Many immune signaling pathways require activation of the Syk tyrosine kinase to link ligation of surface receptors to changes in gene expression. Despite the central role of Syk in these pathways, the Syk activation process remains poorly understood. In this work we quantitatively characterized the molecular mechanism of Syk activation in vitro using a real time fluorescence kinase assay, mutagenesis, and other biochemical techniques. We found that dephosphorylated full-length Syk demonstrates a low initial rate of substrate phosphorylation that increases during the kinase reaction due to autophosphorylation. The initial rate of Syk activity was strongly increased by either pre-autophosphorylation or binding of phosphorylated immune tyrosine activation motif peptides, and each of these factors independently fully activated Syk. Deletion mutagenesis was used to identify regions of Syk important for regulation, and residues 340 -356 of the SH2 kinase linker region were identified to be important for suppression of activity before activation. Comparison of the activation processes of Syk and Zap-70 revealed that Syk is more readily activated by autophosphorylation than Zap-70, although both kinases are rapidly activated by Src family kinases. We also studied Syk activity in B cell lysates and found endogenous Syk is also activated by phosphorylation and immune tyrosine activation motif binding. Together these experiments show that Syk functions as an "OR-gate" type of molecular switch. This mechanism of switch-like activation helps explain how Syk is both rapidly activated after receptor binding but also sustains activity over time to facilitate longer term changes in gene expression.Syk is a tyrosine kinase that functions immediately downstream of antigen receptors in immune cells including B lymphocytes, mast cells, and macrophages (1-3). The central role of Syk in cell types associated with disorders such as rheumatoid arthritis and allergic rhinitis suggests that strategies to block Syk activation may have therapeutic benefit (4 -6). After receptor ligation and phosphorylation, Syk becomes localized to immune receptors and proceeds to phosphorylate downstream targets leading to Ca 2ϩ mobilization, initiation of the extracellular signal-regulated kinase and p38 mitogen-activated protein kinase cascades, and activation of transcription factors such as NF-B (7). Although increased Syk activity is known to be indispensable for each of these cellular events, a molecular-level understanding of the steps leading to Syk activation has not been clearly defined.The Syk domain structure consists of an N-terminal pair of Src homology 2 (SH2) 2 domains separated by an inter-SH2 linker, an SH2-domain-kinase linker, and a C-terminal kinase domain. Recruitment of Syk to immune receptors involves binding of the tandem SH2 domains of Syk to motifs in the receptor known as immune tyrosine activation motifs (ITAMs), which are two YXXL sequences typically separated by 7-12 intervening residues (8, 9). Syk is known to have multiple ...

Bruton's tyrosine kinase (BTK) is a member of the Tec nonreceptor tyrosine kinase family that is involved in regulating B cell proliferation. To better understand the enzymatic mechanism of the Tec family of kinases, the kinetics of BTK substrate phosphorylation were characterized using a radioactive enzyme assay. We first examined whether autophosphorylation regulates BTK activity. Western blotting with a phosphospecific antibody revealed that BTK rapidly autophosphorylates at Tyr 551 within its activation loop in vitro. Examination of a Y551F BTK mutant indicated that phosphorylation of Tyr 551 causes a 10-fold increase in BTK activity. We then proceeded to characterize the steady state kinetic mechanism of BTK. Varying the concentrations of ATP and S1 peptide (biotin-Aca-AAAEEIY-GEI-NH 2 ) revealed that BTK employs a ternary complex mechanism with K mATP ‫؍‬ 84 ؎ 20 M and K mS1 ‫؍‬ 37 ؎ 8 M. Inhibition studies were also performed to examine the order of substrate binding. The inhibitors ADP and staurosporine were both found to be competitive with ATP and non-competitive with S1, indicating binding of ATP and S1 to BTK is either random or ordered with ATP binding first. Negative cooperativity was also found between the S1 and ATP binding sites. Unlike ATP site inhibitors, substrate analog inhibitors did not inhibit BTK at concentrations less than 1 mM, suggesting that BTK may employ a "substrate clamping" type of kinetic mechanism whereby the substrate K d is weaker than K m . This investigation of BTK provides the first detailed kinetic characterization of a Tec family kinase. Bruton's tyrosine kinase (BTK)2 is a non-receptor tyrosine kinase that plays an essential role in B cells and other hematopoietic cell types (1-4). The importance of BTK is evident from the fact that mutations in BTK cause X-linked agammaglobulinemia, a human disease that causes defects in both B cell maturation and mature B cell function (5, 6). A BTK point mutation in mice results in a similar, albeit milder, B cell deficiency known as X-linked immunodeficiency (7). The indispensable requirement of BTK for B cell function in humans makes BTK a promising target for treatment of inflammatory diseases that involve inappropriate B cell activation.The BTK-mediated signaling events that link B cell receptor ligation to downstream signaling have been well characterized (8, 9). Upon ligation of the B cell receptor, Src family kinases associated with the receptor, such as Lyn, are the first enzymes activated. This activation results in phosphorylation of tyrosine residues that become docking sites for other kinases including phosphatidylinositol 3-kinase. Once recruited, phosphatidylinositol 3-kinase phosphorylates phosphatidylinositol 4,5-bisphosphate to form phosphatidylinositol 3,4,5-bisphosphate. Formation of phosphatidylinositol 3,4,5-bisphosphate then recruits BTK to the plasma membrane via interaction with its pleckstrin homology domain. Upon recruitment to the membrane, BTK is activated via phosphorylation. BTK proceeds to phosphorylat...

A small molecule inhibitor of NF-B-dependent cytokine expression was discovered that blocked tumor necrosis factor (TNF) ␣-induced IB␣ degradation in MM6 cells but not the degradation of ␤-catenin in Jurkat cells. Ro106-9920 blocked lipopolysaccharide (LPS)-dependent expression of TNF␣, interleukin-1␤, and interleukin-6 in fresh human peripheral blood mononuclear cells with IC 50 values below 1 M. Ro106-9920 also blocked TNF␣ production in a dose-dependent manner following oral administration in two acute models of inflammation (air pouch and LPS challenge). Ro106-9920 was observed to inhibit an ubiquitination activity that does not require ␤TRCP but associates with IB␣ and will ubiquitinate IB␣ S32E,S36E (IB␣ee) specifically at lysine 21 or 22. Ro106-9920 was identified in a cell-free system as a time-dependent inhibitor of IB␣ee ubiquitination with an IC 50 value of 2.3 ؎ 0.09 M. The ubiquitin E3 ligase activity is inhibited by cysteine-alkylating reagents, supported by E2UBCH7, and requires cIAP2 or a cIAP2-associated protein for activity. These activities are inconsistent with what has been reported for SCF ␤TRCP , the putative E3 for IB␣ ubiquitination. Ro106-9920 was observed to be selective for IB␣ee ubiquitination over the ubiquitin-activating enzyme (E1), E2UBCH7, nonspecific ubiquitination of cellular proteins, and 97 other molecular targets. We propose that Ro106-9920 selectively inhibits an uncharacterized but essential ubiquitination activity associated with LPSand TNF␣-induced IB␣ degradation and NF-B activation.

To understand the physiological changes that occur in response to spaceflight, mice are transported to the International Space Station (ISS) and housed for variable periods of time before euthanasia on-orbit or return to Earth. Sample collection under such difficult conditions introduces confounding factors that need to be identified and addressed. We found large changes in the transcriptome of mouse tissues dissected and preserved on-orbit compared with tissues from mice euthanized on-orbit, preserved, and dissected after return to Earth. Changes due to preservation method eclipsed those between flight and ground samples, making it difficult to identify spaceflight-specific changes. Follow-on experiments to interrogate the roles of euthanasia methods, tissue and carcass preservation protocols, and library preparation methods suggested that differences due to preservation protocols are exacerbated when coupled with polyA selection. This has important implications for the interpretation of existing datasets and the design of future experiments.

To understand the physiological changes that occur in response to spaceflight, mice are transported to the International Space Station (ISS) and housed for variable periods of time before euthanasia on-orbit or return to Earth. Sample collection under such difficult conditions introduces confounding factors that need to be identified and addressed. We found large changes in the transcriptome of mouse tissues dissected and preserved on-orbit compared to tissues from mice euthanized on-orbit, preserved, and dissected after return to Earth. Changes due to preservation method eclipsed those between flight and ground samples making it difficult to identify spaceflight-specific changes. Follow-on experiments to interrogate the roles of euthanasia methods, tissue and carcass preservation protocols, and library preparation methods suggested that differences due to preservation protocols are exacerbated when coupled with polyA selection. This has important implications for the interpretation of existing datasets and the design of future experiments.