Molecular basis of MAPK-activated protein kinase 2:p38 assembly

Abstract

p38 MAPK and MAPK-activated protein kinase 2 (MK2) are key components of signaling pathways leading to many cellular responses, notably the proinflammatory cytokine production. The physical association of p38␣ isoform and MK2 is believed to be physiologically important for this signaling. We report the 2.7-Å resolution crystal structure of the unphosphorylated complex between p38␣ and MK2. These protein kinases bind ''head-to-head,'' present their respective active sites on approximately the same side of the heterodimer, and form extensive intermolecular interactions. Among these interactions, the MK2 Ile-366 -Ala-390, which includes the bipartite nuclear localization signal, binds to the p38␣-docking region. This binding supports the involvement of noncatalytic regions to the tight binding of the MK2:p38␣ binary assembly. The MK2 residues 345-365, containing the nuclear export signal, block access to the p38␣ active site. Some regulatory phosphorylation regions of both protein kinases engage in multiple interactions with one another in this complex. This structure gives new insights into the regulation of the protein kinases p38␣ and MK2, aids in the better understanding of their known cellular and biochemical studies, and provides a basis for understanding other regulatory protein-protein interactions involving signal transduction proteins.X-ray structure T he MAPK has been directly associated with proinflammatory cytokine production. Inhibition of p38␣ (and its homolog, p38␤) by inhibitors such as SB203580 and BIRB 796, reduces LPS-induced production of proinflammatory cytokines, including TNF␣ and IL-1, that are implicated in the etiology of chronic inflammation (1-5). Recent genetic evidence suggests that p38␣, not p38␤, is the major p38 isoform involved in the LPS-induced immune response (6). Embryonic stem cells derived from mice that lack the p38␣ function exhibit impaired IL-1 signaling (7). Biological therapies directed against inflammatory cytokines, e.g., anti-TNF␣ and anti-IL-1␤, have also proven effective at ameliorating the symptoms of inflammatory diseases such as arthritis and psoriasis (not anti-IL-1␤-mediated) (8-11).MAPK-activated protein kinase-2 (MK2), the primary substrate of p38␣, is also a key participant in modulating proinflammatory cytokine production (12, 13). MK2 knockout mice (MK2 Ϫ/Ϫ ) challenged with LPS produce significantly less TNF␣, IL-6, IFN␥, and IL-1 than LPS-stimulated wild-type mice (13). MK2Ϫ/Ϫ mice are also resistant to collagen-induced arthritis (14). The transfection of cultured keratinocytes with MK2-specific small interfering RNA led to a significant decrease in MK2 protein expression and a subsequent, significant reduction in the production of TNF␣, . The catalytic activity of MK2 has further been shown to be necessary for TNF␣ production (16). Taken together, these data suggest that the inhibition of the p38␣ pathway, particularly MK2, is desirable for the potential treatment of chronic inflammation.In resting cells, p38␣ forms a physiological complex with MK2, w...

Activation and Function of the MAPKs and Their Substrates, the MAPK-Activated Protein Kinases

“…5A), Ser272 within subdomain X of the kinase domain, and Thr334 located in a hinge region that controls MK2 autoinhibition (21). The crystal structure of MK2 has been resolved (394) and suggests that Thr334 phosphorylation may serve as a switch for MK2 nuclear import and export (236). Upon activation, phosphorylation of Thr334 is thought to release the autoinhibitory helix from the core of the kinase domain, thereby exposing the NES and promoting nuclear export (104).…”

“…Consistent with this, the CD motif in p38 kinases contains more contiguous acidic residues than ERK1/2, suggesting that specific electrostatic interactions between charged residues in CD and D domains provide specificity. Elucidation of the three-dimensional structure of the p38␣/MK2 complex (361,394) demonstrated that a specific Lys residue, present only in MK2 and MK3, specifically interacts with the acidic ED domain of p38␣.…”

Crystal Structure of the Ca2+/Calmodulin-dependent Protein Kinase Kinase in Complex with the Inhibitor STO-609

“…7) by superposing the crystal structures of CaMKK␤ (this study), CaMKI (PDB code 1A06) (17), and PKB (PDB code 1GZN) (47) onto the structure of p38 MAPK⅐MAPK-activated kinase 2 complex (PDB code 2OZA) (48). These structural models suggested that the negatively charged residues in the ␣D helix of CaMKI (Asp 105 and Glu 109 in rat sequence), which are conserved in CaMKIV but not in PKB (Phe 239 and Arg 243 ), may interact with the RP-insert of CaMKK␤.…”

Structural Basis for the Regulation of the Mitogen-activated Protein (MAP) Kinase p38α by the Dual Specificity Phosphatase 16 MAP Kinase Binding Domain in Solution

“…protein phosphatase 2A (PP2A)). In particular, KIM-PTPs and DUSPs share a common MAPK interaction/anchoring site on MAPKs (13-15, 17, 19, 23, 39, 40), a site also used by MAPKKs (15,41) and substrates (42)(43)(44). A comprehensive understanding of how the activity of MAPKs is finely controlled by their MAPK regulatory proteins can only be obtained by understanding how these proteins interact at a molecular level as it is the small structural and energetic differences between these proteins that ensure pathway fidelity.…”