Technical Notes: RSK1, Phosphorylation at S380

The 90 kDa ribosomal protein S6 kinases (RSK1-4) are a family of broadly expressed serine/threonine kinases activated in response to mitogenic stimuli, including growth factors and tumor-promoting phorbol esters.1 The RSK family is positioned downstream of the Raf-MEK-ERK signal transduction module. RSKs are directly phosphorylated by extracellular signal-regulated kinase-1 (ERK1) and ERK2,2 mitogen-activated protein kinases (MAPKs) that are activated by the dual-specificity MAPK kinases MEK1 and MEK2. Active RSKs appear to play a major role in transcriptional regulation, translocating to the nucleus and phosphorylating c-Fos and CREB.3,4

A highly conserved feature common to all RSK family members is the presence of tandem catalytic domains, the amino-terminal kinase domain (NTD), and the carboxyl-terminal kinase domain (CTD).2,5 The NTD phosphorylates exogenous substrates, while the CTD is involved in autophosphorylation. Current models suggest that RSK activation requires the binding of active ERK1/2 to a docking site at the extreme carboxyl-terminus of RSK.6 Docked ERK phosphorylates RSK1 at a number of sites, including T573 in the activation loop of the CTD. The CTD then autophosphorylates S380, a site in the central hydrophobic region. Phosphorylation of RSK1 in this regulatory linker region is critical for maximal activity,2 creating a phospho-serine docking site that recruits PDK1.7 PDK1 phosphorylates S221 within the activation loop of the NTD, fully activating RSK1. The importance of ERK in phosphorylating RSK1 has been demonstrated with the compound U0126, a MEK1/2 inhibitor.8

Figure 1. Total amounts of phosphorylated RSK1, as quantified by the Phospho-RSK1 (S380) DuoSet IC ELISA (R&D Systems, Cat. # DYC892B), are consistent with the amounts of phosphorylated RSK1 determined by qualitative Western blot analysis. HeLa cells were treated with 200 nM PMA for the indicated times. Following cell lysis, RSK1 phosphorylated at S380 was quantified with the DuoSet IC. The same lysates were also immunoblotted (inset) with either phospho-RSK (S380) (R&D Systems, Cat. # AF889) or RSK1 (R&D Systems, Cat. # AF992) antibodies. Figure 2. Both the quantification and specificity of the Phospho-RSK1 (S380) DuoSet IC ELISA were demonstrated by using cells pretreated with the MEK1/2 inhibitor U0126, which indirectly inhibits phosphorylation of RSK1 at S380. HeLa cells were incubated with no additions or with 200 nM PMA for 20 minutes, with or without either 2 or 50 µM U0126. Cells were lysed and RSK1 phosphorylated at S380 was quantified with the DuoSet IC. The same lysates were also immunoblotted (inset) with either phospho-RSK (S380) or RSK1 antibodies.

References

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  2. Dalby, K. et al. (1998) J. Biol. Chem. 273:1496.
  3. Chen, R-H. et al. (1993) Proc. Natl. Acad. Sci. USA 90:10952.
  4. Xing, J. et al. (1996) Science 273:959.
  5. Bjorbaek, C. et al. (1995) J. Biol. Chem. 270:18848.
  6. Gavin, A-C. & A. Nebreda (1999) Curr. Biol. 9:281.
  7. Frodin, M. et al. (2000) EMBO J. 19:2924.
  8. Favata, M. et al. (1998) J. Biol. Chem. 273:18623.