RECOMBINANT IL-6 ACTIVATES P42 AND P44 MITOGEN-ACTIVATED PROTEIN-KINASES IN THE IL-6 RESPONSIVE B-CELL LINE, AF-10

IL-6 is a multi-functional cytokine that utilizes 80-kDa ligand-bindin g and 130-kDa signal-transducing subunits to stimulate diverse cellula r responses. Although IL-6R ligation has been associated with tyrosine protein phosphorylation and activation of an unidentified serine/thre onine kinase, very little is known about the intermediary signaling ev ents between the cell membrane and the nucleus. rIL-6 treatment of the human B cell line, AF-10, induced MAP kinase (mitogen-activated prote in kinase) activity as determined by in vitro phosphorylation of micro tubule-associated protein-2 (MAP-2) and the synthetic peptide APRTPGGR R, corresponding to amino acids 95-98 of bovine myelin basic protein. The kinetics of the response was rapid and dependent on the dose of rI L-6. The response was cytokine specific, did not require the presence of extracellular Ca2+, and was minimally affected by the presence of s taurosporine. MAP kinase activation in AF-10 cells occurred in paralle l with appearance of 42- and and 44-kDa tyrosine phosphoproteins (p42 and p44). Moreover, MAP kinase activation was diminished when AF-10 ce lls were stimulated with rIL-6 in the presence of tyrosine protein kin ase inhibitors, genistein and geldanomycin. p42 and p44 co-electrophor esed on SDS-PAGE with extracellular signal-related kinase (ERK)-2, and ERK-1, respectively; both are members of the ERK family. In addition to p42MAPK and p44MAPK, rIL-6 also activated a MAP-2 kinase that elute d at a lower salt concentration (20 to 60 mM NaCl, peak I) from Mono-Q resin than p42MAPK (120 to 180 mM NaCl, peak II). The identify of thi s kinase is unknown but it is not an MBP kinase or a protein that exhi bits immunoreactivity with anti-ERK antisera. In another IL-6-responsi ve B cell line, SKW6.4, rIL-6-activated peak I MAP-2 kinase but failed to activate ERK-2. The protein kinase C agonist, PMA, did, however, a ctivate ERK-2 in SKW6.4 cells. These results show that the pleiotrophi c cytokine, IL-6, activates p42MAPK/ERK-2 and at least one other serin e/threonine kinase in B cell lines.