Activation of an S6 kinase from human placenta by autophosphorylation.
A number of protein kinases have been shown to undergo autophosphorylation, but few have demonstrated a coordinate increase or decrease in enzymatic activity as a result. Described here is a novel S6 kinase isolated from human placenta which autoactivates through autophosphorylation in vitro. This S6/H4 kinase, purified in an inactive state, exhibited 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-32P]ATP, and reacted with an antibody to the conserved APE domain of the cAMP-dependent protein kinase. The protein did not cochromatograph with p70 S6 kinase and did not cross-react with an anti-p70 kinase antibody. The synthetic peptide S6-21, histone H4, and myelin basic protein were phosphorylated by the purified S6/H4 kinase. Mild digestion of the inactive S6/H4 kinase with trypsin generated a 40-kDa fragment, as determined by SDS-polyacrylamide gel electrophoresis. The trypsin treatment was necessary, but not sufficient, to fully activate the kinase. Subsequent incubation of the trypsin-treated S6 kinase with MgATP resulted in the rapid autophosphorylation of the 40-kDa fragment along with a coordinate increase in kinase activity. The autophosphorylation of the 40-kDa protein was positively correlated with MgATP incubation time and an increase in activity toward the S6-21 peptide, histone H4, and myelin basic protein. Taken together, these data support the hypothesis that this previously uncharacterized S6 kinase belongs to a unique family of protein kinases which utilize autophosphorylation as part of their in vivo activation mechanism.
Dennis
P B
PB
Department of Biological Sciences, University of North Texas, Denton 76203.
Masaracchia
R A
RA
D000595:Amino Acid Sequence; D000906:Antibodies; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D005260:Female; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010455:Peptides / Q000138:chemical synthesis / Q000276:immunology; D010766:Phosphorylation; D010920:Placenta / Q000201:enzymology*; D011247:Pregnancy; D017346:Protein Serine-Threonine Kinases / Q000302:isolation & purification / Q000378:metabolism*; D019893:Ribosomal Protein S6 Kinases; D013379:Substrate Specificity; D014357:Trypsin / Q000378:metabolism
D000906:Antibodies; D010455:Peptides; D017346:Protein Serine-Threonine Kinases; D019893:Ribosomal Protein S6 Kinases; D014357:Trypsin