Structural basis for activation of the autoinhibitory C-terminal kinase domain of p90 RSK2

Abstract

The X-ray structure at 2.0-Å resolution of the p90 ribosomal S6 kinase 2 C-terminal kinase domain revealed a C-terminal autoinhibitory αL-helix that was embedded in the kinase scaffold and determines the inactive kinase conformation. We suggest a mechanism of activation through displacement of the αL-helix and rearrangement of the conserved residue Glu500, as well as the reorganization of the T-loop into the active conformation.The 90-kDa ribosomal S6 kinase 2 (RSK2) is broadly expressed in response to growth factors, peptide hormones, neurotransmitters, chemokines and other stimuli1 -3. RSK2 is a serine/ threonine kinase containing two distinct catalytically functional kinase domains connected by a linker region4 , 5. The C-terminal domain (CTD) phosphorylates the linker region 6 and regulates the N-terminal domain, which phosphorylates various substrates3 , 4 , 7 ,8 . No defined structure of RSK2 or of either kinase domain has been reported. However, sequence alignment with Ca 2+ /calmodulin-dependent kinase suggested the existence of an autoinhibitory helix outside the CTD RSK2 protein kinase domain, and its autoinhibitory role has been demonstrated in vivo 9 . Efficient activation of RSK2 requires interaction with extracellular signal-regulated protein kinases (ERKs) at a docking site in the RSK2 C terminus (residues 726-735) 10, 11 and subsequent phosphorylation of Thr577 in the CTD T-activation loop12.The CTD RSK2 adopted a classical bilobal kinase fold with an accessible catalytic cleft ( Fig. 1 and Supplementary Fig. 1 online). The C-terminal segment (residues 696-710) formed another αL-helix, located underneath the catalytic cleft and embedded in the kinase scaffold. It occupied a 'cradle' shaped by the αF and αG two-helix junction. The area of the αL-helix surface buried in the 'cradle' was ~800 Å 2 (~50% of the total area) and was composed mostly NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript (~80%) of nonpolar atoms. The position of the αL-helix in the 'cradle' was stabilized by one hydrogen bond (2.7 Å) between Tyr707 and Ser603 (αF-helix).Superimposition of the structures of the RSK2 CTD and active cyclic AMP-dependent protein kinase (PKA) bound to an ATP analog and an inhibitory peptide ( Fig. 2a and Supplementary Fig. 2a online) showed that the CTD had a normal arrangement of the N and C lobes with a slightly more open cleft conformation than found in PKA. Comparing the CTD and PKA invariant residues showed that Asp539 (RD-motif), Asn544 (catalytic loop) and Asp561 (DFGmotif), as well as the ionic pair between Glu463 (αC-helix) and Lys451 (β3-strand), essentially adopted the same conformation in both the CTD and PKA and fit the requirements for optimal phospho-transfer 13 . Despite extensive efforts to cocrystallize the protein with an unhydrolyzable ATP analog, we could not obtain ATP-bound crystals, suggesting that the CTD adopts an inactive conformation.A notable difference between CTD RSK and active PKA entailed the position of the αD-helix, which was shi...

Structural Basis of Ribosomal S6 Kinase 1 (RSK1) Inhibition by S100B Protein

“…Moreover, the helical element from structure C was found to be analogous to the surface-oriented side of the bound inhibitory helix of the previously solved RSK1 structures (Fig. 5C) (13,14,32). We propose that structure C captured the S100B-RSK1 complex in a state where the interaction between the S100B and the RSK1 C-terminal segment may be such as in an S100 inhibited complex with full-length RSK1.…”

“…The first step of MAPKAPK activation is activation loop (AL) phosphorylation by its cognate MAPK. Next, the autoinhibitory helix is extruded by the phosphorylated AL via an unknown mechanism (14). Although most MAPKAPK proteins contain a single catalytic domain, the RSK subfamilies are tandem kinases; in addition to their C-terminal CaMK-type domain (CTKD), they have an N-terminal AGC-type kinase domain (NTKD).…”

Regulation of Protein Kinase A Activity by p90 Ribosomal S6 Kinase 1

“…Thus, besides the phospho-Ser-732 other surrounding residues must play a critical role in association with PKAc. Although the structure of the C-terminal kinase domain of RSK2 was recently reported (22), the extreme C terminus remains unresolved. Moreover, the structure of the C terminus of RSK1 is not known.…”

Multiple myeloma phosphotyrosine proteomic profile associated with FGFR3 expression, ligand activation, and drug inhibition

“…However, RSK2 pY707, identified previously as downstream of FGFR1 (36), was found diminished more than 20-fold by PD173074 and stimulated by FGF1 (Table 1). Y707 is a key residue in the autoinhibition of the C-terminal kinase domain of RSK2 (37), and structural analysis suggests its phosphorylation would activate RSK2 (38). Therefore, RSK2 appears tightly regulated by FGFR3 through distinct mechanisms involving phosphorylations at Y529 (35) and Y707.…”