A membrane-bound protein kinase from rabbit reticulocytes is an active form of multipotential S6 kinase.
An active ribosomal protein S6 kinase has been highly purified from the membranes of rabbit reticulocytes by chromatography of the Triton X-100 extract on DEAE-cellulose, SP-Sepharose Fast Flow, and by FPLC on Mono Q and Superose-12. The S6 kinase elutes around 40 000 daltons upon gel filtration on Superose-12 or Sephacryl S-200. It has a subunit molecular weight of 40-43 kDa as determined by protein kinase activity following denaturation/renaturation in SDS-polyacrylamide gels containing S6 peptide. It also phosphorylates translational initiation factors eIF-2 and eIF-4F, glycogen synthase, histone 1, histone 2B, myelin basic protein, but not prolactin, skeletal myosin light chain, histone 4, tubulin, and casein. Apparent Km values have been determined to be 15 microM for ATP, 1.2 microM for S6 and 10 microM for S6 peptide. Two-dimensional tryptic phosphopeptide mapping shows the same sites on S6 are phosphorylated as those identified previously with proteolytically activated multipotential S6 kinase from rabbit reticulocytes, previously denoted as protease activated kinase II. Examination of relative rates of phosphorylation and kinetic constants of synthetic peptides based on previously identified phosphorylation sites, indicates a minimum substrate recognition sequence to be arginine at the n - 3 position. Based on these characteristics, including molecular weight and an expanded substrate specificity, the membrane S6 kinase can be distinguished from the p90 (Type I) and p70 (Type II) S6 kinases, and from protein kinase C and the catalytic subunit of cAMP-dependent protein kinase.
Bensen
E S
ES
Department of Biochemistry, University of California, Riverside, CA 92521, USA.
Umphress
J L
JL
Traugh
J A
JA
Pinna
L A
LA
Tuazon
P T
PT
D000595:Amino Acid Sequence; D000818:Animals; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D004910:Erythrocyte Membrane / Q000201:enzymology*; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017830:Octoxynol; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010449:Peptide Mapping; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D010748:Phosphopeptides / Q000737:chemistry / Q000302:isolation & purification; D011494:Protein Kinases / Q000097:blood* / Q000737:chemistry / Q000302:isolation & purification; D017346:Protein Serine-Threonine Kinases / Q000097:blood* / Q000737:chemistry / Q000302:isolation & purification; D011817:Rabbits; D012156:Reticulocytes / Q000201:enzymology*; D019893:Ribosomal Protein S6 Kinases; D013379:Substrate Specificity
D046911:Macromolecular Substances; D010446:Peptide Fragments; D010455:Peptides; D010748:Phosphopeptides; D017830:Octoxynol; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; D019893:Ribosomal Protein S6 Kinases