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the structure and function of ion channel and transporter proteins; performs basic molecular biology techniques … other over expression systems, antibody purification and membrane protein crystallization; assists in the preparation… Read more here: the structure and function of ion channel and transporter proteins; performs basic molecular biology techniques … other over expression systems, antibody purification and membrane protein crystallization; assists in the preparation… Read more here:
Methods Mol Biol. 2012; 876: 1-15 Ligand binding by the extracellular domain of receptor kinases leads to phosphorylation and activation of the cytoplasmic domain of these important membrane-bound signaling proteins. To thoroughly characterize receptor kinase function, it is essential to identify specific phosphorylation sites by mass spectrometry. In this chapter, we summarize an efficient protein purification and modification protocol to prepare receptor kinases for liquid chromatography/tandem mass spectrometry analysis. Both recombinant receptor kinase cytoplasmic domains expressed in bacteria and full-length receptor kinase proteins expressed in living plant tissue are considered, and multiple methods of mass spectrometry are described that allow optimal identification of phosphorylated peptides of both in vitro- and in vivo-derived samples. See the original post here:
J Biol Chem. 2012 Apr 23; T-cell receptor (TCR)-induced T-cell activation is a critical event in adaptive immune responses. The engagement of TCR complex by antigen along with the activation of the costimulatory receptors trigger a cascade of intracellular signaling, in which caspase recruitment domain-containing membrane-associated guanylate kinase 1 (CARMA1) is a crucial scaffold protein. Upon stimulation, CARMA1 recruits downstream molecules including B-cell CLL/lymphoma 10 (Bcl10), mucosa associated lymphoid tissue lymphoma translocation gene 1 (MALT1), Bcl10 and TRAF6 to assemble a specific TCR-induced signalosome that triggers NF-κB and JNK activation. In this report, we identified protein kinase C delta (PKCδ) as a CARMA1-associated protein by a biochemical affinity purification approach. PKCδ interacted with CARMA1 in TCR stimulation-dependent manner in Jurkat T cells. Overexpression of PKCδ inhibited CARMA1-mediated NF-κB activation, whereas knockdown of PKCδ potentiated TCR-triggered NF-κB activation and IL-2 secretion in Jurkat T cells. Reconstitution experiments with PKCδ kinase-dead mutant indicated that the kinase activity of PKCδ was dispensable for its ability to inhibit TCR-triggered NF-κB activation. Furthermore, we found that PKCδ inhibited the interaction between MALT1 and TRAF6, but not the association of CARMA1 with PKCδ, Bcl10, or MALT1. These observations suggest that PKCδ is a negative regulator in T cell activation through inhibiting the assembly of CARMA1 signalosome. Read the rest here: |
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