ACTIVATION OF AN S6 KINASE FROM HUMAN PLACENTA BY AUTOPHOSPHORYLATION

A number of protein kinases have been shown to undergo autophosphoryla tion, but few have demonstrated a coordinate increase or decrease in e nzymatic activity as a result. Described here is a novel S6 kinase iso lated from human placenta which autoactivates through autophosphorylat ion in vitro. This S6/H4 kinase, purified in an inactive state, exhibi ted a molecular mass of 60 kDa as estimated by SDS-polyacrylamide gel electrophoresis. The 60-kDa protein underwent autophosphorylation, was labeled by 8-azido-[alpha-P-32]ATP, and reacted with an antibody to t he conserved APE domain of the cAMP-dependent protein kinase. The prot ein did not cochromatograph with p70 S6 kinase and did not cross-react with an anti-p70 kinase antibody. The synthetic peptide S6-21, histon e H4, and myelin basic protein were phosphorylated by the purified S6/ H4 kinase. Mild digestion of the inactive S6/H4 kinase with trypsin ge nerated a 40-kDa fragment, as determined by SDS-polyacrylamide gel ele ctrophoresis. The trypsin treatment was necessary, but not sufficient, to fully activate the kinase. Subsequent incubation of the trypsin-tr eated S6 kinase with MgATP resulted in the rapid autophosphorylation o f the 40-kDa fragment along with a coordinate increase in kinase activ ity. The autophosphorylation of the 40-kDa protein was positively corr elated with MgATP incubation time and an increase in activity toward t he S6-21 peptide, histone H4, and myelin basic protein. Taken together , these data support the hypothesis that this previously uncharacteriz ed S6 kinase belongs to a unique family of protein kinases which utili ze autophosphorylation as part of their in vivo activation mechanism.