Characterization of the activation domain of the Rad53 checkpoint kinase

Rad53 protein, the yeast orthologue of the human checkpoint kinase Chk2, presents two highly conserved phosphorylatable threonine residues (T354 and T358) in the activation domain, whose phosphorylation is critical to allow the activation of the kinase. In this study we found that Rad53 protein variants in which alanine and/or aspartate replace the threonine residues 354 and/or 358 do not retain kinase activity and do not undergo auto-phosphorylation, leading to defect in the checkpoint response and iper-sensitivity to DNA damage and DNA replication stress agents. Interestingly, we found that the rad53-T358D mutation severely affects the kinase activity and causes accumulation of the S129-phosphorylated isoform of histone H2A, even during an unperturbed cell cycle, thus indicating the accumulation of spontaneous DNA breaks. We further found that the protein level of Sml1, which is the physiological inhibitor of ribonucleotide reductase, remains high during DNA replication in rad53-T358D cells, suggesting that an inadequate pool of dNTPs in checkpoint defective cells causes the accumulation of spontaneous DNA breaks.

In conclusion, our results indicate that phosphorylation of both T354 and T358 residues strongly influences the catalytic activity of Rad53 also in unperturbed cell cycles, and support the notion that Rad53 is essential to preserve genome integrity, by controlling the level of Sml1 and the functionality of ribonucleotide reductase.     

Cdc5 blocks in vivo rad53 activity,but not in situ activity (ISA)

DNA damage promotes the activation of a signal transduction cascade referred to as the DNA damage checkpoint. This pathway initiates with the Mec1/ATR kinase, which then phosphorylates the Rad53/Chk2 kinase. Mec1 phosphorylation of Rad53 is then thought to promote Rad53 autophosphorylation, ultimately leading to a fully active Rad53 molecule that can go on to phosphorylate substrates important for DNA damage resistance. In the absence of DNA repair, this checkpoint is eventually downregulated in a Cdc5-dependent process referred to as checkpoint adaptation. Recently, we showed that overexpression of Cdc5 leads to checkpoint inactivation and loss of the strong electrophoretic shift associated with Rad53 inactivation. Interestingly, this same overexpression did not strongly inhibit Rad53 autophosphorylation activity as measured by the in situ assay (ISA). The ISA involves incubating the re-natured Rad53 protein with γ ³²P labeled ATP after electrophoresis and western blotting. Using a newly identified Rad53 target, we show that despite strong ISA activity, Rad53 does not maintain phosphorylation of this substrate. We hypothesize that, during adaptation, Rad53 may be in a unique state in which it maintains some Mec1 phosphorylation but does not have the auto-phosphorylations required for full activity towards exogenous substrates.Key words: DNA damage, checkpoint, adaptation, CDC5, RAD53, ISA     

The phosphorylation network for efficient activation of the DNA replication checkpoint in fission yeast

Protein phosphorylation is the hallmark of checkpoint activation. Hundreds of targets of checkpoint kinases have been identified recently by genome-wide investigations. However, the complete picture of a phosphorylation network required for activation of a checkpoint pathway has not been available. The DNA replication checkpoint in Schizosaccharomyces pombe contains two major protein kinases, the sensor kinase Rad3 and the effector kinase Cds1, with the latter mediating most of the checkpoint functions. We show here that when DNA replication is arrested, efficient activation of Cds1 requires five phosphorylations that cooperate in a parallel or a sequential manner. Phosphorylation of a threonine residue (Thr(11)) in Cds1 by Rad3 occurs at a basal level in the absence of three other parallel Rad3-dependent phosphorylations on the mediator Mrc1 and Rad9 in the checkpoint clamp complex. However, the three parallel Rad3-dependent phosphorylations are all required for efficient phosphorylation of Thr(11) in Cds1 by Rad3. Phosphorylation of Thr(11) has been shown previously to promote autophosphorylation of Thr(328) in the kinase domain of Cds1, which directly activates the enzyme, leading to full activation of the checkpoint pathway. Interestingly, phosphorylation of Mrc1 by Rad3 does not require the phosphorylation of Rad9, suggesting that activation of the sensor kinase Rad3 in the replication checkpoint of fission yeast may involve a different mechanism.     

Activation segment exchange: a common mechanism of kinase autophosphorylation?

The crystal structure of the kinase domain from human checkpoint kinase 2 (Chk2) has shown, for the first time, the reciprocal exchange of activation segments between two adjacent molecules and provides the molecular basis for understanding the observed mode of Chk2 kinase activation via trans-autophosphorylation. With further examples of activation segment exchanged kinase domains now publicly available (i.e. Ste20-like kinase, Ser/Thr kinase 10 and Death-associated protein kinase 3), we suggest that this phenomenon represents a common mechanism of activation amongst a particular subset of protein kinases, that is, those that are dimeric (either transiently or constitutively), that undergo activation by autophosphorylation and that have activation segment amino acid sequences that do not resemble those of their substrate consensus sequence.     

The Arabidopsis thaliana SERK1 Kinase Domain Spontaneously Refolds to an Active State In Vitro

Auto-phosphorylating kinase activity of plant leucine-rich-repeat receptor-like kinases (LRR-RLK''s) needs to be under tight negative control to avoid unscheduled activation. One way to achieve this would be to keep these kinase domains as intrinsically disordered protein (IDP) during synthesis and transport to its final location. Subsequent folding, which may depend on chaperone activity or presence of interaction partners, is then required for full activation of the kinase domain. Bacterially produced SERK1 kinase domain was previously shown to be an active Ser/Thr kinase. SERK1 is predicted to contain a disordered region in kinase domains X and XI. Here, we show that loss of structure of the SERK1 kinase domain during unfolding is intimately linked to loss of activity. Phosphorylation of the SERK1 kinase domain neither changes its structure nor its stability. Unfolded SERK1 kinase has no autophosphorylation activity and upon removal of denaturant about one half of the protein population spontaneously refolds to an active protein in vitro. Thus, neither chaperones nor interaction partners are required during folding of this protein to its catalytically active state.     

Mechanistic link between PKR dimerization, autophosphorylation, and eIF2alpha substrate recognition

The antiviral protein kinase PKR inhibits protein synthesis by phosphorylating the translation initiation factor eIF2alpha on Ser51. Binding of double-stranded RNA to the regulatory domains of PKR promotes dimerization, autophosphorylation, and the functional activation of the kinase. Herein, we identify mutations that activate PKR in the absence of its regulatory domains and map the mutations to a recently identified dimerization surface on the kinase catalytic domain. Mutations of other residues on this surface block PKR autophosphorylation and eIF2alpha phosphorylation, while mutating Thr446, an autophosphorylation site within the catalytic-domain activation segment, impairs eIF2alpha phosphorylation and viral pseudosubstrate binding. Mutational analysis of catalytic-domain residues preferentially conserved in the eIF2alpha kinase family identifies helix alphaG as critical for the specific recognition of eIF2alpha. We propose an ordered mechanism of PKR activation in which catalytic-domain dimerization triggers Thr446 autophosphorylation and specific eIF2alpha substrate recognition.     

Aurora-A kinase Ser349 phosphorylation is required during Xenopus laevis oocyte maturation

Xenopus laevis Aurora-A is phosphorylated in vivo onto three amino acids: Ser53, Thr295 and Ser349. The activation of the kinase depends on its autophosphorylation on Thr295 within the T-loop. The phosphorylation of Ser53 by still unknown kinase(s) prevents its degradation. The present work focused on the regulation of Aurora-A function via Ser349 phosphorylation. Mutagenesis of Ser349 to alanine (S349A) had few impact in vitro on the capability of the kinase to autophosphorylate as well as on its activity. These data in addition to in gel kinase assays and site-specific proteolytic digestion experiments prove that Ser349 is clearly neither a primary autophosphorylation site, nor an autophosphorylation site depending on the priming phosphorylation of Thr295. Using specific antibodies, we also show that the phosphorylation of Aurora-A Ser349 is a physiological event during Xenopus oocyte maturation triggered by progesterone. A peak of phosphorylation paralleled the decrease of Aurora activity observed between meiosis I and II. In response to progesterone, X. laevis stage VI oocytes microinjected with the Aurora-A S349A mutant proceeded normally to germinal vesicle breakdown (GVBD), but degenerated rapidly soon after. Since phosphorylation of Ser349 is responsible for a decrease in kinase activity, our results suggest that a down-regulation of Aurora-A activity involving Ser349 phosphorylation is required in the process of maturation.     

Mechanism of Dun1 activation by Rad53 phosphorylation in Saccharomyces cerevisiae

Despite extensive studies, the molecular mechanism of DNA damage checkpoint activation remains incompletely understood. To better dissect this mechanism, we developed an activity-based assay for Dun1, a downstream DNA damage check-point kinase in yeast, using its physiological substrate Sml1. Using this assay, we confirmed the genetic basis of Dun1 activation. Rad53 was found to be directly responsible for Dun1 activation. We reconstituted the activation of Dun1 by Rad53 and found that phosphorylation of Thr-380 in the activation loop of Dun1 by Rad53 is responsible for Dun1 activation. Interestingly, phosphorylation of the evolutionarily conserved Thr-354 in the activation loop of Rad53 is also important for the regulation of Rad53 activity. Thus, this conserved mode of activation loop phosphorylation appears to be a general mechanism for the activation of Chk2 family kinases.     

Multiple phosphorylation of Rad9 by CDK is required for DNA damage checkpoint activation

The DNA damage checkpoint controls cell cycle arrest in response to DNA damage, and activation of this checkpoint is in turn cell cycle-regulated. Rad9, the ortholog of mammalian 53BP1, is essential for this checkpoint response and is phosphorylated by the cyclin-dependent kinase (CDK) in the yeast Saccharomyces cerevisiae. Previous studies suggested that the CDK consensus sites of Rad9 are important for its checkpoint activity. However, the precise CDK sites of Rad9 involved have not been determined. Here we show that CDK consensus sites of Rad9 function in parallel to its BRCT domain toward checkpoint activation, analogous to its fission yeast ortholog Crb2. Unlike Crb2, however, mutation of multiple rather than any individual CDK site of Rad9 is required to completely eliminate its checkpoint activity in vivo. Although Dpb11 interacts with CDK-phosphorylated Rad9, we provide evidence showing that elimination of this interaction does not affect DNA damage checkpoint activation in vivo, suggesting that additional pathway(s) exist. Taken together, these findings suggest that the regulation of Rad9 by CDK and the role of Dpb11 in DNA damage checkpoint activation are more complex than previously suggested. We propose that multiple phosphorylation of Rad9 by CDK may provide a more robust system to allow Rad9 to control cell cycle-dependent DNA damage checkpoint activation.     

Aurora A kinase activation: Different means to different ends

Aurora A is a serine/threonine kinase essential for mitotic entry and spindle assembly. Recent molecular studies have revealed the existence of multiple, distinct mechanisms of Aurora A activation, each occurring at specific subcellular locations, optimized for cellular context, and primed by signaling events including phosphorylation and oxidation.

IntroductionDuring mitosis, almost 30% of the proteome is modified by the transfer of phosphate to serine, threonine, or tyrosine residues. These phosphorylation events are responsible for the profound transformation of cellular architecture and physiology that occurs as cells progress through mitosis. Pivotal protein kinases responsible for the massive increase in protein phosphorylation as cells transit into mitosis include Aurora A kinase (AURKA), Aurora B kinase, Polo-like kinase 1 (Plk1), and Cyclin-dependent kinase 1 (Cdk1)/Cyclin A/B complexes. Their precise and coordinated activation critically defines the G2-M transition.Overexpression and aberrant activation of AURKA have been functionally linked to oncogenic transformation through centrosome amplification, aneuploidy, and chromosomal instability. Beyond its pivotal role in mitotic cell division, AURKA has numerous nonmitotic functions in tumorigenesis. AURKA thus represents a critical “druggable target” in cancer, controlling key oncogenic pathways associated with drug resistance and poor patient outcome.AURKA activation is unexpectedly complex, and a number of different mechanisms have been described, including autophosphorylation of its activation segment and binding to a variety of allosteric modulators, which recruit and locally activate AURKA at specific subcellular localizations (Fig. 1). Recent findings show that these allosteric modulators activate AURKA through surprisingly distinct mechanisms, each acting at different subcellular locations to trigger a unique event in response to different upstream signals. We summarize current views of these activation mechanisms and speculate on the reasons underlying this complexity.Open in a separate windowFigure 1.Model of AURKA activation during cell cycle progression. Left panel: AURKA is activated during the G2/M transition by binding to Bora phosphorylated on its M3 motif by Cyclin A-Cdk1 (CycA-Cdk1). Phospho-Bora (pBora) binds unphosphorylated inactive AURKA (in gray, N and C denote the N-lobe and the C-lobe, respectively) via its M1, M2 (dark blue), and phospho-M3 (green) motifs. Binding of phospho-Bora turns on the catalytic activity of AURKA (red) by substituting in trans the phosphoregulatory site on Thr288, leading to mitotic entry. P denotes phosphate. Middle panel: AURKA is activated at centrosomes via Cep192-dependent oligomerization and oxidation of Cys290 by ROS. NEDB, nuclear envelope breakdown. Right panel: AURKA is activated at spindle microtubules by Tpx2 binding through M1 and M2 motifs and by autophosphorylation at Thr288.Autophosphorylation of the activation segment and binding to the allosteric regulator Targeting protein for Xklp2 (Tpx2) synergize to locally activate AURKA at microtubulesIn eukaryotes, the transfer of phosphate from ATP to protein substrates is mediated by the protein kinase domain, a bilobal catalytic entity. The protein kinase domain possesses a complicated structure, with many flexible parts but a highly restricted catalytic mechanism (i.e., there is only one way to transfer phosphate). This affords great opportunity for the diversification of how each kinase turns on and off (Endicott et al., 2012). Like the majority of eukaryotic protein kinases, AURKA is regulated by phosphorylation of a conserved residue, Thr288, within a flexible element of the kinase domain termed the activation segment. This event leads to a reorganization of the active site that is required, but not sufficient, for full catalytic activation. This is in sharp contrast to many other protein kinases, where phosphorylation of the activation segment is sufficient for maximal catalytic activation. Similar to the closely related AGC family kinases, AURKA has evolved a dependency for its full activation on the binding of an allosteric modulator to its smaller N-terminal kinase lobe (Leroux et al., 2018). This event positions or stabilizes structural elements in the kinase active site that are not sufficiently aligned by activation segment phosphorylation alone. For most AGC family kinases, such as the exemplars PKA and AKT, the allosteric modulator represents a linear peptide sequence contained within the protein kinase itself, but distal to the protein kinase domain. In contrast, in the case of AURKA, the allosteric modulator is presented by an entirely separate protein.The best characterized allosteric modulator of AURKA is the microtubule-binding protein Txp2. Upon nuclear envelope breakdown, Tpx2 is released by RAN-GTP from importins, which then allows it to concurrently recruit and activate AURKA at microtubules to promote mitotic spindle assembly. Tpx2 uses its first N-terminal 43 amino acids to activate AURKA, in a manner synergistic with activation segment phosphorylation, by binding across the N-lobe of the AURKA kinase domain (Fig. 1). Notably, in the absence of Tpx2 binding and activation segment phosphorylation, AURKA retains marginal but detectable protein kinase activity. Binding of Tpx2 alone boosts AURKA catalytic function modestly (15-fold) while autophosphorylation alone boosts catalytic function substantially (157-fold). However, the action of both events translates into a 448-fold enhancement in activity relative to the fully repressed state (Dodson and Bayliss, 2012). This coordination of maximal activation with the recruitment of AURKA to microtubules may serve a double duty to minimize spurious phosphorylation of proteins elsewhere in the cell.Autophosphorylation and binding to Tpx2 trigger conformational changes in AURKA that are sufficiently large to be probed by time-resolved fluorescence energy transfer approaches (Ruff et al., 2018). Time-resolved fluorescence energy transfer revealed that the activation segment of AURKA adopts a wide range of conformations in solution. Notably, binding of Tpx2 to AURKA locks an inward conformation of a catalytic element termed the DFG motif (the Asp in the Asp-Phe-Gly motif coordinates a magnesium ion required for ATP binding) by rigidifying an inherently flexible helix αC (Ruff et al., 2018). In contrast, Thr288 phosphorylation promotes a large conformational change in the activation segment that enables the binding of peptide substrates. Both Tpx2 binding and autophosphorylation are required for AURKA function at microtubules. This transient “doubly activated” form of AURKA is not detectable at spindle microtubules in normal cells due to the action of the AURKA-directed protein phosphatase 6 (PP6), which specifically dephosphorylates Tpx2-bound AURKA on the activation segment.Phospho-Bora activates unphosphorylated AURKA in the cytoplasm to trigger mitotic entryIn a manner thematically similar to how Tpx2 binding and AURKA autophosphorylation synergize to activate AURKA at microtubules, recent work revealed that a phosphorylated form of Bora activates cytoplasmic AURKA during mitotic commitment (Tavernier et al., 2021).Commitment to mitosis is tightly coordinated with DNA replication to preserve genome integrity. Commitment is achieved by a tightly choreographed biochemical tug-of-war between mitotic kinases and phosphatases (PPases). To this end, AURKA activates Plk1 by phosphorylating its activation segment at Thr210. In turn, Plk1 promotes activation of the Cdk1/Cyclin B complex by phosphorylating both negative and positive regulators of Cdk1 to trigger mitotic entry. As AURKA lies at the top of this mitotic kinase cascade, the key question that arises is how is AURKA initially activated in G2?As noted above, AURKA can autophosphorylate its own activation segment at Thr288, but this form of the enzyme is rapidly dephosphorylated by counteracting PPases in G2. As dephosphorylation maintains AURKA in an inactive state, how then does AURKA overcome the repressive effect of PPases to activate Plk1?AURKA activation during mitotic commitment is critically dependent on the evolutionarily conserved protein Bora, following its own phosphorylation on a key regulatory site on Ser112. This event is essential for the phosphorylation of Plk1 on Thr210 by AURKA in vitro and for timely mitotic entry in vivo, both in Xenopus egg extracts (Vigneron et al., 2018) and in human cells (Tavernier et al., 2021). Remarkably, phospho-Bora binds to and potently activates AURKA lacking phosphorylation of its activation segment, suggesting the possibility that the phosphate on S112 of Bora may physically and/or functionally substitute for the phosphorylated activation segment on AURKA.Dissection of how Bora binds AURKA revealed at least two motifs in Bora, denoted M1 and M2, with weak similarity to AURKA-binding elements in Tpx2^1–43. Both motifs are required for the binding and activating function of Bora on AURKA, and notably, the essential Ser112 phospho-regulatory site (in the sequence Pro-Ser-Pro, denoted motif M3) lies immediately C-terminal to binding motif M2. By analogy to the mechanism of action of Tpx2, Bora motif M1 likely binds in an extended manner parallel to the top surface of helix αC, whereas motif M2 adopts a helical conformation and binds parallel to the bottom surface of helix αC. As Bora motif M3 is immediately adjacent to motif M2, this binding mode would orient the Ser112 phospho-moiety of motif M3 in close proximity to a constellation of positively charged residues that normally engage the phosphate moiety of the phosphorylated activation segment of AURKA (Fig. 1; Tavernier et al., 2021). This mode of action elegantly allows phospho-Bora to allosterically activate AURKA during mitotic commitment, when AURKA itself is catalytically repressed by dephosphorylation. The precise atomic details of how phospho-Bora binds and activates AURKA and whether other protein kinases use analogous mechanisms for activation remain to be determined.Since phosphorylation of Bora Ser112 is essential for its ability to activate AURKA and commit cells to mitosis, the upstream kinase responsible for this regulatory event represents a critical component of the AURKA activation puzzle. This function is performed by Cyclin A–Cdk1, which is active in S-G2 and known to promote mitotic entry. Consistent with this model, Bora phosphorylated on S112 is sufficient to promote mitotic commitment in Xenopus egg extracts depleted of Cyclin A (Vigneron et al., 2018). In human cells, Cyclin A–Cdk1 is confined to the nucleus during S phase, but at the S/G2 transition it is abruptly exported to the cytoplasm, allowing it to phosphorylate Bora (Silva Cascales et al., 2021). As such, Bora acts as a bridge linking Cyclin A–Cdk1 activity to the activation of the mitotic kinase cascade. Why phospho-Bora can persist under conditions that disfavor AURKA activation by autophosphorylation remains an open question worthy of further investigation. Possibilities include that the phospho-Ser112 residue is a suboptimal PPase substrate or that it is protected from dephosphorylation by cis-activating factors.Redox regulation of AURKA during mitosisRecent work indicates that AURKA activity is also regulated by oxidative signaling with both stimulatory and inhibitory outcomes. While autophosphorylation of AURKA on Thr288 is largely neutralized in the cytoplasm and at spindle microtubules by counteracting PPases, it is readily detected at centrosomes. Centrosomal AURKA autoactivation is stimulated as a consequence of Cep192-mediated oligomerization and AURKA autophosphorylation, but the underlying mechanism was poorly understood. New studies reveal that oxidative modification of a conserved cysteine residue, Cys290, located in the activation segment of the kinase domain promotes AURKA autophosphorylation during mitosis when AURKA is oligomerized.While biochemical studies using purified proteins in the absence of an oligomerizing agent revealed that oxidative modification of Cys290 inhibited AURKA kinase activity (Byrne et al., 2020; Tsuchiya et al., 2020), cell treatment with oxidizing agents such as H₂0₂ increased AURKA phosphorylation on Thr288 (Wang et al., 2017; Tsuchiya et al., 2020). This increase in Thr288 phosphorylation was accompanied by dimerization of AURKA in a manner sensitive to reducing agents such as DTT. This result hinted that disulfide bond formation between AURKA monomers might be involved in promoting AURKA trans-autophosphorylation. Consistent with this hypothesis, a crystal structure of an AURKA kinase domain obtained under disulfide bond–promoting conditions revealed a face-to-face dimer orientation of the kinase domain stabilized by a Cys290–Cys290 disulfide bond (Lim et al., 2020). In this configuration, the active site of the AURKA kinase domain adopts a productive conformation predicted to support substrate phosphorylation. Given the inherent flexibility of the activation segment itself, this face-to-face configuration of the kinase domain was also predicted to support AURKA trans-autophosphorylation on Thr288. Follow-up biochemical studies proved that the Cys290–Cys290 disulfide–linked configuration of the AURKA kinase domain is indeed compatible with trans-autophosphorylation on Thr288.An interesting feature of the Cys290-dependent activation mechanism of AURKA is the requirement for Cep192. Presumably, the ability of Cep192 to recruit and oligomerize AURKA favors the formation of the Cys290–Cys290 disulfide bond between kinase domains (Fig. 1). In support of this model, oxidation-induced Cys290–Cys290 disulfide cross-linking of AURKA could also be recapitulated in Xenopus extracts by the addition of bivalent antibodies directed at AURKA.At subcellular locations beyond centrosomes, where AURKA is not dimeric, oxidation of the activation segment would be expected to have an opposite effect on protein kinase activity. For instance, a crystal structure of monomeric AURKA covalently bound to Coenzyme A (CoAlation), a major regulator of cellular metabolism that contains both nucleotide and thiol moieties, revealed that AURKA CoAlation robustly inhibits kinase activity (Tsuchiya et al., 2020) through an ATP-competitive mechanism. Competitive binding is achieved by the nucleotide moiety of Coenzyme A engaging the nucleotide-binding pocket of ATP, while the reactive pantetheine thiol moiety forms a disulfide bond with Cys290. This dual anchoring of Coenzyme A to AURKA imparts not only affinity but also specificity toward kinase inhibition. Supporting the possibility that Coenzyme A can exert a potent inhibitory effect on AURKA under physiological conditions, microinjection of CoA into mouse oocytes caused abnormal spindles and chromosome misalignment, phenotypes typically observed upon AURKA inactivation.Interestingly, when bound to AURKA, Tpx2 exerts a protective effect against inhibition by CoAlation. Given that Bora is predicted to bind AURKA similar to Tpx2 (Tavernier et al., 2021), this could allow Bora to also protect AURKA from inhibition by CoAlation. Together, these results suggest that each distinct cellular pool of AURKA will respond differently to oxidation signals.Reactive oxygen species (ROS) are emerging as important signaling molecules. ROS and oxidative stress have been shown to increase during G2 and M phases in an otherwise unperturbed asynchronous cell cycle (Patterson et al., 2019), suggesting that oxidative modification of biomolecules, including AURKA, might regulate mitotic progression. Likewise, H₂O₂ locally released by mitochondria, where a pool of AURKA has been recently shown to localize (Bertolin et al., 2018), is implicated in symmetry breaking and polarity establishment in early Caenorhabditis elegans embryos (De Henau et al., 2020). It will be particularly exciting to determine whether AURKA, which also plays a role in setting up embryo polarity, is regulated by redox signaling in this specific context.Concluding remarksAURKA is activated by a growing list of mechanisms, with each acting at specific stages of the cell cycle and subcellular location. The ability to monitor which specific mechanism is at play at any one time in vivo presents a particular challenge. The activation state of AURKA is often measured by the use of a phospho-specific antibody targeting the phosphorylated Thr288 epitope. However, this has limited effectiveness to detect AURKA activated by Tpx2 at spindle microtubules because of the transient nature of the phospho-Thr288 epitope at this location. Furthermore, in the case of cytoplasmic AURKA activation by Bora, phosphorylation at Thr288 is not required for kinase activation. A live fluorescence energy transfer sensor has been reported for the phosphorylation status of AURKA on Thr288 that detects conformational changes induced by Thr288 phosphorylation rather than the phosphorylation motif itself (Bertolin et al., 2016). If the binding of Tpx2 to phosphorylated AURKA and the binding of phospho-Bora to dephosphorylated AURKA induces similar conformational changes to those induced by autophosphorylation, then this could represent a more generally applicable assay for monitoring the activation state of AURKA.Why is the activation of AURKA so complex? We speculate that the major reason for this complexity is related to kinase action at distinct times and in spatially distinct locations (Fig. 1). Bora acts in the cytoplasm before mitotic entry, and Cep192 acts at the centrosome before and likely after mitotic entry, whereas Tpx2 acts on spindle microtubules after mitotic entry. Thus, the allosteric regulators and their own upstream controllers (e.g., Cyclin A/Cdk1 for Bora) direct AURKA activity to execute distinct functions. In some ways, this complexity of activation represents a different solution than the one adopted by Plk1 or Protein Phosphatase 1 (PP1), which also acts at distinct time points and subcellular locations. Both Plk1 and PP1 employ docking motifs that are post-translationally controlled to dictate their time and sites of action, as well as their substrate specificity. As deeper insights are gained into the control of mitotic kinases and PPases, it will be intriguing to see what additional solutions have evolved to address the challenge of temporally and spatially restricted actions.We end by noting that the different mechanisms described above for AURKA activation are associated with specific pathologies. Doubly activated AURKA, with bound Tpx2 and Thr288 phosphorylation, is not detected on microtubules in normal cells due to the action of PP6. However, this form of AURKA is readily detected in melanoma cells bearing PP6 mutations, which gives rise to pathological chromosome instability and DNA damage (Hammond et al., 2013). Bora is overexpressed in multiple cancer types, including ovarian cancer, where it plays a pro-oncogenic role (Parrilla et al., 2020), and there is significant evidence for ROS signaling, which is involved in centrosomal AURKA activation by Cep192, contributing to a number of disease states. Finally, AURKA itself is overexpressed in numerous cancers associated with drug resistance and poor patient outcome. However, the clinical utility of AURKA inhibitors to date has been limited, likely because of essential roles of AURKA in multiple events in the cell cycle. We posit that the discovery that AURKA is activated through a variety of mechanisms to execute distinct events may afford opportunities to develop drugs targeting a subset of the biological functions of AURKA and hence enable more precise tuning of the therapeutic window.     

The Parkinson’s disease kinase LRRK2 autophosphorylates its GTPase domain at multiple sites

Mutations in leucine-rich repeat kinase 2 (LRRK2) are a common cause of inherited Parkinson’s disease (PD). The protein is large and complex, but pathogenic mutations cluster in a region containing GTPase and kinase domains. LRRK2 can autophosphorylate in vitro within a dimer pair, although the significance of this reaction is unclear. Here, we mapped the sites of autophosphorylation within LRRK2 and found several potential phosphorylation sites within the GTPase domain. Using mass spectrometry, we found that Thr1343 is phosphorylated and, using kinase dead versions of LRRK2, show that this is an autophosphorylation site. However, we also find evidence for additional sites in the GTPase domain and in other regions of the protein suggesting that there may be multiple autophosphorylation sites within LRRK2. These data suggest that the kinase and GTPase activities of LRRK2 may exhibit complex autoregulatory interdependence.     

Active role for nibrin in the kinetics of atm activation

The Atm protein kinase is central to the DNA double-strand break response in mammalian cells. After irradiation, dimeric Atm undergoes autophosphorylation at Ser 1981 and dissociates into active monomers. Atm activation is stimulated by expression of the Mre11/Rad50/nibrin complex. Previously, we showed that a C-terminal fragment of nibrin, containing binding sites for both Mre11 and Atm, was sufficient to provide this stimulatory effect in Nijmegen breakage syndrome (NBS) cells. To discriminate whether nibrin's role in Atm activation is to bind and translocate Mre11/Rad50 to the nucleus or to interact directly with Atm, we expressed an Mre11 transgene with a C-terminal NLS sequence in NBS fibroblasts. The Mre11-NLS protein complexed with Rad50, localized to the nucleus in NBS fibroblasts, and associated with chromatin. However, Atm autophosphorylation was not stimulated in cells expressing Mre11-NLS, nor were downstream Atm targets phosphorylated. To determine whether nibrin-Atm interaction is necessary to stimulate Atm activation, we expressed nibrin transgenes lacking the Atm binding domain in NBS fibroblasts. The nibrin DeltaAtm protein interacted with Mre11/Rad50; however, Atm autophosphorylation was dramatically reduced after irradiation in NBS cells expressing the nibrin DeltaAtm transgenes relative to wild-type nibrin. These results indicate that nibrin plays an active role in Atm activation beyond translocating Mre11/Rad50 to the nucleus and that this function requires nibrin-Atm interaction.     

Substrates enhance autophosphorylation and activation of p21-activated protein kinase gamma-PAK in the absence of activation loop phosphorylation.

The p21-activated protein kinase gamma-PAK from rabbit, expressed in insect cells, is activated following binding of Cdc42(GTPgammaS). The rate of autophosphorylation is increased fivefold and the protein kinase activity 13-fold, as measured with the synthetic heptapeptide (AKRESAA). The mutant K278R, where the invariant lysine in the catalytic site is replaced by arginine, shows neither autophosphorylation nor activity. Replacement of the conserved threonine in the catalytic domain with alanine (T402A) reduces autophosphorylation and protein kinase activity to 1% that of the wild-type gamma-PAK, indicating autophosphorylation of Thr402 in the activation loop is essential for protein kinase activity. In contrast, certain protein substrates such as histone 2B, histone 4 and myelin basic protein, stimulate both autophosphorylation and protein kinase activity to levels similar to those observed with Cdc42(GTPgammaS). This substrate-level activation does not require autophosphorylation of Thr402 in the activation loop. As shown with T402A, the protein kinase activity with histone 4 is similar to that observed with recombinant wild-type gamma-PAK. Basic proteins or peptides which are not substrates of gamma-PAK, such as histone 1 and polylysine, do not stimulate autophosphorylation or activity. Other substrates such as the Rous sarcoma virus protein NC are phosphorylated by gamma-PAK following activation by Cdc42(GTPgammaS), but are not phosphorylated by T402A. The data suggest that some substrates can override the requirement for Cdc42(GTPgammaS), by activating gamma-PAK directly.     

Phosphorylation at Ser724 of the ER stress sensor IRE1α governs its activation state and limits ER stress–induced hepatosteatosis

Inositol-requiring enzyme 1 (IRE1) is an evolutionarily conserved sensor of endoplasmic reticulum (ER) stress and mediates a key branch of the unfolded protein response in eukaryotic cells. It is an ER-resident transmembrane protein that possesses Ser/Thr protein kinase and endoribonuclease (RNase) activities in its cytoplasmic region. IRE1 is activated through dimerization/oligomerization and autophosphorylation at multiple sites, acting through its RNase activity to restore the functional capacity of the ER. However, it remains poorly defined in vivo how the autophosphorylation events of endogenous IRE1 govern its dynamic activation and functional output. Here, we generated a mouse model harboring a S724A knock-in mutation (Ern1^S724A/S724A) and investigated the importance of phosphorylation at Ser⁷²⁴ within the kinase activation loop of murine IRE1α. We found that in mouse embryonic fibroblast cells and in primary hepatocytes, S724A mutation resulted in markedly reduced IRE1α autophosphorylation in parallel with blunted activation of its RNase activity to catalyze X-box binding protein 1 (Xbp1) mRNA splicing. Furthermore, ablation of IRE1α phosphorylation at Ser⁷²⁴ exacerbated ER stress–induced hepatic steatosis in tunicamycin-treated Ern1^S724A/S724A mice. This was accompanied by significantly decreased hepatic production of spliced XBP1 protein but increased CCAAT-enhancer–binding protein homologous protein (CHOP) level, along with suppressed expression of key metabolic regulators of fatty acid β-oxidation and lipid secretion. These results demonstrate a critical role of phosphorylation at Ser⁷²⁴ of IRE1α in dynamically controlling its kinase activity, and thus its autophosphorylation state, which is coupled to activation of its RNase activity in counteracting hepatic steatosis under ER stress conditions.     

Autophosphorylation of threonine 485 in the activation loop is essential for attaining eIF2alpha kinase activity of HRI

In heme deficiency, protein synthesis is inhibited by the activation of the heme-regulated eIF2alpha kinase (HRI) through its multiple autophosphorylation. Autophosphorylation sites in HRI were identified in order to investigate their functions. We found that there were eight major tryptic phosphopeptides of HRI activated in heme deficiency. In this report we focused on the role of autophosphorylation at Thr483 and Thr485 in the activation loop of HRI. Disruption of the autophosphorylation of Thr485, but not Thr483, resulted in a lower autokinase activity and locked Thr485Ala HRI in a hypophosphorylated state. Most importantly, autophosphorylation of Thr485, but not Thr483, was essential for attaining eIF2alpha kinase activity of HRI. In addition, autophosphorylation of Thr485 was necessary for arsenite-induced activation of the eIF2alpha kinase activity of HRI, while autophosphorylation at Thr483 was not required for activation by arsenite. The function of Thr490, another conserved Thr residue in the activation loop of HRI, was also investigated. Mutations of Thr490 to either Ala or Asp resulted in reduced autokinase activity and loss of eIF2alpha kinase activity in heme deficiency or upon arsenite treatment. Since Thr490 was not identified as an autophosphorylated site, it is likely that Thr490 itself might be critical for the catalytic activity of HRI. Importantly, Thr485 was very poorly phosphorylated in Thr490 mutant HRI. Collectively, our results demonstrate that autophosphorylation of Thr485 is essential for the hyperphosphorylation and activation of HRI and is required for the acquisition of the eIF2alpha kinase activity.     

ATR autophosphorylation as a molecular switch for checkpoint activation

The ataxia telangiectasia-mutated and Rad3-related (ATR) kinase is a master checkpoint regulator safeguarding the genome. Upon DNA damage, the ATR-ATRIP complex is recruited to sites of DNA damage by RPA-coated single-stranded DNA and activated by an elusive process. Here, we show that ATR is transformed into a hyperphosphorylated state after DNA damage, and that a single autophosphorylation event at Thr 1989 is crucial for ATR activation. Phosphorylation of Thr 1989 relies on RPA, ATRIP, and ATR kinase activity, but unexpectedly not on the ATR stimulator TopBP1. Recruitment of ATR-ATRIP to RPA-ssDNA leads to congregation of ATR-ATRIP complexes and promotes Thr 1989 phosphorylation in trans. Phosphorylated Thr 1989 is directly recognized by TopBP1 via the BRCT domains 7 and 8, enabling TopBP1 to engage ATR-ATRIP, to stimulate the ATR kinase, and to facilitate ATR substrate recognition. Thus, ATR autophosphorylation on RPA-ssDNA is a molecular switch to launch robust checkpoint response.     

Allosteric effects mediate CHK2 phosphorylation of the p53 transactivation domain

The tumour suppressor p53 is a tetrameric protein that is phosphorylated in its BOX-I transactivation domain by checkpoint kinase 2 (CHK2) in response to DNA damage. CHK2 cannot phosphorylate small peptide fragments of p53 containing the BOX-I motif, indicating that undefined determinants in the p53 tetramer mediate CHK2 recognition. Two peptides derived from the DNA-binding domain of p53 bind to CHK2 and stimulate phosphorylation of full-length p53 at Thr 18 and Ser 20, thus identifying CHK2-docking sites. CHK2 can be fully activated in trans by the two p53 DNA-binding-domain peptides, and can phosphorylate BOX-I transactivation-domain fragments of p53 at Thr 18 and Ser 20. Although CHK2 has a basal Ser 20 kinase activity that is predominantly activated towards Thr 18, CHK1 has constitutive Thr 18 kinase activity that is predominantly activated in trans towards Ser 20. Cell division cycle 25C (CDC25C) phosphorylation by CHK2 is unaffected by the p53 DNA-binding-domain peptides. The CHK2-docking site in the BOX-V motif is the smallest of the two CHK2 binding sites, and mutating certain amino acids in the BOX-V peptide prevents CHK2 activation. A database search identified a p53 BOX-I-homology motif in p21^WAF1 and although CHK2 is inactive towards this protein, the p53 DNA-binding-domain peptides induce phosphorylation of p21^WAF1 at Ser 146. This provides evidence that CHK2 can be activated allosterically towards some substrates by a novel docking interaction, and identify a potential regulatory switch that may channel CHK2 into distinct signalling pathways in vivo.     

Identification of novel phosphorylation sites required for activation of MAPKAP kinase-2.

MAP kinase-activated protein (MAPKAP) kinase-2 is activated in vivo by reactivating kinase (RK), a MAP kinase (MAPK) homologue stimulated by cytokines and cellular stresses. Here we show that in vitro RK phosphorylates human GST-MAPKAP kinase-2 at Thr25 in the proline-rich N-terminal region Thr222 and Ser272 in the catalytic domain and Thr334 in the C-terminal domain. Using novel methodology we demonstrate that activation of MAPKAP kinase-2 requires the phosphorylation of any two of the three residues Thr222, Ser272 and Thr334. Ser9, Thr25, Thr222, Ser272, Thr334 and Thr338 became 32P-labelled in stressed KB cells and labelling was prevented by the specific RK inhibitor SB 203580, establishing that RK phosphorylates Thr25, Thr222, Ser272 and Thr334 in vivo. The 32P-labelling of Thr338 is likely to result from autophosphorylation. GST-MAPKAP kinase-2 lacking the N-terminal domain was inactive, but activated fully when phosphorylated at Thr222, Ser272 and Thr334 by p42 MAPK or RK. In contrast, full-length GST-MAPKAP kinase-2 was phosphorylated at Thr25 (and not activated) by p42 MAPK, suggesting a role for the N-terminal domain in specifying activation by RK in vivo. The mutant Asp222/Asp334 was 20% as active as phosphorylated MAPKAP kinase-2, and this constitutively active form may be useful for studying its physiological roles.     

Regulatory mechanism of Dictyostelium myosin light chain kinase A

In this study, we examined the activation mechanism of Dictyostelium myosin light chain kinase A (MLCK-A) using constitutively active Ca2+/calmodulin-dependent protein kinase kinase as a surrogate MLCK-A kinase. MLCK-A was phosphorylated at Thr166 by constitutively active Ca2+/calmodulin-dependent protein kinase kinase, resulting in an approximately 140-fold increase in catalytic activity, using intact Dictyostelium myosin II. Recombinant Dictyostelium myosin II regulatory light chain and Kemptamide were also readily phosphorylated by activated MLCK-A. Mass spectrometry analysis revealed that MLCK-A expressed by Escherichia coli was autophosphorylated at Thr289 and that, subsequent to Thr166 phosphorylation, MLCK-A also underwent a slow rate of autophosphorylation at multiple Ser residues. Using site-directed mutagenesis, we show that autophosphorylation at Thr289 is required for efficient phosphorylation and activation by an upstream kinase. By performing enzyme kinetics analysis on a series of MLCK-A truncation mutants, we found that residues 283-288 function as an autoinhibitory domain and that autoinhibition is fully relieved by Thr166 phosphorylation. Simple removal of this region resulted in a significant increase in the kcat of MLCK-A; however, it did not generate maximum enzymatic activity. Together with the results of our kinetic analysis of the enzymes, these findings demonstrate that Thr166 phosphorylation of MLCK-A by an upstream kinase subsequent to autophosphorylation at Thr289 results in generation of maximum MLCK-A activity through both release of an autoinhibitory domain from its catalytic core and a further increase (15-19-fold) in the kcat of the enzyme.     

Phosphorylation-dependent activation of TAK1 mitogen-activated protein kinase kinase kinase by TAB1

TAK1 is a mitogen-activated protein kinase kinase kinase (MAP3K) that is involved in the c-Jun N-terminal kinase/p38 MAPKs and NF-kappaB signaling pathways. Here, we characterized the molecular mechanisms of TAK1 activation by its specific activator TAB1. Autophosphorylation of two threonine residues in the activation loop of TAK1 was necessary for TAK1 activation. Association with TAK1 and induction of TAK1 autophosphorylation required the C-terminal 24 amino acids of TAB1, but full TAK1 activation required additional C-terminal Ser/Thr rich sequences. These results demonstrated that the association between the kinase domain of TAK1 and the C-terminal TAB1 triggered the phosphorylation-dependent TAK1 activation mechanism.