Phosphorylation of p90 ribosomal S6 kinase (RSK) regulates extracellular signal-regulated kinase docking and RSK activity

Abstract

Stimulation of the Ras/extracellular signal-regulated kinase (ERK) pathway can modulate cell growth, proliferation, survival, and motility. The p90 ribosomal S6 kinases (RSKs) comprise a family of serine/threonine kinases that lie at the terminus of the ERK pathway. Efficient RSK activation by ERK requires its interaction through a docking site located near the C terminus of RSK, but the regulation of this interaction remains unknown. In this report we show that RSK1 and ERK1/2 form a complex in quiescent HEK293 cells that transiently dissociates upon mitogen stimulation. Complex dissociation requires phosphorylation of RSK1 serine 749, which is a mitogen-regulated phosphorylation site located near the ERK docking site. Using recombinant RSK1 proteins, we find that serine 749 is phosphorylated by the N-terminal kinase domain of RSK1 in vitro, suggesting that ERK1/2 dissociation is mediated through RSK1 autophosphorylation of this residue. Consistent with this hypothesis, we find that inactivating mutations in the RSK1 kinase domains disrupted the mitogen-regulated dissociation of ERK1/2 in vivo. Analysis of different RSK isoforms revealed that RSK1 and RSK2 readily dissociate from ERK1/2 following mitogen stimulation but that RSK3 remains associated with active ERK1/2. RSK activity assays revealed that RSK3 also remains active longer than RSK1 and RSK2, suggesting that prolonged ERK association increased the duration of RSK3 activation. These results provide new evidence for the regulated nature of ERK docking interactions and reveal important differences among the closely related RSK family members.

FIG. 1.

Regulated association between RSK1 and ERK1/2. (A) HEK293 cells were transfected with wt avian RSK1, RSK-CTT (CTT), or control vector (pRK7); serum starved for 16 to 18 h; and stimulated with EGF for the indicated times. Anti-HA RSK1 immunoprecipitates (IP) were immunoblotted for ERK1/2 association, for RSK1 using anti-avian epitope antibodies, and for phosphorylated RSK1 Thr590 (pT590) using phosphospecific antibodies. The cell lysates were probed for ERK1/2 to measure MAPK-ERK pathway activation. (B) HEK293 cells were transfected as for panel A. Where indicated, the cells were pretreated (+) with 5 μM U0126 for 30 min prior to stimulation with EGF (50 ng/ml) for an additional 10 min. Anti-HA RSK1 immunoprecipitates and whole-cell lysates were blotted as for panel A. (C) HEK293 cells were stimulated for 10 min with EGF (50 ng/ml), phorbol myristate acetate (PMA) (100 ng/ml), or FBS (10%) (serum), and analyzed as for panel A.

FIG. 2.

Mutational analysis of the ERK docking region. HEK293 cells were transfected with control vector (pRK7), wt RSK1 (WT), RSK-CTT (CTT), and full-length RSK1 with point mutations in the C-terminal region (L739A, R742A, R743A, K745A, K746A, L747A, and S749A) as shown in the schematic (A). (B) Cells were serum starved for 16 to 18 h and stimulated with EGF (50 ng/ml) for 10 min where indicated (+). Anti-HA RSK1 immunoprecipitates (IP) were immunoblotted for ERK1/2 association and for RSK1 using anti-avian epitope antibodies. The cell lysates were probed for ERK1/2 to measure MAPK-ERK pathway activation. The phosphotransferase activities of wt RSK1 and RSK1 mutants were determined by kinase assays using GST-S6 as a substrate. Phosphorimager analysis of GST-S6 was performed, and typical results are shown as a histogram (n = 3). NTKD, N-terminal kinase domain; CTKD, C-terminal kinase domain; circled Ps, regulated phosphorylation sites.

FIG. 3.

Mutational analysis of RSK1 serine 749. (A) HEK293 cells were transfected with RSK1 containing an alanine mutation on Ser749, RSK-CTT (CTT), or control vector (pRK7); serum starved for 16 to 18 h; and stimulated with EGF for the indicated times. Anti-HA RSK1 immunoprecipitates (IP) were blotted for ERK1/2 association and for RSK1 using anti-avian epitope antibodies. The cell lysates were probed for ERK1/2 to measure MAPK-ERK pathway activation. (B) HEK293 cells were transfected with RSK1 containing an alanine (S749A), aspartic acid (S749D), or glutamic acid (S749E) mutation, serum starved for 16 to 18 h, and stimulated with FBS (10%) (serum) for 10 min where indicated (+). Anti-HA RSK1 immunoprecipitates and whole-cell lysates were blotted as for panel A. The phosphotransferase activities of all RSK1 mutants were determined by kinase assays using GST-S6 as a substrate. Phosphorimager analysis of GST-S6 was performed, and typical results are shown as a histogram (n = 4).

FIG. 4.

The RSK1 N-terminal kinase domain phosphorylates serine 749. HEK293 cells were transfected with wt RSK1 (WT), kinase-inactive alleles (K112R or K464R), double-kinase-inactive RSK1 (K112/464R), or control vector (pRK7); serum starved for 16 to 18 h; and stimulated with EGF for 10 min where indicated (+). Anti-HA RSK1 immunoprecipitates were assayed for phosphotransferase activity toward recombinant kinase-inactive (K464R) GST-RSK1^386-752 with alanine substitutions for Ser398 and Thr590 (A) or Ser398, Thr590, and Ser749 (B). Kinase reaction products were subjected to SDS-PAGE, and ³²P incorporation was assessed by autoradiography and analyzed using a Phosphorimager. Typical results are shown as a histogram (n = 2).

FIG. 5.

ERK interaction is regulated by RSK1 autophosphorylation. (A and C) HEK293 cells were transfected with wt RSK1 (WT), kinase-inactive alleles (K112R, K464R, or AAA/D1), double-kinase-inactive RSK1 (K112/464R), or control vector (pRK7); serum starved for 16 to 18 h; and stimulated with EGF for 10 min where indicated (+). Anti-HA-RSK1 immunoprecipitates (IP) were blotted for ERK1/2 association and for RSK1 using anti-avian epitope antibodies. The cell lysates were probed for ERK1/2 to measure MAPK-ERK pathway activation. The phosphotransferase activities of all RSK1 mutants were determined by kinase assays using GST-S6 as a substrate. Phosphorimager analysis of GST-S6 was performed, and typical results are shown as a histogram (n = 2). (B) HEK293 cells were transfected with double-kinase-inactive RSK1 (K112/464R), RSK-CTT (CTT), or control vector (pRK7); serum starved for 16 to 18 h; and stimulated with EGF for the indicated times. Anti-HA RSK1 immunoprecipitates and lysates were immunoblotted as for panel A.

FIG. 6.

Differential regulation of ERK docking to RSK isoforms. (A) Alignments of the C termini of avian (av) and human (h) RSK1, murine (m) RSK2, human RSK3 and RSK4, and human MSK1 and MSK2. The boxed residues indicate the ERK and/or p38 docking sites, the dashed box indicates residues that modulate ERK interaction with RSK1, the arrow indicates the conserved serine that lies within an RSK/MSK consensus phosphorylation sequence, and the asterisks indicate the C termini of the Rsk proteins. (B) HEK293 cells were transfected with wt avian and human RSK1, murine RSK2, human RSK3, or control vector (pRK7); serum starved for 16 to 18 h; and stimulated with EGF for 10 min where indicated (+). Anti-HA RSK immunoprecipitates (IP) were blotted for ERK1/2 association and for RSK1 to -3 using anti-HA antibodies. The cell lysates were probed for ERK1/2 to measure MAPK-ERK pathway activation. The phosphotransferase activities of all RSK isoforms were determined by kinase assays using GST-S6 as a substrate. Phosphorimager analysis of GST-S6 was performed, and typical results are shown as a histogram (n = 3). (C) HEK293 cells were transfected with hRSK3, RSK-CTT (CTT), or control vector (pRK7); serum starved for 16 to 18 h; and stimulated with EGF for the indicated times. Anti-HA RSK3 immunoprecipitates and lysates were immunoblotted as for panel B.

FIG. 7.

Activation kinetics of RSK isoforms. HEK293 cells were serum starved and stimulated for 0, 10, 30, 60, 90, or 120 min with EGF (50 ng/ml). The phosphotransferase activities of all immunoprecipitated RSK isoforms were determined by kinase assays using GST-S6 as a substrate. Phosphorimager analysis of GST-S6 was performed, and typical results are shown as a histogram (n = 3). Activation levels are shown as percentages of maximum activation at 10 min of EGF stimulation for each RSK construct. Analyses were made among human RSK1, murine RSK2, and human RSK3 (A) and between wt avian RSK1 and RSK1 S749A (B). Each histogram represents the average plus standard error of three independent experiments.