engleski

Phosphorylation of immunoreceptor tyrosine based activation motifs (ITAMs) by tyrosine kinases, members of the Src and Syk families in vitro

The immunoreceptor tyrosine-based activation motifs (ITAM) play a central role in transmembrane signal transduction in hematopoetic cells. T cell receptor (TCR) is a multi-subunit receptor consisting of Tiαβ heterodimer (responsible for antigen recognition) and CD3 subunits (responsible for signal transduction) built of CD3ζ2, CD3γε and CD3δε dimmers. The T-cell receptor complex contains a total of ten ITAMs within CD3 γ, δ, ε and ζ chain. Kinases of Src (lck, fyn) and Syk (ZAP-70) families are involved in initiation of TCR mediated signalling. It is known that lck is not directly associated with ITAMs but has an important role in their phosphorylation and initiation of TCR signalling. On the other hand, fyn kinase is directly associated with TCR and in the absence of lckmay take over only few of its functions. Phosphorylation of ITAMs is essential also for the activation of ZAP-70 tyrosine kinase. Binding to phosphorylated ITAMs and activation of ZAP-70 by its phosphorylation is one of the first biochemical events and an important step in the activation of T-cells. ZAP-70 binds with its two SH2 domains to the phosphorylated tyrosine residues of ITAMs, primarly of the CD3 ζ subunit. After ZAP-70 is anchored to the plasma membrane it can be phosphorylated by lck and perhaps fyn. To study the phosphorylation of ITAMs, they were cloned from Jurkat T-cell line and subsequently expressed and purified as GST-ITAM fusion proteins. Seven fusion proteins containing ITAM sequences of CD3 γ, δ, ε and ζ subunits are incubated with purified lck, fyn, lyn or ZAP-70 kinases. Ability of these kinases to phosphorylate different ITAM is determined by in vitro kinase assay using γ-33-P-ATP. Differences in ability of tested kinases to phosphorylate ITAMs will be discussed.

TCR; CD3; ITAM; lck; fyn; lyn; ZAP-70 kinases

nije evidentirano