Veillette. in T cells. Through cell fractionation studies and analyses of genetically altered mice, we founded that PTPs such as PEP and SHP-1 are unlikely to be involved in the dephosphorylation of PAG in T cells. However, the transmembrane PTP CD45 seems to play an Rabbit Polyclonal to OR2T10 important role in this process. Taken collectively, these data provide firm evidence that PAG is definitely a bona fide bad regulator of T-cell activation as a result of its capacity to recruit Csk. They also suggest that the inhibitory function of PAG in T cells is definitely suppressed by CD45. Lastly, they support the idea that dephosphorylation of proteins on tyrosine residues is critical for the initiation of T-cell activation. T-cell activation is initiated by the connection of the T-cell receptor (TCR) for antigens with antigenic peptides complexed to major histocompatibility complex molecules (37). TCR engagement by antigens causes the tyrosine phosphorylation of a short sequence, the immunoreceptor tyrosine-based activation motif, present in the TCR-associated CD3- subunits (7, 23). Such immunoreceptor tyrosine-based activation motifs function by orchestrating the sequential activation of the Src-related protein tyrosine kinases (PTKs) Lck and FynT, which initiate TCR signaling, followed by that of the Zap-70/Syk PTKs, which amplify NVP-ADW742 the response (7). These numerous PTKs induce tyrosine phosphorylation of several polypeptides, including the transmembrane adaptor LAT, the adaptor SLP-76, and enzymatic effectors such as phospholipase C (PLC)- (9, 24, 27, 28). Protein tyrosine phosphorylation consequently prospects to the activation of several other signaling pathways, which ultimately induce effector functions. PAG (phosphoprotein associated with glycosphingolipid-enriched microdomains), or Cbp (Csk-binding protein), is an 80-kDa transmembrane protein indicated in all cell types, including T cells (2, 20). It possesses a short extracellular website (16 to 18 amino acids), a single transmembrane section, and a long intracytoplasmic website bearing multiple tyrosine-based motifs (9 in mouse PAG and 10 in human being PAG). Interestingly, PAG/Cbp (hereafter termed PAG) is concentrated in membrane microdomains referred to as lipid rafts. These microdomains are rich in signaling molecules such as Src-related PTKs, LAT, G proteins, and glycophosphatidyl inositol-linked receptors and are NVP-ADW742 proposed to play an important part in cell signaling (19, 23). Earlier studies exposed that PAG is definitely tyrosine phosphorylated in unstimulated human being T cells, seemingly as a result of the action of Src kinases (2, 32). This phosphorylation is definitely accompanied from the association of PAG with significant amounts of Csk, a Src homology 2 (SH2) domain-bearing NVP-ADW742 cytoplasmic PTK indicated ubiquitously (23, 30, 35). Interestingly, PAG becomes rapidly dephosphorylated and is dissociated from Csk in response to activation of human being T cells (2, 17, 32). Since Csk inhibits Src-related enzymes by phosphorylating their inhibitory tyrosine [tyrosine (Y) 505 in Lck; Y528 in FynT], it was proposed that PAG might mediate a negative transmission in lipid rafts which is definitely aimed at avoiding or terminating T-cell activation. In support of this idea, transient overexpression of PAG was reported to cause an inhibition (2-collapse) of TCR-mediated activation of nuclear element of triggered T cells and interleukin-2 (IL-2) production in Jurkat T cells (2, 17). Whether a similar inhibitory function is present for endogenous PAG molecules indicated in normal T cells was not established. The mechanism of PAG-mediated inhibition remains to be clarified. While inactivation of Src kinases by PAG-associated Csk molecules is definitely a plausible explanation, other possibilities exist. Along these lines, it is noteworthy the cytoplasmic website of PAG bears several protein-protein connection motifs, including the aforementioned tyrosines, proline-rich motifs, and a carboxyl-terminal PDZ-binding sequence. While one of the tyrosines, tyrosine 314.