Phosphorylation of actin-binding protein 280 by growth factors is mediated by p90 ribosomal protein S6 kinase.
Although Ras-related small GTPases are believed to control cell proliferation and motility through activation of protein kinase cascades, little is known about the intracellular protein targets of activated kinases. Here we show that the p90 ribosomal S6 kinase 2 (RSK2) phosphorylates actin-binding protein (ABP-280) in intact rat 3Y1 fibroblasts. Growth factors such as fetal calf serum, epidermal growth factor, phorbol 12-myristate 13-acetate, and lysophosphatidic acid stimulate the phosphorylation of serine residues in ABP-280 in quiescent 3Y1 cells. Extracts from 3Y1 cells prepared after stimulation by lysophosphatidic acid, fetal calf serum, and epidermal growth factor retain activated protein kinase activity(s) toward ABP-280 in vitro. ABP kinase activities in lysates from lysophosphatidic acid-stimulated 3Y1 cells can be fractionated by MonoQ anion exchange column chromatography into three peaks having ABP kinase activities. One (ABP kinase peak 1) coelutes with the peak of RSK2 as judged by immunoblotting and S6 peptide kinase assays. Two-dimensional phosphopeptide maps show RSK2 phosphorylated ABP-280 to be phosphorylated at the same site(s) as those stimulated by growth factors in vivo. Incubation of ABP kinase peak 1 fractionated from unstimulated cells with activated ERK2 activates latent ABP kinase activity. These results show RSK2 to phosphorylate ABP-280 in vivo.
Ohta
Y
Y
Division of Experimental Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA.
Hartwig
J H
JH
D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000502:physiology; D004815:Epidermal Growth Factor / Q000494:pharmacology; D005260:Female; D006133:Growth Substances / Q000494:pharmacology*; D008246:Lysophospholipids / Q000494:pharmacology*; D008840:Microfilament Proteins / Q000378:metabolism*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000502:physiology*; D051381:Rats; D011916:Rats, Inbred F344; D019893:Ribosomal Protein S6 Kinases
D006133:Growth Substances; D008246:Lysophospholipids; D008840:Microfilament Proteins; D004815:Epidermal Growth Factor; D017346:Protein Serine-Threonine Kinases; D019893:Ribosomal Protein S6 Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases