Tyrosine-phosphorylated proteins

Since then, a plethora of tyrosine-phosphorylated proteins has been discovered. Originally, tyrosine phosphorylation was believed to be involved primarily in regulating cell proliferation, since many oncogene products and growth factor receptors are protein tyrosine kinases (PTKs). However, it has become clear that tyrosine phosphorylation is involved in regulating a variety of cellular processes. In fact, the nervous system contains a large variety of PTKs and protein tyrosine phosphatases (PTPs), and some of these are exclusively expressed in neuronal tissues. Figure 24-1 shows the... 

FIGURE 24-1 Phosphotyrosine staining of a hippocampal neuron. A cultured rat hippocampal pyramidal neuron is stained with an antiphosphotyrosine antibody and detected by a secondary antibody conjugated to rhodamine. The staining reflects the presence of tyrosine-phosphorylated proteins throughout the neuron, including the cell body and synaptic regions. 

Moon, I. S., Apperson, M. L. and Kennedy, M. B. The major tyrosine-phosphorylated protein in the postsynaptic density fraction is N-methyl-D-aspartate receptor subunit 2B. Proc. Natl Acad. Sci. U.S.A. 91 3954-3958,1994. 

Saxton, T.M., Oostween, I., BowteU, D., Aebersold R., and Gold, M.R., 1994, B cell antigen receptor crosshnking induces phosphorylation of the ras activators SHC and mSOSl as well as the assembly of complexes containing SHC, Grb2 and SOSl and a 145 kDa tyrosine phosphorylated protein. J. Immunol. 153 623-636. 

Smit, L., de Vries-Smits, A.M.M., Bos, J.L., and Borst, J., 1994, B cell antigen receptor stimulation induces formation ofaShc-Grb2 complex containing multiple tyrosine-phosphorylated proteins. 7. Biol. Chem. 269 20209-20212... 

Donovan, J. A., R. L. Wange, W. Y. Langdon, and L. E. Samelson. The protein product of the c-cbl protooncogene is the 120-kDa tyrosine-phosphorylated protein in Jurkat cells activated via the T cell antigen receptor. J Biol. Chem. 269 22921-22924.1994. 

Wilson, P. D., Geng, L., Li, X., and Burrow, C. R. (1999). The PKD1 gene product, polycystin-1, is a tyrosine-phosphorylated protein that colocalizes with alpha2belal -intecrrin in focal clusters in adherent renal epithelia. Lab. Invest. 79, 1311-1323. 

Nakanishi T, Ando E, Furuta M, Tsunasawa S, Nishimura O (2007) Direct on-membrane peptide mass fingerprinting with MALDI-MS of tyrosine-phosphorylated protein detected by immunostaining. J Chromatogr B 847 24-29. doi 10.1016/j.jchromb.2006.08.024... 

PTK, a family of kinases that regulate phosphotyrosine (PT) levels including the Src family found in the cytoplasm and are associated with the cytoskeleton. Other members of this family include ppl25FAK, FAK,PTK, pl20, a tyrosine-phosphorylated protein that is similar in sequence to cadherin-binding factors, an actin filament associated protein (AFAP-110), and contactin. 

Kavanaugh, W. M. and Williams, L. T. An alternative to SH2 domains for binding tyrosine-phosphorylated proteins. Science, 266,1862-1865, 1994. 

H- Sabe, A. Hata, M. Okada, H. Nakagawa, and H. Hanafusa. Analysis of the binding of the Src homology 2 domain of Csk to tyrosine-phosphorylated proteins in the suppression and mitotic activation of c-Stc. Proc Natl Acad Sci, USA, 91 (9), 3984-3988, 1994. 

Otto, H., Dreger, M., Bengtsson, L. andHucho, F. (2001) Identification of tyrosine-phosphorylated proteins associated with the nuclear envelope. Eur. J. Biochem. 268, 420-428. 

Signaling proteins form complexes with other molecules using conserved motifs or domains, which are modular in the sense that the domain by itself typically is sufficient for its function (7, 8). Protein-protein interaction domains include those responsible for binding to phosphorylated receptors and other tyrosine-phosphorylated proteins (Src homology 2 and phospho-tyrosine binding domains) or to proline-rich protein... 

Post-kinase Signal Transduction Tyrosine-phosphorylated Proteins. 34... 

POST-KINASE SIGNAL TRANSDUCTION TYROSINE-PHOSPHORYLATED PROTEINS... 

For a long time the search for tyrosine-phosphorylated proteins which might serve as a substrate for the insulin receptor kinase was unsuccessful. White... 

PBMC are a mixed cell population, only 40 % of which constitute T cells. While with flow cytometry, effects of inhibitors on T cells (or even subsets of T cells) can be separated and recorded, PBMC cannot be used to verify such effects in immunoblot assays, as tyrosine-phosphorylated proteins from non-T cells may mask the effect of the inhibitor. Therefore, primary human T cells are purified by negative selection from freshly isolated PBMC see Subheading 3.7) using the StemSep Human T Cell Enrichment Kit. Follow the selection protocol step-by-step according to the vendor s instructions. Expect to recover >1x10 T cells from 7X 10 purified PBMC (from 50 mL buffy coat). 

Yin, T., Yasukawa, K., Taga, T., Kishimoto, T. and Yang, Y.C. (1994) Identification of a 130-kilodaIton tyrosine-phosphorylated protein induced by interIeukin-41 as JAK2 tyrosine kinase, which associates with gpl30 signal transducer. Exp. Hematol. 22 467-472. 

Tyrosine kinases have been implicated in Ca +-sensitization largely as the result of the inhibitory effects of tyrosine kinase inhibitors, genistein and vanadate, and the correlation between tyrosine phosphorylation and vanadate- or agonist-induced contractions (Di Salvo et al. 1993a,b, 1994, 1997 Steusloff et al. 1995). However, the tyrosine-phosphorylated proteins have yet to be directly linked to a known contractile regulatory mechanism i.e., MLCK, SMPP-IM) and evaluation of the relationships between tyrosine phosphorylation and modulation of Ca +-sensitization of contraction in intact muscle is somewhat complicated by concurrent changes in cytosolic Ca (Di Salvo et al. 1994,1997), possibly as the result of activation of PLC-y (Marrero et al. 1994) or Gaq.n (Umemori et al. 1997). 

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