PAF priming of human PMNs activates a preassembled P38 MAPK module involving RAC

Abstract Mitogen activated protein kinase (MAPK) signaling constructs are three-tiered kinase modules. The initial kinase, an MKKK, may be activated by interaction with a small GTPase (i.e., RAC). P21-activated kinase (PAK) is an MKKK activated by RAC. Recent evidence suggests that platelet activating factor (PAF) priming of PMNs for superoxide production involves rapid RAC/P38 MAPK activation. Recognizing the rapid priming by PAF, we hypothesize that a preassembled P38 MAPK module involving RAC is activated by PAF stimulation of human PMNs. Methods: Human PMNs were obtained and stimulated with 2 μM PAF. PAK pull downs were performed on PMN lysates (1.25 × 107 cells) whereby; cellular PAK and its adherent proteins were collected. Proteins were separated and immunoblotted for RAC, total P38 and activated (phosphorylated) P38. Results: In resting PMNs there was very little activated RAC, as determined by its binding to PAK. PAF stimulation increased the amount of activated RAC bound to and precipitated with PAK. P38 precipitated with PAK in resting and PAF stimulated PMNs. The quantity of total P38 bound to PAK did not change following PAF stimulation. In resting PMNs, very little PAK bound P38 was active. PAF stimulation increased the amount of active P38 bound to and precipitated with PAK. See figure.Conclusions: PAK, an MKKK, is bound to P38 MAPK in human PMNs at rest and following PAF stimulation. RAC activation occurs rapidly following stimulation. Activated RAC binds to, and activates PAK. RAC/PAK activation corresponds to the activation of the PAK bound P38 MAPK. These results indicate the existence of a preformed PAK-MKK- P38 MAPK complex in human PMNs, involving RAC. These findings represent new insights into MAPK module signaling in human PMNs during PAF priming. Download : Download high-res image (35KB) Download : Download full-size image