No abstract

Apoptosis signal-regulating kinases (ASK1-3) are apical kinases of the p38 and JNK MAP kinase pathways. They are activated by diverse stress stimuli, including reactive oxygen species, cytokines, and osmotic stress; however, a molecular understanding of how ASK proteins are controlled remains obscure. Here, we report a biochemical analysis of the ASK1 kinase domain in conjunction with its N-terminal thioredoxin-binding domain, along with a central regulatory region that links the two. We show that in solution the central regulatory region mediates a compact arrangement of the kinase and thioredoxin-binding domains and the central regulatory region actively primes MKK6, a key ASK1 substrate, for phosphorylation. The crystal structure of the central regulatory region reveals an unusually compact tetratricopeptide repeat (TPR) region capped by a cryptic pleckstrin homology domain. Biochemical assays show that both a conserved surface on the pleckstrin homology domain and an intact TPR region are required for ASK1 activity. We propose a model in which the central regulatory region promotes ASK1 activity via its pleckstrin homology domain but also facilitates ASK1 autoinhibition by bringing the thioredoxin-binding and kinase domains into close proximity. Such an architecture provides a mechanism for control of ASK-type kinases by diverse activators and inhibitors and demonstrates an unexpected level of autoregulatory scaffolding in mammalian stress-activated MAP kinase signaling.itogen-activated protein (MAP) kinase cascades transmit signals from membrane-associated receptors to intracellular targets to effect changes in cellular behavior. They form a hierarchical system in which activated upstream kinases (MAP3Ks) phosphorylate intermediate MAP kinase kinases (MAP2Ks), which in turn phosphorylate terminal MAP kinases, primarily ERK, p38, and JNK and their isoforms (1). Extensive studies have focused on the activation of RAS-RAF-MEK upstream in the ERK pathway and provided fertile ground for the discovery of new therapeutics (2). In contrast to the ERK pathway, which primarily promotes cellular proliferation, JNK and p38 phosphorylate a range of substrates to promote inflammation and cell death (1, 3). In addition, cross-regulation among the p38, JNK, and ERK pathways is important for the efficacy of various cancer therapies that are in use or in development (4, 5). Molecular details on the more diverse upstream regulation of the p38 and JNK pathways are currently less clear, however.Apoptosis signal-regulating kinases (ASK1-3) are MAP3Ks that trigger cellular responses to redox stress and inflammatory cytokines (6, 7) and play vital roles in innate immunity and viral infection (8-11). When activated, ASK1-3 activate JNK and p38 via phosphorylation of MAP2Ks (MKK3/4/6/7) (12). The key initiator role of ASK1-3 in this pathway means that either too much or too little ASK activity can have pathological effects. For instance, inhibiting ASK1 is beneficial against gastric cancer (13,14), but inactivating mutations in ASK1...

Despite continuing progress in kinesin enzyme mechanochemistry and emerging understanding of the cargo recognition machinery, it is not known how these functions are coupled and controlled by the -helical coiled coils encoded by a large component of kinesin protein sequences. Here, we combine computational structure prediction with single-particle negative-stain electron microscopy to reveal the coiled-coil architecture of heterotetrameric kinesin-1 in its compact state. An unusual flexion in the scaffold enables folding of the complex, bringing the kinesin heavy chain-light chain interface into close apposition with a tetrameric assembly formed from the region of the molecule previously assumed to be the folding hinge. This framework for autoinhibition is required to uncover how engagement of cargo and other regulatory factors drives kinesin-1 activation.

The cargo-binding capabilities of cytoskeletal motor proteins have expanded during evolution through both gene duplication and alternative splicing. For the light chains of the kinesin-1 family of microtubule motors, this has resulted in an array of carboxyl-terminal domain sequences of unknown molecular function. Here, combining phylogenetic analyses with biophysical, biochemical, and cell biology approaches, we identify a highly conserved membrane-induced curvature-sensitive amphipathic helix within this region of a subset of long kinesin light-chain paralogs and splice isoforms. This helix mediates the direct binding of kinesin-1 to lipid membranes. Membrane binding requires specific anionic phospholipids, and it contributes to kinesin-1–dependent lysosome positioning, a canonical activity that, until now, has been attributed exclusively the recognition of organelle-associated cargo adaptor proteins. This leads us to propose a protein-lipid coincidence detection framework for kinesin-1–mediated organelle transport.

ERK1 and ERK2 (ERK1/2) are the primary effector kinases of the RAS-RAF-MEK-ERK signaling pathway. A variety of substrates and regulatory partners associate with ERK1/2 through distinct D-peptide- and DEF-docking sites on their kinase domains. While understanding of D-peptides that bind to ERK1/2 has become increasingly clear over the last decade, only more recently have structures of proteins interacting with other binding sites on ERK1/2 become available. PEA-15 is a 130-residue ERK1/2 regulator that engages both the D-peptide- and DEF-docking sites of ERK kinases, and directly sequesters the ERK2 activation loop in various different phosphorylation states. Here we describe the methods used to derive crystallization-grade complexes of ERK2-PEA-15, which may also be adapted for other regulators that associate with the activation loop of ERK1/2.

The cargo-binding capabilities of cytoskeletal motor proteins have expanded during evolution through both gene duplication and alternative splicing. For the light chains of the kinesin-1 family of microtubule motors, this has resulted in an array of carboxy-terminal domain sequences of unknown molecular function. Here, combining phylogenetic analyses with biophysical, biochemical and cell biology approaches we identify a highly conserved membrane-induced curvature-sensitive amphipathic helix within this region of a newly defined subset of long kinesin light chain paralogues and splice isoforms. This helix mediates the direct binding of kinesin-1 to lipid membranes. Membrane binding requires specific anionic phospholipids and is important for kinesin-1 dependent lysosome positioning, a canonical activity that until now has been attributed exclusively the recognition of organelle-associated cargo adaptor proteins. This leads us to propose a new protein-lipid coincidence detection framework for kinesin-1 mediated organelle transport.

Despite continuing progress in kinesin enzyme mechanochemistry and emerging understanding of the cargo recognition machinery, it is not known how these functions are coupled and controlled by the alpha-helical coiled coils encoded by a large component of kinesin protein sequences. Here, we combine computational structure prediction with single-particle negative stain electron microscopy to reveal the coiled-coil architecture of heterotetrameric kinesin-1, in its compact state. An unusual flexion in the scaffold enables folding of the complex, bringing the kinesin heavy chain-light chain interface into close apposition with a tetrameric assembly formed from the region of the molecule previously assumed to be the folding hinge. This framework for autoinhibition is required to uncover how engagement of cargo and other regulatory factors drive kinesin-1 activation.Summary statementIntegration of computational structure prediction with electron microscopy reveals the coiled-coil architecture of the autoinhibited compact conformer of the microtubule motor, kinesin-1.

No abstract