Pb. Dennis et Ra. Masaracchia, ACTIVATION OF AN S6 KINASE FROM HUMAN PLACENTA BY AUTOPHOSPHORYLATION, The Journal of biological chemistry, 268(26), 1993, pp. 19833-19841
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.