The tyrosine kinase Syk (relative molecular mass 72,000), which is widely expressed in haematopoietic cells, becomes associated with and activated by engagement of the B-cell antigen receptor. Furthermore, it has been implicated in signalling through the receptors for interleukin-2 (IL-2), granulocyte colony-stimulating factor (G-CSF) and Fc, the T cell receptor, as well as through receptors for several platelet agonists. A homologous kinase, ZAP-70, is crucial in signalling through the T-cell receptor and in T-cell development. Using homologous recombination in embryonic stem cells, we created mice null for the syk gene which showed petechiae in utero and died shortly after birth. Irradiated mice reconstituted with Syk-deficient fetal liver showed a block in B-cell development at the pro-B to pre-B cell transition, consistent with a key role for Syk in pre-B-cell receptor signalling. Despite the production of small numbers of immature B cells, Syk-deficient radiation chimaeras failed to accumulate mature B cells, indicating a possible role for this protein in the production or maintenance of mature B cells. In addition, whereas the development of alpha beta T cells proceeded normally, Syk-deficient mice showed impaired development of thymocytes using the V gamma 3 variable region gene (V gamma 3+ thymocytes). Finally, we show that Syk is not required for signalling through the IL-2 and G-CSF receptors.

The B cell receptor (BCR) transduces antigen binding into alterations in the activity of intracellular signaling pathways through its ability to recruit and activate the cytoplasmic protein-tyrosine kinase Syk. The recruitment of Syk to the receptor, its activation and its subsequent interactions with downstream effectors are all regulated by its phosphorylation on tyrosine. This review discusses our current understanding of how this phosphorylation regulates the activity of Syk and its participation in signaling through the BCR.

Force-displacement (F-ZIn recent years interest has increased in the measurement of the viscoelastic properties of soft biological samples motivated by their correlation with disease, differentiation, or cellular transformation [1][2][3][4] . A large variety of methods have been introduced for measuring cellular mechanical properties including micropipette aspiration 5,6 , stretching or compression between two microplates [7][8][9] , optical tweezers 10,11 , and magnetic twisting cytometry 12,13 . However, indentation with the atomic force microscope (AFM) remains one of the most popular methods for probing the nanoscale properties of soft samples like cells, tissues and hydrogels [14][15][16][17] . In the AFM, the elastic properties of live cells are usually evaluated from force versus displacement (F-Z) curves. Then the Hertz model or its modifications are applied to the approach part of the F-Z curve to extract Young's modulus (E Hertz ), the elastic parameter used for characterization of the sample's mechanical properties 18,19 . However, such models assume a purely elastic nature of the sample, while in reality most biological samples are viscoelastic. Viscoelasticity is revealed in a clear hysteresis between the approach and retraction parts of curves 20 ; the indentation speed dependence of E Hertz values extracted from force curves with the Hertz model 11 ; the observations of force relaxation at constant indentation depth and the creep at constant loading force 21 . Approaches other than the standard F-Z curves are usually used to obtain the viscoelastic properties of samples with AFM in both the time [22][23][24] and frequency domains [25][26][27][28][29][30] . These generally require modifications in the AFM apparatus and/or in the data acquisition protocol. Equally importantly, each approach has its own sets of measurement uncertainties.If a standard F-Z curve could also be used to quantify viscoelastic properties, it would allow one standard method with well quantified uncertainties 31 to be used for both viscoelasticity and elasticity measurements. This has not been possible to date, we believe, due to the lack of a mathematical/computational framework that allows the post-processing of force-displacement data to extract the relevant viscoelastic constitutive parameters.

Syk is a cytoplasmic protein-tyrosine kinase well known for its ability to couple immune cell receptors to intracellular signaling pathways that regulate cellular responses to extracellular antigens and antigen-immunoglobulin complexes of particular importance to the initiation of inflammatory responses. Thus, Syk is an attractive target for therapeutic kinase inhibitors designed to ameliorate symptoms and consequences of acute and chronic inflammation. Its more recently recognized role as a promoter of cell survival in numerous cancer cell types ranging from leukemia to retinoblastoma has attracted considerable interest as a target for a new generation of anticancer drugs. This review discusses the biological processes in which Syk participates that have made this kinase such a compelling drug target. Keywordsprotein kinase inhibitor; tyrosine-phosphorylation; fostamatinib; allergic asthma; rheumatoid arthritis; leukemia Spleen tyrosine kinaseThe central role played by Syk (spleen tyrosine kinase) in the immune system in mediating inflammatory responses, coupled with its more recently identified association with malignancy, has made this kinase a popular target for the development of therapeutic agents. A search of the patent literature reveals over 70 filings describing the development of small molecular inhibitors of Syk for the treatment of multiple disease states ranging from arthritis and asthma to leukemia and lymphoma.Syk catalyzes the phosphorylation of proteins on tyrosines located at sites rich with acidic amino acids [1]. Interestingly, at least two substrates, CD79a [2] and the Ikaros transcription factor [3], have been reported to be phosphorylated by Syk on serine, suggesting that Syk may access a wider distribution of substrates than originally thought. The Syk protein comprises a pair of Src homology 2 (SH2) domains at the N-terminus that are joined to each © 2014 Elsevier Ltd. All rights reserved.Corresponding author: Geahlen, R.L. (geahlen@purdue.edu). Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Figure 1). Aromatic amino acids in linker A, linker B, the catalytic domain and the extreme C-terminus participate in the formation of a "linker-kinase sandwich" (as first identified in the Syk-family member, ) that maintains the enzyme in an autoinhibited conformation [7]. Activation of Syk occurs when the tandem SH2 domains are engaged or when tyrosines participating in the linker-kinase sandwich become phosphorylated. NIH Public AccessSH2 domains are structural motifs that bind phosphotyrosine to promote protein-protein interactions [8]. Each of ...

The ability to obtain in-depth understanding of signaling networks in cells is a key objective of systems biology research. Such ability depends largely on unbiased and reproducible analysis of phosphoproteomes. We present here a novel proteomics tool, polymer-based metal ion affinity capture (PolyMAC), for the highly efficient isolation of phosphopeptides to facilitate comprehensive phosphoproteome analyses. This approach uses polyamidoamine dendrimers multifunctionalized with titanium ions and aldehyde groups to allow the chelation and subsequent isolation of phosphopeptides in a homogeneous environment. Compared with current strategies based on solid phase micro-and nanoparticles, PolyMAC demonstrated outstanding reproducibility, exceptional selectivity, fast chelation times, and high phosphopeptide recovery from complex mixtures. Using the PolyMAC method combined with antibody enrichment, we identified 794 unique sites of tyrosine phosphorylation in malignant breast cancer cells, 514 of which are dependent on the expression of Syk, a protein-tyrosine kinase with unusual properties of a tumor suppressor. The superior sensitivity of PolyMAC allowed us to identify novel components in a variety of major signaling networks, including cell migration and apoptosis. PolyMAC offers a powerful and widely applicable tool for phosphoproteomics and molecular signaling. Molecular & Cellular Proteomics 9:2162-2172, 2010.Reversible phosphorylation of proteins is a major mechanism for the regulation of multiple cellular processes (1, 2). Mass spectrometry-based phosphoproteomics provides a method for the global analysis of protein phosphorylation and molecular signaling in cells (3,4). Despite the great progress that has been made over the past few years, the isolation of phosphopeptides and their analysis by mass spectrometry are still a considerable challenge because of the typically low stoichiometry of protein phosphorylation and the resulting low abundance of phosphopeptides. An early step in any phosphoproteome analysis is the isolation of phosphopeptides, preferably with high efficiency, selectivity, sensitivity, and reproducibility. Currently, there are three major strategies for the isolation of phosphopeptides: antibody-based affinity capture, chemical derivatization of phosphoamino acids, and metal ion-based affinity capture. Antibody-based methods are used mainly for the selective isolation of phosphotyrosinecontaining proteins or peptides (5-8). Chemical derivatization methods begin with the ␤-elimination of phosphates from phosphoserine and phosphothreonine (9) or the formation of phosphoramidates by reactions with amines (10) to selectively immobilize phosphopeptides. Metal ion-based affinity capture techniques use immobilized metal affinity chromatography (IMAC) with Fe(III) (11,12) or Ga(III) (13) and, for the past a few years, more successful metal oxide approaches (i.e. TiO 2 (14, 15) and ZrO 2 (16, 17)) for the selective binding of phosphorylated peptides. Almost all of the current isolation methods are b...