The third conformation of p38α MAP kinase observed in phosphorylated p38α and in solution

MAPKs engage substrates, MAP2Ks, and phosphatases via a docking groove in the C-terminal domain of the kinase. Prior crystallographic studies on the unphosphorylated MAPKs p38α and ERK2 defined the docking groove and revealed long-range conformational changes affecting the activation loop and active site of the kinase induced by peptide. Solution NMR data presented here for unphosphorylated p38α with a MEK3b-derived peptide (p38α/pepMEK3b) validate these findings. Crystallograhic data from doubly phosphorylated active p38α (p38α/T?GY?/pepMEK3b) reveal a structure similar to unphosphorylated p38α/MEK3b, and distinct from phosphorylated p38γ (p38γ/T?GY?) and ERK2 (ERK2/T?EY?). The structure supports the idea that MAP kinases adopt three distinct conformations: unphosphorylated, phosphorylated, and a docking peptide-induced form.     

DEF Pocket in p38α Facilitates Substrate Selectivity and Mediates Autophosphorylation

Signaling processes are primarily promoted by molecular recognition and corresponding protein-protein interactions. One of the key eukaryotic signaling pathways is the MAP kinase cascade involved in vital cellular processes such as cell proliferation, differentiation, apoptosis, and stress response. The principle recognition site of MAP kinases, the common docking (CD) region, forms selective interactions with substrates, upstream activators, and phosphatases. A second docking site, defined as the DEF site interaction pocket (DEF pocket), is formed subsequent to ERK2 and p38α activation. Both crystal structures of p38α in its dually phosphorylated form and of intrinsically active mutants showed the DEF pocket, giving motivation for studying its role in substrate activation and selectivity. Mutating selected DEF pocket residues significantly decreased the phosphorylation levels of three p38α substrates (ATFII, Elk-1, and MBP) with no apparent effect on the phosphorylation of MK2 kinase. Conversely, mutating the CD region gave the opposite effect, suggesting p38α substrates can be classified into DEF-dependent and DEF-independent substrates. In addition, mutating DEF pocket residues decreased the autophosphorylation capability of intrinsically active p38α mutants, suggesting DEF-mediated trans-autophosphorylation in p38α. These results could contribute to understanding substrate selectivity of p38α and serve as a platform for designing p38α-selective DEF site blockers, which partially inhibit p38α binding DEF-dependent substrates, whereas maintaining its other functions intact. In this context, preliminary results using synthetic peptides reveal significant inhibition of substrate phosphorylation by activated p38α.     

Structural Basis of Protein Kinase Cα Regulation by the C-Terminal Tail

Protein kinase C (PKC) isoenzymes are multi-modular proteins activated at the membrane surface to regulate signal transduction processes. When activated by second messengers, PKC undergoes a drastic conformational and spatial transition from the inactive cytosolic state to the activated membrane-bound state. The complete structure of either state of PKC remains elusive. We demonstrate, using NMR spectroscopy, that the isolated Ca²⁺-sensing membrane-binding C2 domain of the conventional PKCα interacts with a conserved hydrophobic motif of the kinase C-terminal region, and we report a structural model of the complex. Our data suggest that the C-terminal region plays a dual role in regulating the PKC activity: activating, through sensitization of PKC to intracellular Ca²⁺ oscillations; and auto-inhibitory, through its interaction with a conserved positively charged region of the C2 domain.     

Scattering of MCF7 Cells by Heregulin ?-1 Depends on the MEK and p38 MAP Kinase Pathway

Heregulin (HRG) β1 signaling promotes scattering of MCF7 cells by inducing breakdown of adherens and tight junctions. Here, we show that stimulation with HRG-β1 causes the F-actin backbone of junctions to destabilize prior to the loss of adherent proteins and scattering of the cells. The adherent proteins dissociate and translocate from cell–cell junctions to the cytosol. Moreover, using inhibitors we show that the MEK1 pathway is required for the disappearance of F-actin from junctions and p38 MAP kinase activity is essential for scattering of the cells. Upon treatment with a p38 MAP kinase inhibitor, adherens junction complexes immediately reassemble, most likely in the cytoplasm, and move to the plasma membrane in cells dissociated by HRG-β1 stimulation. Subsequently, tight junction complexes form, most likely in the cytoplasm, and move to the plasma membrane. Thus, the p38 MAP kinase inhibitor causes a re-aggregation of scattered cells, even in the presence of HRG-β1. These results suggest that p38 MAP kinase signaling to adherens junction proteins regulates cell aggregation, providing a novel understanding of the regulation of cell–cell adhesion.     

p38α MAP kinase phosphorylates RCAN1 and regulates its interaction with calcineurin

RCAN1, also known as DSCR1, is an endogenous regulator of calcineurin, a serine/threonine protein phosphatase that plays a critical role in many physiological processes. In this report, we demonstrate that p38?? MAP kinase can phosphorylate RCAN1 at multiple sites in vitro and show that phospho-RCAN1 is a good protein substrate for calcineurin. In addition, we found that unphosphorylated RCAN1 noncompetitively inhibits calcineurin protein phosphatase activity and that the phosphorylation of RCAN1 by p38?? MAP kinase decreases the binding affinity of RCAN1 for calcineurin. These findings reveal the molecular mechanism by which p38?? MAP kinase regulates the function of RCAN1/calcineurin through phosphorylation.     

Casein Kinase 1α Regulates an MDMX Intramolecular Interaction To Stimulate p53 Binding

MDMX is an important regulator of p53 during embryonic development and malignant transformation. Previous studies showed that casein kinase 1α (CK1α) stably associates with MDMX, stimulates MDMX-p53 binding, and cooperates with MDMX to inactivate p53. However, the mechanism by which CK1α stimulates MDMX-p53 interaction remains unknown. Here, we present evidence that p53 binding by the MDMX N-terminal domain is inhibited by the central acidic region through an intramolecular interaction that competes for the p53 binding pocket. CK1α binding to the MDMX central domain and phosphorylation of S289 disrupts the intramolecular interaction, allowing the N terminus to bind p53 with increased affinity. After DNA damage, the MDMX-CK1α complex is disrupted by Chk2-mediated phosphorylation of MDMX at S367, leading to reduced MDMX-p53 binding. Therefore, CK1α is an important functional partner of MDMX. DNA damage activates p53 in part by disrupting CK1α-MDMX interaction and reducing MDMX-p53 binding affinity.     

Design and synthesis of piperazine-indole p38α MAP kinase inhibitors with improved pharmacokinetic profiles

Derivatives of the 4-fluorobenzyl dimethylpiperazine-indole class of p38α MAP kinase inhibitors are described. Biological evaluation of these compounds focused on maintaining activity while improving pharmacokinetic (PK) properties. Improved properties were observed for structures bearing substitutions on the benzylic methylene.     

Homo-oligomerization and Activation of AMP-activated Protein Kinase Are Mediated by the Kinase Domain ��G-Helix

AMP-activated protein kinase (AMPK) is a heterotrimeric complex playing a crucial role in maintaining cellular energy homeostasis. Recently, homodimerization of mammalian AMPK and yeast ortholog SNF1 was shown by us and others. In SNF1, it involved specific hydrophobic residues in the kinase domain αG-helix. Mutation of the corresponding AMPK α-subunit residues (Val-219 and Phe-223) to glutamate reduced the tendency of the kinase to form higher order homo-oligomers, as was determined by the following three independent techniques in vitro: (i) small angle x-ray scattering, (ii) surface plasmon resonance spectroscopy, and (iii) two-dimensional blue native/SDS-PAGE. Recombinant protein as well as AMPK in cell lysates of primary cells revealed distinct complexes of various sizes. In particular, the assembly of very high molecular mass complexes was dependent on both the αG-helix-mediated hydrophobic interactions and kinase activation. In vitro and when overexpressed in double knock-out (α1^−/−, α2^−/−) mouse embryonic fibroblast cells, activation of mutant AMPK was impaired, indicating a critical role of the αG-helix residues for AMPK activation via its upstream kinases. Also inactivation by protein phosphatase 2Cα was affected in mutant AMPK. Importantly, activation of mutant AMPK by LKB1 was restored by exchanging the corresponding and conserved hydrophobic αG-helix residues of LKB1 (Ile-260 and Phe-264) to positively charged amino acids. These mutations functionally rescued LKB1-dependent activation of mutant AMPK in vitro and in cell culture. Our data suggest a physiological role for the hydrophobic αG-helix residues in homo-oligomerization of heterotrimers and cellular interactions, in particular with upstream kinases, indicating an additional level of AMPK regulation.The maintenance of energy homeostasis is a basic requirement of all living organisms. The AMP-activated protein kinase (AMPK)² is crucially involved in this essential process by playing a central role in sensing and regulating energy metabolism on the cellular and whole body level (1–6). AMPK is also participating in several signaling pathways associated with cancer and metabolic diseases, like type 2 diabetes mellitus, obesity, and other metabolic disorders (7–9).Mammalian AMPK belongs to a highly conserved family of serine/threonine protein kinases with homologs found in all eukaryotic organisms examined (1, 3, 10). Its heterotrimeric structure includes a catalytic α-subunit and regulatory β- and γ-subunits. These subunits exist in different isoforms (α1, α2, β1, β2, γ1, γ2, and γ3) and splice variants (for γ2 and γ3) and can thus assemble to a broad variety of heterotrimeric isoform combinations. The α- and β-subunits possess multiple autophosphorylation sites, which have been implicated in regulation of subcellular localization and kinase activation (11–15). The most critical step of AMPK activation, however, is phosphorylation of Thr-172 within the activation segment of the α-subunit kinase domain. At least two AMPK upstream kinases (AMPKKs) have been identified so far, namely the tumor suppressor kinase LKB1 in complex with MO25 and STRAD (16) and Ca²⁺/calmodulin-dependent protein kinase kinase-2 (CamKK2) (17). Furthermore, the transforming growth factor-β-activated kinase 1 was also shown to activate AMPK using a variety of in vitro approaches (18), but the physiological relevance of these findings remains unclear. Besides direct phosphorylation of Thr-172, AMPK activity is stimulated by the allosteric activator AMP, which can bind to two Bateman domains formed by two pairs of CBS domains within the γ-subunit (19–22). Hereby bound AMP not only allosterically stimulates AMPK but also protects Thr-172 from dephosphorylation by protein phosphatase 2Cα (PP2Cα) and thus hinders inactivation of the kinase (19, 22, 23). Consequently, on the cellular level, AMPK is activated upon metabolic stress increasing the AMP/ATP ratio. Furthermore, AMPK activation can also be induced by several chemical compounds, like nucleoside 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (24) and the anti-diabetic drug Metformin (25–28). In addition, the small molecule compound A-769662 was recently developed as a direct allosteric activator of AMPK (29, 30).Previous work in our groups proposed a model of AMPK regulation by AMP, which incorporates the major functional features and the latest structural information (31). The latter mainly included truncated core complexes of AMPK from different species (32–35). Further valuable structural information is provided by the x-ray structures of the isolated catalytic domains, in particular of the human AMPK α2-subunit (Protein Data Bank code 2H6D) and its yeast ortholog SNF1 (36, 37). The kinase domain of SNF1 is capable of forming homodimers in the protein crystal, as well as in vitro in solution, in a unique way, which has not been observed previously in any other kinase (36). The dimer interface is predominantly formed by hydrophobic interactions of the loop-αG region, also known as subdomain X situated on the large kinase lobe (36, 38, 39), and it mainly involves Ile-257 and Phe-261. Because the T-loop activation segment was buried within the dimer interface, it was suggested that the dimeric state of the SNF1 catalytic domain represents the inactive form of the kinase. Intriguingly, it was shown in our groups by small angle x-ray scattering that AMPK self-organizes in a concentration-dependent manner to form homo-oligomers in solution (31). However, the interface responsible for oligomerization of the AMPK heterotrimer has remained elusive.Here we further investigate the distinct oligomeric states of the AMPK heterotrimer and suggest a possible regulatory function for this process. Most importantly, we provide conclusive evidence for participation of αG-helix residues in the recognition of AMPK by its upstream kinases LKB1 and CamKK2.     

p38α MAP kinase dimers with swapped activation segments and a novel catalytic loop conformation

Many protein kinase functions, including autophosphorylation in trans, require dimerization, possibly by activation segment exchange. Such dimers have been reported for a few autophosphorylating protein kinases, but not for mitogen-activated protein kinases (MAPKs). Activation of MAPKs proceeds not only via the well-characterized action of dual T/Y specificity MAPK kinases, phosphorylating both residues of the MAPK TxY activation loop motif, but also via a noncanonical activation pathway triggered by phosphorylation at Tyr323 and homodimerization. Here, we report the 2. 7-Å-resolution structure of p38α MAPK from Salmo salar in a novel domain-swapped homodimeric form. The tyrosines of the conserved sequence YxAPE anchor the swapped activation segments in a configuration suitable for autophosphorylation in trans and provide a model for the noncanonical pathway. In the dimer, a structural unit containing Tyr323 is formed at a dimerization contact region that stabilizes the HRD catalytic loop in a unique inactive geometry. This feature is consistent with the requirement of Tyr323 phosphorylation for the initiation of the noncanonical pathway. Despite the interacting surface of more than 2600 Å², the dimer is not obligate, as gel filtration shows the dimerization to occur only at relatively high concentrations. The transition from monomer to dimer involves a relatively simple hinged displacement of helix EF and adjacent residues. Thus, dimer formation is likely to be transient, compatible with functional requirements for autophosphorylation, allowing further modulation, for example, by scaffolding mechanisms.     

Synthesis and biological activity of 2H-quinolizin-2-one based p38α MAP kinase inhibitors

The development and synthesis of potent p38α MAP kinase inhibitors containing a 2H-quinolizin-2-one platform is described. Evolution of the 2H-quinolizin-2-one series from an early lead to solving off target activity and pharmacokinetic issues is also discussed.     

p38α MAP Kinase Mediates Hypoxia-Induced Motor Neuron Cell Death: A Potential Target of Minocycline’s Neuroprotective Action

Hypoxia-ischemia (HI) may play a significant role in motor neuron death associated with the pathology of spinal cord injury and, perhaps, amyotrophic lateral sclerosis. The present study employs an in vitro model of HI to investigate the role of a stress kinase pathway, i.e., p38 MAP kinase, in cell death signaling in a motor neuron cell line, i.e., NSC34, subjected to oxygen-glucose deprivation (OGD). Although the neurons were essentially tolerant to either hypoxia (0.2% O₂) or low glucose (1 mM) alone, more than 60% of them died in response to combined low oxygen and low-glucose exposure. Minocycline, a semi-synthetic tetracycline known for its neuroprotective effects in models of neurodegeneration, afforded substantial (∼50%) protection against hypoxic cell death, assessed by lactate dehydrogenase release and flow cytometry, while suppressing OGD-induced p38 MAP kinase activation. An inhibitor of p38 kinase, SB203580, as well as siRNA-mediated down-regulation of p38 kinase elicited an almost complete blockade of OGD-induced cell death. The use of p38 isoform-specific siRNAs further revealed preferential involvement of the α over the β isoform of p38 MAP kinase in hypoxic neuronal cell death in our model.     

Lattice stabilization and enhanced diffraction in human p38α crystals by protein engineering

Mitogen-activated protein (MAP) kinase p38α is activated in response to environmental stress and cytokines, and plays a significant role in inflammatory responses. For these reasons, it is an important target for the treatment of a wide range of inflammatory and autoimmune diseases. The crystals of p38α that we obtained by published procedures were usually small, quite mosaic, and difficult to reproduce and thus posed a difficulty for the intensive high-resolution studies required for a structure-guided drug discovery approach. Based on crystallographic and biochemical evidences, we prepared a single point mutation of a surface cysteine (C162S) and found that it prevents aggregation and improves the homogeneity and stability of the enzyme. This mutation also facilitates the crystallization process and increases the diffracting power of p38α crystals. Surprisingly, we found that the mutation induces a change in the conformation of a nearby surface loop resulting in stronger lattice interactions, consistent with the improved crystal quality. The mutant protein, because of its improved stability and strengthened lattice interactions, thus provides a significantly improved reagent for use in structure-based drug design for this important disease target.     

Structural basis of p38α regulation by hematopoietic tyrosine phosphatase

MAP kinases regulate essential cellular events, including cell growth, differentiation and inflammation. The solution structure of a complete MAPK-MAPK-regulatory protein complex, p38α-HePTP, was determined, enabling a comprehensive investigation of the molecular basis of specificity and fidelity in MAPK regulation. Structure determination was achieved by combining NMR spectroscopy and small-angle X-ray scattering data with a new ensemble calculation-refinement procedure. We identified 25 residues outside of the HePTP kinase interaction motif necessary for p38α recognition. The complex adopts an extended conformation in solution and rarely samples the conformation necessary for kinase deactivation. Complex formation also does not affect the N-terminal lobe, the activation loop of p38α or the catalytic domain of HePTP. Together, these results show how the downstream tyrosine phosphatase HePTP regulates p38α and provide for fundamentally new insights into MAPK regulation and specificity.     

Efficient adult skeletal muscle regeneration in mice deficient in p38β, p38γ and p38δ MAP kinases

Adult skeletal muscle is a very stable tissue containing a small population of myofiber-associated quiescent satellite cells compared with late embryonic/neonatal skeletal muscle, which contains highly proliferating myoblasts and small actively growing myofibers, suggesting that specific regulatory pathways may control myogenesis at distinct developmental stages. The p38 MAPK signaling pathway is central for myogenesis, based on studies using immortalized and neonatal primary myoblasts in vitro. However, the contribution of this pathway to adult myogenesis has never been investigated. Four p38 isoforms (p38α, p38β, p38γ and p38δ) exist in mammalian cells, being p38α and p38γ the most abundantly expressed isoforms in adult skeletal muscle. Given the embryonic/neonatal lethality of p38α-deficient mice, here we investigate the relative contribution of p38β, p38γ and p38δ to adult myogenesis. Regeneration and myofiber growth of adult muscle proceeds with similar efficiency in mice lacking p38β, p38γ and p38δ as in wild-type control mice. In agreement with this, there is no difference in adult satellite cell behavior in vitro among the different genotypes. Importantly, the pattern of p38 activation (ascribed to p38α) remains unperturbed during satellite myogenesis in vitro and adult muscle regeneration in wild type and p38β-, p38γ- and p38δ-deficient mice, rendering p38α as the essential p38 isoform sustaining adult myogenesis. This study constitutes the first analysis addressing the functionality of p38β, p38γ and p38δ in satellite cell-dependent adult muscle regeneration and growth.