinical data and reexamined the histopathology. Immunophenotyping and molecular studies were done using sections from paraffin-embedded formalin-fixed tissue. The most common clinical manifestation was a depressed patch on upper arm. Patients showed good response to variable treatment modalities, but, generally, relapse of panniculitis was noted when treatment was discontinued. Histopathologically, most specimens revealed lymphoplasmacytic lobular panniculitis with epidermal and dermal changes of LE, hyaline fat necrosis, and lymphoid follicles. Immunohistochemistry showed a mixture of T and B cells in dermis and subcutis with a slight preponderance of T cell. Although the polymerase chain reaction analysis of the T-cell receptor-gamma gene rearrangement showed a polyclonal smear in 89.5% of cases, a small portion of specimens demonstrated monoclonality. LEP is a chronic recurrent disease with characteristic features. Its diagnosis is often challenging, and a precise diagnosis is achievable only upon elaborate clinicopathologic correlation and integrated interpretation of all diagnostic criteria.

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