The structure of phosphorylated P38γ is monomeric and reveals a conserved activation-loop conformation

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 ␣G-helix. Mutation of the corresponding AMPK ␣-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 ␣G-helix-mediated hydrophobic interactions and kinase activation. In vitro and when overexpressed in double knock-out (␣1) mouse embryonic fibroblast cells, activation of mutant AMPK was impaired, indicating a critical role of the ␣G-helix residues for AMPK activation via its upstream kinases. Also inactivation by protein phosphatase 2C␣ was affected in mutant AMPK. Importantly, activation of mutant AMPK by LKB1 was restored by exchanging the corresponding and conserved hydrophobic ␣G-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 ␣G-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 ␣-subunit and regulatory ␤-and ␥-subunits. These subunits exist in different isoforms (␣1, ␣2, ␤1, ␤2, ␥1, ␥2, and ␥3) and splice variants (for ␥2 and ␥3) and can thus assemble to a broad variety of heterotrimeric isoform combinations. The ␣-and ␤-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 ␣-subunit kinase domain. At least two AMPK upstream kinases (AMPKKs) have been i...

Section: Discussionmentioning

confidence: 99%

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

Scholz

Suter

Weimann

et al. 2009

“…Furthermore, we describe preliminary results that p38␥ becomes active using similar mutations as well. In addition, we present a structural analysis of the activating mutants based on the currently available three-dimensional models of p38 and ERK2 (12,(41)(42)(43)(44) that provides a plausible mechanism that underlies the activation of the p38 mutants.…”

Section: Mitogen-activated Protein (Map)mentioning

confidence: 99%

“…We conducted a comprehensive structural analysis using the available three-dimensional structural data on MAP kinases in order to propose a mechanism that promotes activation of the mutants. p38 (42)(43)(44) and ERK2 (12, 41) share a similar topology consisting of two kinase lobes (N-terminal and C-terminal domains) that create the catalytic groove in their interface. The MAP kinases are also characterized by an insertion in the C domain and an extension in the C terminus (located in the N domain).…”

Section: P38 Activation May Occur Through Destabilization Of a Hydropmentioning

confidence: 99%

Active Mutants of the Human p38α Mitogen-activated Protein Kinase

Diskin

Askari²,

Capone³

et al. 2004

125

MitogenD176A/F327S ) mutants acquired high intrinsic activity independent of any upstream regulation and reached levels of 10 and 25%, respectively, in reference to the dually phosphorylated wild type p38␣. The active p38 mutants have retained high specificity toward p38 substrates and were inhibited by the specific p38 inhibitors SB-203580 and PD-169316. We also show that similar mutations can render p38␥ active as well. Based on the available structures of p38 and ERK2, we have analyzed the p38 mutants and identified a hydrophobic core stabilized by three aromatic residues, Tyr-69, Phe-327, and Trp-337, in the vicinity of the L16 loop region. Upon activation, a segment of the L16 loop, including Phe-327, becomes disordered. Structural analysis suggests that the active p38 mutants emulate the conformational changes imposed naturally by dual phosphorylation, namely, destabilization of the hydrophobic core. Essentially, the hydrophobic core is an inherent stabilizer that maintains low basal activity level in unphosphorylated p38.

“…4A). Furthermore, this region displays considerable sequence and structural diversity among MAPK family members (23,25,26) yet is highly conserved between ERK1 and ERK2 (Fig. 4B).…”

Section: Mutations Of Pea-15 That Block Erk1/2 Binding Abrogatementioning

confidence: 99%

PEA-15 Binding to ERK1/2 MAPKs Is Required for Its Modulation of Integrin Activation

Chou

Hill

Hsieh

et al. 2003

Activation of Raf-1 suppresses integrin activation, potentially through the activation of extracellular signalregulated kinases 1 and 2 (ERK1/2). However, bulk ERK1/2 activation does not correlate with suppression. PEA-15 reverses suppression of integrin activation and binds ERK1/2. Here we report that PEA-15 reversal of integrin suppression depends on its capacity to bind ERK1/2, indicating that ERK1/2 function is indeed required for suppression. Mutations in either the death effector domain or C-terminal tail of PEA-15 that block ERK1/2 binding abrogated the reversal of integrin suppression. Furthermore, we used ERK/p38 chimeras and site-directed mutagenesis to identify ERK1/2 residues required for binding PEA-15. Mutations of residues that precede the ␣G helix and within the mitogenactivated protein kinase insert blocked ERK2 binding to PEA-15, but not activation of ERK2. These ERK2 mutants blocked the ability of PEA-15 to reverse suppression of integrin activation. Thus, PEA-15 regulation of integrin activation depends on its binding to ERK1/2. To directly test the role of ERK1/2 localization in suppression, we enforced membrane association of ERK1 and 2 by joining a membrane-targeting CAAX box sequence to them. Both ERK1-CAAX and ERK2-CAAX were membrane-localized and suppressed integrin activation. In contrast to suppression by membrane-targeted Raf-CAAX, suppression by ERK1/2-CAAX was not reversed by PEA-15. Thus, ERK1/2 are the Raf effectors for suppression of integrin activation, and PEA-15 reverses suppression by binding ERK1/2.