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 th e membranes of rabbit reticulocytes by chromatography of the Triton X- 100 extract on DEAF-cellulose, SP-Sepharose Fast Row, and by FPLC on M ono Q and Superose-12. The S6 kinase elutes around 40000 daltons upon gel filtration on Superose-12 or Sephacryl S-200. It has a subunit mol ecular weight of 40-43 kDa as determined by protein kinase activity fo llowing denaturation/renaturation in SDS-polyacrylamide gels containin g S6 peptide. It also phosphorylates translational initiation factors eIF-2 and eIF-4F, glycogen synthase, histone 1, histone 2B, myelin bas ic protein, but not prolactin, skeletal myosin light chain, histone 4, tubulin, and casein. Apparent K-m values have been determined to be 1 5 mu M for ATP, 1.2 mu M for S6 and 10 mu M for S6 peptide. Two-dimens ional tryptic phosphopeptide mapping shows the same sites on S6 are ph osphorylated as those identified previously with proteolytically activ ated multipotential S6 kinase from rabbit reticulocytes, previously de noted as protease activated kinase II. Examination of relative rates o f phosphorylation and kinetic constants of synthetic peptides based on previously identified phosphorylation sites, indicates a minimum subs trate 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 fro m the p90 (Type I) and p70 (Type II) S6 kinases, and from protein kina se C and the catalytic subunit of cAMP-dependent protein kinase.