Abstract
Full T cell activation requires antigen recognition (Signal 1) in the context of costimulation (Signal 2). Our group and others have determined that the mammalian Target of Rapamycin (mTOR) plays an important role in integrating costimulatory signals. Specifically, Th1 T cell activation in the absence of mTOR activation leads to tolerance in the form of T cell anergy. mTOR is an evolutionarily-conserved serine/threonine kinase which has been shown to interpret environmental cues in mammalian cells. mTOR is activated by an array of diverse inputs including insulin, amino acids, growth factors and CD28. mTOR signals through two signaling complexes: TORC1 and TORC2. A critical component of TORC1 signaling is the Regulatory Associated Protein of TOR (raptor). One of the central questions in understanding mTOR function is determining how diverse upstream signals can lead to diverse downstream functional consequences. To address this issue in T cells we undertook a proteomic approach to identify novel binding proteins for raptor. Jurkat T cells were either incubated in serum-free media or hyper-activated. Raptor was immunoprecipitated (IP) from the two lysates, separated by SDS-PAGE and silver stained. Next, protein bands that were differentially bound to raptor in the lysates from stimulated versus unstimulated cells were identified. One band located near 90 kDa was excised and using mass spectrometry subsequently determined to be Hsp90, a chaperone protein necessary for the correct folding of many protein “clients”. These findings were next confirmed in primary T cells. Upon activation with anti-CD3 and anti-CD28, IP of raptor led to co-IP of Hsp90 and IP of Hsp90 led to the concomitant precipitation of raptor. To further determine the role of the Hsp90-raptor interaction in T cells we used 17-AAG, an Hsp90 inhibitor. Primary T cells were stimulated in the presence of rapamycin or 17-AAG. Incubation with 17-AAG but not rapamycin led to a decrease in raptor protein levels consistent with the concept that raptor is an Hsp90 client. Functionally, this led to a decrease in TORC1 activation as measured by phosphorylation of S6K-1. More importantly, although17-AAG did not inhibit IL-2 production upon initial stimulation, primary T cells stimulated with anti-CD3 and anti-CD28 in the presence of 17-AAG failed to produce IL-2 upon rechallenge 5 days later; they were anergic. Overall, our findings demonstrate a novel activation-induced interaction between Hsp90 and raptor in T cells. This interaction can regulate the decision between TCR-induced activation and tolerance. Hsp90 inhibitors are currently being evaluated as anti-neoplastic agents, and while such agents do not acutely inhibit T cell function, they may induce anergy in activated Th1 cells. Thus, Hsp90 inhibitors might be incorporated into novel immunosuppressive regimens to treat and prevent GVHD and transplant rejection through the promotion of T cell tolerance.
Macropinocytosis drives T cell growth by sustaining the activation of mTORC1
