Phosphorylation of actin-binding protein 280 by growth factors is mediated by p90 ribosomal protein S6 kinase

Area of Science:

• Cell Biology
• Molecular Biology
• Biochemistry

Background:

• Ras-related small GTPases regulate cell proliferation and motility via protein kinase cascades.
• Intracellular protein targets of activated kinases remain largely uncharacterized.

Purpose of the Study:

• To identify the specific protein targets of activated kinases in cellular signaling.
• To elucidate the role of p90 ribosomal S6 kinase 2 (RSK2) in phosphorylating actin-binding protein (ABP-280).

Main Methods:

• Utilized intact rat 3Y1 fibroblasts for in vivo phosphorylation studies.
• Employed growth factors (fetal calf serum, EGF, PMA, LPA) to stimulate cellular signaling.
• Performed in vitro kinase assays, MonoQ anion exchange chromatography, immunoblotting, S6 peptide kinase assays, and 2D phosphopeptide mapping.

Main Results:

• RSK2 was identified as the kinase responsible for phosphorylating ABP-280 in 3Y1 fibroblasts.
• Growth factors significantly stimulated serine residue phosphorylation of ABP-280.
• RSK2 phosphorylated ABP-280 at the same sites as observed in growth factor-stimulated cells.
• ERK2 activation of RSK2 in vitro suggests a regulatory mechanism.

Conclusions:

• RSK2 directly phosphorylates ABP-280 in vivo, linking GTPase signaling to cytoskeletal regulation.
• This phosphorylation event is stimulated by various growth factors, highlighting its importance in cellular responses.
• The findings provide a molecular link between growth factor signaling, RSK2 activation, and the modification of actin-binding proteins.