Structure of mitogen-activated protein kinase kinase 1 in the DFG-out conformation

Eukaryotic protein kinases contain an Asp-Phe-Gly (DFG) motif, the conformation of which is involved in controlling the catalytic activity, at the N-terminus of the activation segment. The motif can be switched between active-state (DFG-in) and inactive-state (DFG-out) conformations: however, the mechanism of conformational change is poorly understood, partly because there are few reports of the DFG-out conformation. Here, a novel crystal structure of nonphosphorylated human mitogen-activated protein kinase kinase 1 (MEK1; amino acids 38–381) complexed with ATP-γS is reported in which MEK1 adopts the DFG-out conformation. The crystal structure revealed that the structural elements (the αC helix and HRD motif) surrounding the active site are involved in the formation/stabilization of the DFG-out conformation. The ATP-γS molecule was bound to the canonical ATP-binding site in a different binding mode that has never been found in previously determined crystal structures of MEK1. This novel ATP-γS binding mode provides a starting point for the design of high-affinity inhibitors of nonphosphorylated inactive MEK1 that adopts the DFG-out conformation.

BRAFnon-V600E more frequently co-occurs with IDH1/2 mutations in adult patients with gliomas than in patients harboring BRAFV600E but without a survival advantage

Background The effects of BRAFnon-V600E and BRAFV600E on the outcomes and the molecular characteristics of adult glioma patients are unknown and need to be explored, although BRAFV600E has been extensively studied in pediatric glioma. Methods Co-occurring mutations and copy number alterations of associated genes in the MAPK and p53 pathways were investigated using data from The Cancer Genome Atlas (TCGA) public database retrieved by cBioPortal. The prognosis of available adult glioma cohorts with BRAFV600E and BRAFnon-V600E mutations were also investigated. Results Ninety patients with BRAFV600E or BRAFnon-V600E were enrolled in this study, and data from 52 nonredundant patients were investigated. Glioblastoma multiform was the most common cancer type, with BRAFnon-V600E and BRAFV600E. TP53 (56.00% vs. 7.41%), IDH1/2 (36.00% vs. 3.70%), and ATRX (32.00% vs. 7.41%) exhibited more mutations in BRAFnon-V600E than in BRAFV600E, and TP53 was an independent risk factor (56.00% vs. 7.41%). Both BRAFnon-V600E and BRAFV600E frequently overlapped with CDKN2A/2B homozygous deletions (HDs), but there was no significant difference. Survival analysis showed no difference between the BRAFnon-V600E and BRAFV600E cohorts, even after excluding the survival benefit of IDH1/2 mutations and considering the BRAFnon-V600E mutations in the glycine-rich loop (G-loop) and in the activation segment. The estimated mean survival of patients with BRAFnon-V600E & IDH1/2WT with mutations in the G-loop groups was the shortest. Conclusions BRAFnon-V600E exhibited a stronger association with IDH1/2 mutations than BRAFV600E, but no survival advantage was found.

Bora phosphorylation substitutes in trans for T-loop phosphorylation in Aurora A to promote mitotic entry

Polo-like kinase 1 (Plk1) is instrumental for mitotic entry and progression. Plk1 is activated by phosphorylation on a conserved residue Thr210 in its activation segment by the Aurora A kinase (AURKA), a reaction that critically requires the co-factor Bora phosphorylated by a CyclinA/B-Cdk1 kinase. Here we show that phospho-Bora is a direct activator of AURKA kinase activity. We localize the key determinants of phospho-Bora function to a 100 amino acid region encompassing two short Tpx2-like motifs and a phosphoSerine-Proline motif at Serine 112, through which Bora binds AURKA. The latter substitutes in trans for the Thr288 phospho-regulatory site of AURKA, which is essential for an active conformation of the kinase domain. We demonstrate the importance of these determinants for Bora function in mitotic entry both in Xenopus egg extracts and in human cells. Our findings unveil the activation mechanism of AURKA that is critical for mitotic entry.

BRAFnon-V600E more frequently co-occurs with IDH1/2 mutation in adult patients with gliomas than patients harboring BRAFV600E, but without survival advantage

Background The effect of BRAFnon−V600E and BRAFV600E on the outcome and the molecular characteristics in adult glioma patients is unknown and needs to be explored, although BRAFV600E has been extensively studied in pediatric glioma. Methods Co-occurring mutations and copy number alterations of associated genes in the MAPK and p53 pathways were retrieved and investigated using the cBioPortal. The prognosis of available adult glioma cohorts with BRAFV600E and BRAFnon−V600E mutations was also investigated. Results Glioblastoma multiform was the most common cancer type with BRAF non−V600E and BRAFV600E. TP53 (56.00% vs. 7.41%), IDH1/2 (36.00% vs. 3.70%), and ATRX (32.00% vs. 7.41%) exhibited more mutations in BRAFnon−V600E than in BRAFV600E, and TP53 was the independent risk factor (56.00% vs. 7.41%). Both BRAFnon−V600E and BRAFV600E frequently overlapped with CDKN2A/2B homozygous deletions (HD), whereas there was no significant difference. Survival analysis showed no difference between BRAF non−V600E and BRAFV600E cohorts, even excluded the effects of IDH1/2 mutations, and concerned the BRAFnon−V600E mutations in the glycine-rich loop (G-loop) and in the activation segment. The estimated mean survival of BRAFnon−V600E and IDH1/2WT with mutations in the G-loop groups was the shortest. Conclusions BRAF non−V600E exhibited a higher association with IDH1/2 mutation than BRAFV600E, but no survival advantage was found.

Aurora A regulation by reversible cysteine oxidation reveals evolutionarily conserved redox control of Ser/Thr protein kinase activity

Reactive oxygen species (ROS) are physiological mediators of cellular signaling and play potentially damaging roles in human diseases. In this study, we found that the catalytic activity of the Ser/Thr kinase Aurora A was inhibited by the oxidation of a conserved cysteine residue (Cys290) that lies adjacent to Thr288, a critical phosphorylation site in the activation segment. Cys is present at the equivalent position in ~100 human Ser/Thr kinases, a residue that we found was important not only for the activity of human Aurora A but also for that of fission yeast MAPK-activated kinase (Srk1) and PKA (Pka1). Moreover, the presence of this conserved Cys predicted biochemical redox sensitivity among a cohort of human CAMK, AGC, and AGC-like kinases. Thus, we predict that redox modulation of the conserved Cys290 of Aurora A may be an underappreciated regulatory mechanism that is widespread in eukaryotic Ser/Thr kinases. Given the key biological roles of these enzymes, these findings have implications for understanding physiological and pathological responses to ROS and highlight the importance of protein kinase regulation through multivalent modification of the activation segment.

Tyr198 is the Essential Autophosphorylation Site for STK16 Localization and Kinase Activity

STK16, reported as a Golgi localized serine/threonine kinase, has been shown to participate in multiple cellular processes, including the TGF-β signaling pathway, TGN protein secretion and sorting, as well as cell cycle and Golgi assembly regulation. However, the mechanisms of the regulation of its kinase activity remain underexplored. It was known that STK16 is autophosphorylated at Thr185, Ser197, and Tyr198 of the activation segment in its kinase domain. We found that STK16 localizes to the cell membrane and the Golgi throughout the cell cycle, but mutations in the auto-phosphorylation sites not only alter its subcellular localization but also affect its kinase activity. In particular, the Tyr198 mutation alone significantly reduced the kinase activity of STK16, abolished its Golgi and membrane localization, and affected the cell cycle progression. This study demonstrates that a single site autophosphorylation of STK16 could affect its localization and function, which provides insights into the molecular regulatory mechanism of STK16’s kinase activity.

Checkpoint signaling and error correction require regulation of the MPS1 T-loop by PP2A-B56

During mitosis, the formation of microtubule–kinetochore attachments is monitored by the serine/threonine kinase monopolar spindle 1 (MPS1). MPS1 is recruited to unattached kinetochores where it phosphorylates KNL1, BUB1, and MAD1 to initiate the spindle assembly checkpoint. This arrests the cell cycle until all kinetochores have been stably captured by microtubules. MPS1 also contributes to the error correction process rectifying incorrect kinetochore attachments. MPS1 activity at kinetochores requires autophosphorylation at multiple sites including threonine 676 in the activation segment or “T-loop.” We now demonstrate that the BUBR1-bound pool of PP2A-B56 regulates MPS1 T-loop autophosphorylation and hence activation status in mammalian cells. Overriding this regulation using phosphomimetic mutations in the MPS1 T-loop to generate a constitutively active kinase results in a prolonged mitotic arrest with continuous turnover of microtubule–kinetochore attachments. Dynamic regulation of MPS1 catalytic activity by kinetochore-localized PP2A-B56 is thus critical for controlled MPS1 activity and timely cell cycle progression.

MARK4 protein can explore the active-like conformations in its non-phosphorylated state

Microtubule affinity-regulating kinase 4 (MARK4) is a Ser/Thr protein kinase, best known for its role in phosphorylating microtubule associated proteins, causing their detachment from microtubules. In the current study, the non-phosphorylated conformation of the activation loop was modeled in a structure representing the enzymatically inactive form of this protein, and its dynamics were evaluated through a 100 ns initial all-atom simulation, which was prolonged by another 2 μs. Although the activation loop was folding on itself and was leaning toward ATP site in the initial modeled structure, soon after the initiating the simulation, this loop stretched away from the ATP binding site and stably settled in its new position for the rest of simulation time. A network of hydrogen bonds, mainly between the activation segment residues, αC-helix and the catalytic loop reinforced this conformation. Interestingly, several features of active kinase conformation such as formation of R-spine, Glu106-Lys88 salt-bridge, and DFG-In motif were observed during a considerable number of trajectory frames. However, they were not sustainably established during the simulation time, except for the DFG-In motif. Consequently, this study introduces a stable conformation of the non-phosphorylated form of MARK4 protein with a partially stretched activation loop conformation as well as partial formation of R-spine, closely resembling the active kinase.

Checkpoint signaling and error correction require regulation of the MPS1 T-loop by PP2A-B56

During mitosis, the formation of microtubule-kinetochore attachments is monitored by the serine/threonine kinase Mono-Polar Spindle 1 (MPS1). MPS1 is recruited to unattached kinetochores where it phosphorylates KNL1, BUB1 and MAD1 to initiate the spindle checkpoint. This arrests the cell cycle until all kinetochores have been stably captured by microtubules. MPS1 also contributes to the error correction process rectifying incorrect kinetochore attachments. MPS1 activity at kinetochores requires auto-phosphorylation at multiple sites including T676 in the activation segment or “T-loop”. We now demonstrate that a BUBR1-bound pool of PP2A-B56 regulates MPS1 T-loop autophosphorylation and hence activation status in mammalian cells. Overriding this regulation using phospho-mimetic mutations in the MPS1 T-loop to generate a constitutively active kinase results in a prolonged mitotic arrest with continuous turn-over of microtubule-kinetochore attachments. Dynamic regulation of MPS1 catalytic activity by kinetochore-localized PP2A-B56 is thus critical for controlled MPS1 activity and timely cell cycle progression.