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Christine A Cartwright - One of the best experts on this subject based on the ideXlab platform.
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RACK1: a novel substrate for the Src protein-Tyrosine kinase
Oncogene, 2002Co-Authors: Betty Y Chang, Robert A. Harte, Christine A CartwrightAbstract:RACK1 is one of a group of PKC-interacting proteins collectively called RACKs ( R eceptors for A ctivated C - K inases). Previously, we showed that RACK1 also interacts with the Src Tyrosine kinase, and is an inhibitor of Src activity and cell growth. PKC activation induces the intracellular movement and co-localization of RACK1 and Src, and the Tyrosine phosphorylation of RACK1. To determine whether RACK1 is a Src substrate, we assessed phosphorylation of RACK1 by various Tyrosine kinases in vitro , and by kinase-active and inactive mutants of Src in vivo . We found that RACK1 is a Src substrate. Moreover, Src activity is necessary for both the Tyrosine phosphorylation of RACK1 and the binding of RACK1 to Src's SH2 domain that occur following PKC activation. To identify the Tyrosine(s) on RACK1 that is phosphorylated by Src, we generated and tested a series of RACK1 mutants. We found that Src phosphorylates RACK1 on Tyr 228 and/or Tyr 246, highly-conserved Tyrosines located in the sixth WD repeat that interact with Src's SH2 domain. We think that RACK1 is an important Src substrate that signals downstream of growth factor receptor Tyrosine kinases and is involved in the regulation of Src function and cell growth.
Reuben P Siraganian - One of the best experts on this subject based on the ideXlab platform.
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once phosphorylated Tyrosines in carboxyl terminus of protein Tyrosine kinase syk interact with signaling proteins including tula 2 a negative regulator of mast cell degranulation
Journal of Biological Chemistry, 2012Co-Authors: Rodrigo Orlandini De Castro, Juan Zhang, Jacqueline R Groves, Emilia Alina Barbu, Reuben P SiraganianAbstract:Abstract Activation of the high affinity IgE-binding receptor (FcϵRI) results in the Tyrosine phosphorylation of two conserved Tyrosines located close to the COOH terminus of the protein-Tyrosine kinase Syk. Synthetic peptides representing the last 10 amino acids of the tail of Syk with these two Tyrosines either nonphosphorylated or phosphorylated were used to precipitate proteins from mast cell lysates. Proteins specifically precipitated by the phosphorylated peptide were identified by mass spectrometry. These included the adaptor proteins SLP-76, Nck-1, Grb2, and Grb2-related adaptor downstream of Shc (GADS) and the protein phosphatases SHIP-1 and TULA-2 (also known as UBASH3B or STS-1). The presence of these in the precipitates was further confirmed by immunoblotting. Using the peptides as probes in far Western blots showed direct binding of the phosphorylated peptide to Nck-1 and SHIP-1. Immunoprecipitations suggested that there were complexes of these proteins associated with Syk especially after receptor activation; in these complexes are Nck, SHIP-1, SLP-76, Grb2, and TULA-2 (UBASH3B or STS-1). The decreased expression of TULA-2 by treatment of mast cells with siRNA increased the FcϵRI-induced Tyrosine phosphorylation of the activation loop Tyrosines of Syk and the phosphorylation of phospholipase C-γ2. There was parallel enhancement of the receptor-induced degranulation and activation of nuclear factor for T cells or nuclear factor κB, indicating that TULA-2, like SHIP-1, functions as a negative regulator of FcϵRI signaling in mast cells. Therefore, once phosphorylated, the terminal Tyrosines of Syk bind complexes of proteins that are positive and negative regulators of signaling in mast cells.
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Conformational changes induced in the protein Tyrosine kinase p72syk by Tyrosine phosphorylation or by binding of phosphorylated immunoreceptor Tyrosine-based activation motif peptides.
Molecular and cellular biology, 1996Co-Authors: Teruaki Kimura, Hiroshi Sakamoto, Ettore Appella, Reuben P SiraganianAbstract:A critical event in signaling in immune cells is the interaction of Syk or ZAP-70 protein Tyrosine kinases with multisubunit receptors that contain an approximately 18-amino-acid domain called the immunoreceptor Tyrosine-based activation motif (ITAM). Tyrosine-phosphorylated Syk from activated cells was in a conformation different from that in nonstimulated cells as demonstrated by changes in immunoreactivity. The addition of Tyrosine-diphosphorylated ITAM peptides resulted in a similar conformational change in Syk from nonactivated cells. The peptides based on FcepsilonRIgamma were more active than those based on Fcepsilon RIbeta. In vitro autophosphorylation of Syk was dramatically enhanced by the addition of the diphosphorylated ITAM peptides. The conformational change and the enhanced autophosphorylation required the presence of both phosphorylated Tyrosines on the same molecule. These conformational changes in Syk by Tyrosine phosphorylation or binding to diphosphorylated ITAM could be critical for Syk activation and downstream propagation of intracellular signals.
Betty Y Chang - One of the best experts on this subject based on the ideXlab platform.
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RACK1: a novel substrate for the Src protein-Tyrosine kinase
Oncogene, 2002Co-Authors: Betty Y Chang, Robert A. Harte, Christine A CartwrightAbstract:RACK1 is one of a group of PKC-interacting proteins collectively called RACKs ( R eceptors for A ctivated C - K inases). Previously, we showed that RACK1 also interacts with the Src Tyrosine kinase, and is an inhibitor of Src activity and cell growth. PKC activation induces the intracellular movement and co-localization of RACK1 and Src, and the Tyrosine phosphorylation of RACK1. To determine whether RACK1 is a Src substrate, we assessed phosphorylation of RACK1 by various Tyrosine kinases in vitro , and by kinase-active and inactive mutants of Src in vivo . We found that RACK1 is a Src substrate. Moreover, Src activity is necessary for both the Tyrosine phosphorylation of RACK1 and the binding of RACK1 to Src's SH2 domain that occur following PKC activation. To identify the Tyrosine(s) on RACK1 that is phosphorylated by Src, we generated and tested a series of RACK1 mutants. We found that Src phosphorylates RACK1 on Tyr 228 and/or Tyr 246, highly-conserved Tyrosines located in the sixth WD repeat that interact with Src's SH2 domain. We think that RACK1 is an important Src substrate that signals downstream of growth factor receptor Tyrosine kinases and is involved in the regulation of Src function and cell growth.
Michael Reth - One of the best experts on this subject based on the ideXlab platform.
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synthetic biology of b cell activation understanding signal amplification at the b cell antigen receptor using a rebuilding approach
Biological Chemistry, 2019Co-Authors: Yogesh Kulathu, Christa Zuern, Jianying Yang, Michael RethAbstract:Upon activation of the B cell antigen receptor (BCR), the spleen Tyrosine kinase (Syk) and the Src family kinase Lyn phosphorylate Tyrosines of the immunoreceptor Tyrosine-based activation motif (ITAM) of Igα and Igβ which further serve as binding sites for the SH2 domains of these kinases. Using a synthetic biology approach, we dissect the roles of different ITAM residues of Igα in Syk activation. We found that a leucine to glycine mutation at the Y+3 position after the first ITAM Tyrosine prevents Syk binding and activation. However, a pre-activated Syk can still phosphorylate this Tyrosine in trans. Our data show that the formation of a Syk/ITAM initiation complex and trans-ITAM phosphorylation is crucial for BCR signal amplification. In contrast, the interaction of Lyn with the first ITAM Tyrosine is not altered by the leucine to glycine mutation. In addition, our study suggests that an ITAM-bound Syk phosphorylates the non-ITAM Tyrosine Y204 of Igα only in cis. Collectively, our reconstitution experiments suggest a model whereby first trans-phosphorylation amplifies the BCR signal and subsequently cis-phosphorylation couples the receptor to downstream signaling elements.
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Syk is a dual-specificity kinase that self-regulates the signal output from the B-cell antigen receptor
Proceedings of the National Academy of Sciences of the United States of America, 2010Co-Authors: Beate Heizmann, Michael Reth, Simona InfantinoAbstract:Upon B-cell activation, the signaling subunits Ig-α and Ig-β of the B-cell antigen receptor become phosphorylated not only on Tyrosines but also on serine residues. Using a specific antibody, we show that serine 197 (S197) in the cytoplasmic tail of Ig-α is phosphorylated upon B-cell antigen receptor activation, and that this modification inhibits the signal output of the B-cell antigen receptor. Surprisingly, we found that the well-known protein Tyrosine kinase Syk (spleen Tyrosine kinase) phosphorylates S197 on Ig-α, thus not only activating but also inhibiting signaling from the B-cell antigen receptor. This finding identifies Syk as a dual-specificity kinase and establishes a previously unexplored paradigm for the self-regulation of biological signaling processes.
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analysis of novel phospho itam specific antibodies in a s2 reconstitution system for tcr cd3 signalling
Immunology Letters, 2010Co-Authors: Elaine P Dopfer, Michael Reth, Barbara Schopf, Christine Louisditsully, Eva Dengler, Kerstin Hohne, Andrea Klescova, Marek Prouza, Miloslav Suchanek, Wolfgang W A SchamelAbstract:Abstract The T cell antigen receptor (TCR–CD3) complex contains 12 different cytoplasmic Tyrosines, each of which is part of an immunoreceptor Tyrosine-based activation motif and thus occurs in similar sequence context. Since phosphorylation of individual Tyrosines can be correlated with the quality of the T cell response, monitoring their phosphorylation is important. We thus generated novel antibodies against phospho-Tyrosines of the TCR–CD3 complex and tested the specificity in a synthetic biology approach. We utilized the Drosophila S2 reconstitution system testing several kinases and stimulation conditions that lead to optimal phosphorylation of the TCR–CD3 subunit ζ. Expressing TCR–CD3 subunits and Tyrosine mutants thereof we tested the specificity of the novel antibodies in Western blot and immunopurification experiments. In particular, we generated and characterized the monoclonal antibody EM-26 that specifically recognizes phosphorylation of the membrane proximal Tyrosine of ζ (phospho-ζY1) and antisera raised against the first and the second phospho-Tyrosine of CD3ɛ (phospho-ɛY1 and phospho-ɛY2).
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amplification of b cell antigen receptor signaling by a syk itam positive feedback loop
Molecular Cell, 2002Co-Authors: Veronique Rolli, Maike Gallwitz, Thomas Wossning, Alexandra Flemming, Wolfgang W A Schamel, Christa Zurn, Michael RethAbstract:We have established a protocol allowing transient and inducible coexpression of many foreign genes in Drosophila S2 Schneider cells. With this powerful approach of reverse genetics, we studied the interaction of the protein Tyrosine kinases Syk and Lyn with the B cell antigen receptor (BCR). We find that Lyn phosphorylates only the first Tyrosine whereas Syk phosphorylates both Tyrosines of the BCR immunoreceptor Tyrosine-based activation motif (ITAM). Furthermore, we show that Syk is a positive allosteric enzyme, which is strongly activated by the binding to the phosphorylated ITAM Tyrosines, thus initiating a positive feedback loop at the receptor. The BCR-dependent Syk activation and signal amplification is efficiently counterbalanced by protein Tyrosine phosphatases, the activity of which is regulated by H(2)O(2) and the redox equilibrium inside the cell.
Robert A. Harte - One of the best experts on this subject based on the ideXlab platform.
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RACK1: a novel substrate for the Src protein-Tyrosine kinase
Oncogene, 2002Co-Authors: Betty Y Chang, Robert A. Harte, Christine A CartwrightAbstract:RACK1 is one of a group of PKC-interacting proteins collectively called RACKs ( R eceptors for A ctivated C - K inases). Previously, we showed that RACK1 also interacts with the Src Tyrosine kinase, and is an inhibitor of Src activity and cell growth. PKC activation induces the intracellular movement and co-localization of RACK1 and Src, and the Tyrosine phosphorylation of RACK1. To determine whether RACK1 is a Src substrate, we assessed phosphorylation of RACK1 by various Tyrosine kinases in vitro , and by kinase-active and inactive mutants of Src in vivo . We found that RACK1 is a Src substrate. Moreover, Src activity is necessary for both the Tyrosine phosphorylation of RACK1 and the binding of RACK1 to Src's SH2 domain that occur following PKC activation. To identify the Tyrosine(s) on RACK1 that is phosphorylated by Src, we generated and tested a series of RACK1 mutants. We found that Src phosphorylates RACK1 on Tyr 228 and/or Tyr 246, highly-conserved Tyrosines located in the sixth WD repeat that interact with Src's SH2 domain. We think that RACK1 is an important Src substrate that signals downstream of growth factor receptor Tyrosine kinases and is involved in the regulation of Src function and cell growth.
