Ribosomal S6 kinase 2 inhibition by a potent C-terminal repressor domain is relieved by mitogen-activated protein-extracellular signal-regulated kinase kinase-regulated phosphorylation

Ribosomal S6 kinase 2 (S6K2) is a recently identified serine/threonine prot ein kinase that phosphorylates the 40 S ribosomal protein S6 in vitro. S6K2 is highly homologous to S6K1 in the core kinase and Linker regulatory doma ins but differs from S6K1 in the N- and C-terminal regions and is different ly localized primarily to the nucleus because of a C-terminal nuclear local ization signal unique to S6K2. We have recently demonstrated that S6K2 is r egulated similarly to S6K1 by the mammalian target of rapamycin pathway and by multiple PI3-K pathway effecters in vivo. However, deletion of the C-te rminal domain of S6K2 enhances kinase activity, whereas analogous deletion of S6K1 is inhibitory, Here, we characterize the S6K2 C-terminal motifs tha t confer this differential regulation. We demonstrate that the inhibitory e ffects of the S6K2 C-terminal domain are only partly attributable to the nu clear localization signal but that three C-terminal proline-directed potent ial mitogen-activated protein kinase phosphorylation sites are critical med iators of this inhibitory effect. Site-specific mutation of these sites to alanine completely desensitizes S6K2 to activating inputs, whereas mutation to aspartic acid to mimic phosphorylation results in an activated enzyme w hich is hypersensitive to activating inputs. Pretreatment of cells with the mitogen-activated protein-extracellular signal-regulated kinase kinase (ME K) inhibitor U0126 inhibited S6K2 activation to a greater extent than S6K1. Furthermore, S6K2 mutants with C-terminal deletion or acidic phosphorylati on site mutations displayed greatly reduced U0126 sensitivity. Thus, MEK-de pendent inputs to C-terminal phosphorylation sites appear to be essential f or relief of S6K2 inhibition but less critical for activation of S6K1. Thes e data suggest a mechanism by which weak PI3-K agonists can regulate S6 pho sphorylation and selective translation in the presence of mitogen-activated protein kinase signaling.