Abstracts (complete list) - Wissenschaft Online

Michal Smida, Anita Posevitz-Fejfar, Burkhart Schraven, Jonathan A. Lindquist
PAG downregulation results in increased proximal signaling
but reduced functional outcomes in primary T cells
PAG (the phosphoprotein associated with glycosphingolipid-enriched microdomains) is a
transmembrane adaptor protein that recruits Csk (C-terminal Src kinase) to the plasma
membrane, where it then phosphorylates inhibitory tyrosines within Src kinases.
Recently, we have also shown that PAG negatively regulates Ras activation by recruiting
RasGAP. Thus, PAG functions as a negative regulator of both Src kinases and Ras. Since
PAG knockout mice possess no apparent phenotype, the importance of PAG as a
negative regulator has been questioned. Therefore we investigated PAG function in
primary human T cells and in Jurkat T cells using RNA interference.
Suppression of PAG expression leads to an enhancement of kinase activity in both Fyn
and Lck, which is reflected by increased phosphorylation of their activatory tyrosines. As
a consequence, we detect enhanced basal tyrosine phosphorylation in resting cells,
which is also more sustained upon TCR stimulation. Additionally, we have investigated
specific proteins involved in the proximal signaling. We observed an enhanced activation
of ZAP70 and PLC gamma and a 5-fold increase in Ras activation. However, IL-2
promotor activity appears to be decreased and also the proliferation of PAG deficient
cells was reduced, as measured by both CFSE labeling and thymidine incorporation.
Thus, although the removal of a negative regulator leads to enhanced proximal
signaling, this does not necessarily lead to an increase in proliferation or IL-2
production, but rather may potentiate other outcomes of T-cell activation, e.g. cell
death.