Fibroblast growth factor treatment of Swiss 3T3 cells activates a subunit S6 kinase that phosphorylates a synthetic peptide substrate.
Exposure of quiescent cultures of Swiss 3T3-D1 cells to bovine brain acidic fibroblast growth factor (FGF) enhanced phosphorylation of a 31-kDa protein tentatively identified as 40S ribosomal subunit S6 (S6). Soluble extracts from FGF-treated as compared with quiescent fibroblasts exhibited up to 3-fold higher kinase activity towards S6 in exogenously added rat liver 40S ribosomes and a synthetic peptide, RRLSSLRA. This peptide was patterned after a phosphorylation site sequence in S6 and was phosphorylated with an apparent Km corresponding to 0.18 mM. Optimal activation of the S6 kinase with pure mitogen at 10 ng/ml occurred within 15 to 20 min exposure to FGF. Half-maximal stimulation of the FGF-induced S6 kinase was attained with FGF at 0.4 ng/ml. The S6 kinase in crude extracts utilized both [gamma-32P]ATP (apparent Km congruent to 6-8 microM) and [gamma-32P]GTP (apparent Km congruent to 3 microM), but the ability to utilize GTP was lost after partial purification of the kinase. The FGF-stimulated kinase had an apparent Mr of about 95,000 as determined by chromatography on Sephacryl S300 but appeared to be retarded on TSK 400 HPLC columns, since it eluted with an apparent Mr of 29,000. Treatment of Swiss 3T3 cells with the tumor promoter phorbol 12-myristate 13-acetate (PMA) activated the FGF-stimulated S6 kinase. However, protein kinase C was not required to mediate the FGF activation of the S6 kinase, as FGF still evoked a two-fold activation of the S6 kinase in phorbol ester-pretreated, protein kinase C-depleted cells.
Pelech
S L
SL
Olwin
B B
BB
Krebs
E G
EG
D000818:Animals; D002478:Cells, Cultured; D004789:Enzyme Activation; D005346:Fibroblast Growth Factors / Q000494:pharmacology*; D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D051379:Mice; D008970:Molecular Weight; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D012269:Ribosomal Proteins / Q000378:metabolism
D046911:Macromolecular Substances; D012269:Ribosomal Proteins; D005346:Fibroblast Growth Factors; D006160:Guanosine Triphosphate; D011494:Protein Kinases