Cloning of the mitogen-activated S6 kinase from rat liver reveals an enzyme of the second messenger subfamily.
Recently we reported the purification of a mitogen-activated S6 kinase from Swiss mouse 3T3 fibroblasts and rat liver. The rat liver protein was cleaved with cyanogen bromide or trypsin and 17 of the resulting peptides were sequenced. DNA primers were generated from 3 peptides that had homology to sequences of the conserved catalytic domain of protein kinases. These primers were used in the polymerase chain reaction to obtain a 0.4-kilobase DNA fragment. This fragment was either radioactively labeled and hybridized to Northern blots of poly(A)+ mRNA or used to screen a rat liver cDNA library. Northern blot analysis revealed four transcripts of 2.5, 3.2, 4.0, and 6.0 kilobases, and five S6 kinase clones were obtained by screening the library. Only two of the clones, which were identical, encoded a full-length protein. This protein had a molecular weight of 56,160, which correlated closely to that of the dephosphorylated kinase determined by SDS/PAGE. The catalytic domain of the kinase resembles that of other serine/threonine kinases belonging to the second messenger subfamily of protein kinases.
Kozma
S C
SC
Friedrich Miescher Institute, Basel, Switzerland.
Ferrari
S
S
Bassand
P
P
Siegmann
M
M
Totty
N
N
Thomas
G
G
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods; D004622:Embryo, Mammalian; D004789:Enzyme Activation; D015723:Gene Library; D008099:Liver / Q000201:enzymology*; D051379:Mice; D008934:Mitogens / Q000494:pharmacology; D008969:Molecular Sequence Data; D005810:Multigene Family; D011061:Poly A / Q000235:genetics / Q000302:isolation & purification; D016133:Polymerase Chain Reaction / Q000379:methods; D011494:Protein Kinases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D012333:RNA, Messenger; D051381:Rats; D015183:Restriction Mapping; D019893:Ribosomal Protein S6 Kinases; D015290:Second Messenger Systems; D012689:Sequence Homology, Nucleic Acid
D008934:Mitogens; D012333:RNA, Messenger; D011061:Poly A; D012313:RNA; D011494:Protein Kinases; D019893:Ribosomal Protein S6 Kinases