ACTIVATION OF P38(MAPK) MKK3, AND MKK4 BY TNF-ALPHA IN MOUSE BONE-MARROW-DERIVED MACROPHAGES

TNF-alpha regulates the expression of many proinflammatory and profibr ogenic gene products in macrophages, and hence plays a vital role in c ontrolling the inflammatory response. We have shown previously that ex posure of macrophages to TNF-alpha stimulates the activation of member s of the mitogen-activated protein kinase (MAPK) family. In this study , we have investigated the mechanism of activation of the p38(mapk) by TNF-alpha in mouse bone marrow-derived macrophages. Exposure to TNF-a lpha resulted in the activation of p38(mapk), as measured by 1) the tr ans-phosphorylation of recombinant activating transcription factor-2 s ubstrate by immunoprecipitated p38(mapk), and 2) specific tyrosine pho sphorylation of immunoprecipitated p38(mapk). In addition, selective l igation of the TNF-LU receptor CD120a (p55) with human TNF-alpha was s ufficient to induce p38(mapk) activation. Using an in vitro kinase ass ay with recombinant kinase-inactive p38(mapk) as substrate in the pres ence of [gamma-P-32]ATP, the upstream kinases MKK3 (mitogen-activated protein kinase kinase 3) and MKK4 were found to be activated in respon se to TNF-alpha. These findings suggest that TNF-alpha transiently pho sphorylates and activates the three members of the MAPK family, namely p42(mapk/erk2), p46 c-jun amino-terminal kinase/stress-activated prot ein kinase (JNK/SAPK), and p38(mapk) following cross-linking of CD120a (p55), and that MKK3 and MKK4 are capable of phosphorylating p38(mapk ).