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Fragile X Mental Retardation protein (FMRP), widely known for its role in hereditary intellectual disability, is an RNA-binding protein (RBP) that controls translation of select mRNAs. We discovered that endoplasmic reticulum (ER) stress induces phosphorylation of FMRP on a site that is known to enhance translation inhibition of FMRP-bound mRNAs. We show ER stress-induced activation of Inositol requiring enzyme-1 (IRE1), an ER-resident stress-sensing kinase/endoribonuclease, leads to FMRP phosphorylation and to suppression of macrophage cholesterol efflux and apoptotic cell clearance (efferocytosis). Conversely, FMRP deficiency and pharmacological inhibition of IRE1 kinase activity enhances cholesterol efflux and efferocytosis, reducing atherosclerosis in mice. Our results provide mechanistic insights into how ER stress-induced IRE1 kinase activity contributes to macrophage cholesterol homeostasis and suggests IRE1 inhibition as a promising new way to counteract atherosclerosis., (© 2022 The Authors. Published under the terms of the CC BY 4.0 license.)

MAP/microtubule-affinity regulating kinases (MARK1-4) are members of the AMPK family of Ser/Thr-specific kinases, which phosphorylate substrates at consensus LXRXXSXXXL motifs. Within microtubule-associated proteins, MARKs also mediate phosphorylation of variant KXGS or ζXKXGSXXNΨ motifs, interfering with the ability of tau and MAP2/4 to bind to microtubules. Here we show that, although MARKs and the closely related salt-inducible kinases (SIKs) phosphorylate substrates with consensus AMPK motifs comparably, MARKs are more potent in recognizing variant ζXKXGSXXNΨ motifs on cellular tau. In studies to identify regions of MARKs that confer catalytic activity towards variant sites, we found that the C-terminal kinase associated-1 (KA1) domain in MARK1-3 mediates binding to microtubule-associated proteins CLASP1/2; but this interaction is dispensable for ζXKXGSXXNΨ phosphorylation. Mutational analysis of MARK2 revealed that the N-terminal kinase domain of MARK2 is sufficient for phosphorylation of both consensus and variant ζXKXGSXXNΨ sites. Within this domain, the KLDpT activation loop motif promotes MARK2 activity both intracellularly and in vitro, but has no effect on SIK2 activity. As KLDpT is conserved in all vertebrates MARKs, we conclude that this sequence is crucial for MARK-dependent regulation of cellular polarity., Competing Interests: The authors have declared that no competing interests exist.

We recently demonstrated that the circadian clock component CRY2 is an essential cofactor in the SCF FBXL3 -mediated ubiquitination of c-MYC. Because our demonstration that CRY2 recruits phosphorylated substrates to SCF FBXL3 was unexpected, we investigated the scope of this role by searching for additional substrates of FBXL3 that require CRY1 or CRY2 as cofactors. Here, we describe an affinity purification mass spectrometry (APMS) screen through which we identified more than one hundred potential substrates of SCF FBXL3+CRY1/2 , including the cell cycle regulated Tousled-like kinase, TLK2. Both CRY1 and CRY2 recruit TLK2 to SCF FBXL3 , and TLK2 kinase activity is required for this interaction. Overexpression or genetic deletion of CRY1 and/or CRY2 decreases or enhances TLK2 protein abundance, respectively. These findings reinforce the idea that CRYs function as co-factors for SCF FBXL3 , provide a resource of potential substrates, and establish a molecular connection between the circadian and cell cycle oscillators via CRY-modulated turnover of TLK2.

Doublecortin-like kinase 1 (DCLK1) is a member of the neuronal microtubule-associated doublecortin (DCX) family and functions in multiple stages of neural development including radial migration and axon growth of cortical neurons. DCLK1 is suggested to play the roles in part through its protein kinase activity, yet the kinase substrates of DCLK1 remain largely unknown. Here we have identified MAP7D1 (microtubule-associated protein 7 domain containing 1) as a novel substrate of DCLK1 by using proteomic analysis. MAP7D1 is expressed in developing cortical neurons, and knockdown of MAP7D1 in layer 2/3 cortical neurons results in a significant impairment of callosal axon elongation, but not of radial migration, in corticogenesis. We have further defined the serine 315 (Ser 315) of MAP7D1 as a DCLK1-induced phosphorylation site and shown that overexpression of a phosphomimetic MAP7D1 mutant in which Ser 315 is substituted with glutamic acid (MAP7D1 S315E), but not wild-type MAP7D1, fully rescues the axon elongation defects in Dclk1 knockdown neurons. These data demonstrate that DCLK1 phosphorylates MAP7D1 on Ser 315 to facilitate axon elongation of cortical neurons. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 419-437, 2017., (© 2016 Wiley Periodicals, Inc.)

The second messenger cAMP stimulates cellular gene expression via the PKA-mediated phosphorylation of the transcription factor CREB and through dephosphorylation of the cAMP-responsive transcriptional coactivators (CRTCs). Under basal conditions, CRTCs are phosphorylated by members of the AMPK family of Ser/Thr kinases and sequestered in the cytoplasm via a phosphorylation-dependent association with 14-3-3 proteins. Increases in cAMP promote the dephosphorylation and nuclear translocation of CRTCs, where they bind to CREB and stimulate relevant target genes. Although they share considerable sequence homology, members of the CRTC family exert non-overlapping effects on cellular gene expression through as yet unidentified mechanisms. Here we show that the three CRTCs exhibit distinct patterns of 14-3-3 binding at three conserved sites corresponding to S70, S171, and S275 (in CRTC2). S171 functions as the gatekeeper site for 14-3-3 binding; it acts cooperatively with S275 in stabilizing this interaction following its phosphorylation by the cAMP-responsive SIK and the cAMP-nonresponsive MARK kinases. Although S171 contains a consensus recognition site for phosphorylation by AMPK family members, S70 and S275 carry variant motifs (MNTGGS275LPDL), lacking basic residues that are otherwise critical for SIK/MARK recognition as well as 14-3-3 binding. Correspondingly, the activity of these motifs differs between CRTC family members. As the variant (SLPDL) motif is present and apparently phosphorylated in other mammalian proteins, our studies suggest that the regulation of cellular targets by AMPK family members is more extensive than previously appreciated.

Signaling via the inducible costimulator ICOS fuels the stepwise development of follicular helper T cells (TFH cells). However, a signaling pathway unique to ICOS has not been identified. We found here that the kinase TBK1 associated with ICOS via a conserved motif, IProx, that shares homology with the tumor-necrosis-factor receptor (TNFR)-associated factors TRAF2 and TRAF3. Disruption of this motif abolished the association of TBK1 with ICOS, TRAF2 and TRAF3, which identified a TBK1-binding consensus. Alteration of this motif in ICOS or depletion of TBK1 in T cells severely impaired the differentiation of germinal center (GC) TFH cells and the development of GCs, interfered with B cell differentiation and disrupted the development of antibody responses, but the IProx motif and TBK1 were dispensable for the early differentiation of TFH cells. These results reveal a previously unknown ICOS-TBK1 signaling pathway that specifies the commitment of GC TFH cells.

Endoplasmic reticulum (ER)-associated degradation (ERAD) represents a principle quality control mechanism to clear misfolded proteins in the ER; however, its physiological significance and the nature of endogenous ERAD substrates remain largely unexplored. Here we discover that IRE1α, the sensor of the unfolded protein response (UPR), is a bona fide substrate of the Sel1L-Hrd1 ERAD complex. ERAD-mediated IRE1α degradation occurs under basal conditions in a BiP-dependent manner, requires both the intramembrane hydrophilic residues of IRE1α and the lectin protein OS9, and is attenuated by ER stress. ERAD deficiency causes IRE1α protein stabilization, accumulation and mild activation both in vitro and in vivo. Although enterocyte-specific Sel1L-knockout mice (Sel1L(ΔIEC)) are viable and seem normal, they are highly susceptible to experimental colitis and inflammation-associated dysbiosis, in an IRE1α-dependent but CHOP-independent manner. Hence, Sel1L-Hrd1 ERAD serves a distinct, essential function in restraint of IRE1α signalling in vivo by managing its protein turnover.

Quantification of proteomes by mass spectrometry has proven to be useful to study human pathology recapitulated in cellular or animal models of disease. Enriching and quantifying newly synthesized proteins (NSPs) at set time points by mass spectrometry has the potential to identify important early regulatory or expression changes associated with disease states or perturbations. NSP can be enriched from proteomes by employing pulsed introduction of the noncanonical amino acid, azidohomoalanine (AHA). We demonstrate that pulsed introduction of AHA in the feed of mice can label and identify NSP from multiple tissues. Furthermore, we quantitate differences in new protein expression resulting from CRE-LOX initiated knockout of LKB1 in mouse livers. Overall, the PALM strategy allows for the first time in vivo labeling of mouse tissues to differentiate protein synthesis rates at discrete time points.

The plant hormone abscisic acid (ABA) controls growth and development and regulates plant water status through an established signaling pathway. In the presence of ABA, pyrabactin resistance/regulatory component of ABA receptor proteins inhibit type 2C protein phosphatases (PP2Cs). This, in turn, enables the activation of Sucrose Nonfermenting1-Related Protein Kinases2 (SnRK2). Open Stomata1 (OST1)/SnRK2.6/SRK2E is a major SnRK2-type protein kinase responsible for mediating ABA responses. Arabidopsis (Arabidopsis thaliana) expressing an epitope-tagged OST1 in the recessive ost1-3 mutant background was used for the copurification and identification of OST1-interacting proteins after osmotic stress and ABA treatments. These analyses, which were confirmed using bimolecular fluorescence complementation and coimmunoprecipitation, unexpectedly revealed homo- and heteromerization of OST1 with SnRK2.2, SnRK2.3, OST1, and SnRK2.8. Furthermore, several OST1-complexed proteins were identified as type 2A protein phosphatase (PP2A) subunits and as proteins involved in lipid and galactolipid metabolism. More detailed analyses suggested an interaction network between ABA-activated SnRK2-type protein kinases and several PP2A-type protein phosphatase regulatory subunits. pp2a double mutants exhibited a reduced sensitivity to ABA during seed germination and stomatal closure and an enhanced ABA sensitivity in root growth regulation. These analyses add PP2A-type protein phosphatases as another class of protein phosphatases to the interaction network of SnRK2-type protein kinases., (© 2015 American Society of Plant Biologists. All Rights Reserved.)

Oncogene-induced senescence is an important tumor-suppressing defense mechanism. However, relatively little is known about the signaling pathway mediating the senescence response. Here, we demonstrate that a multifunctional acetyltransferase, Tip60, plays an essential role in oncogenic ras-induced senescence. Further investigation reveals a cascade of posttranslational modifications involving p38, Tip60, and PRAK, three proteins that are essential for ras-induced senescence. Upon activation by ras, p38 induces the acetyltransferase activity of Tip60 through phosphorylation of Thr158; activated Tip60 in turn directly interacts with and induces the protein kinase activity of PRAK through acetylation of K364 in a manner that depends on phosphorylation of both Tip60 and PRAK by p38. These posttranslational modifications are critical for the prosenescent function of Tip60 and PRAK, respectively. These results have defined a signaling pathway that mediates oncogene-induced senescence, and identified posttranslational modifications that regulate the enzymatic activity and biological functions of Tip60 and PRAK., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Scaffold-assisted signaling cascades guide cellular decision-making. In budding yeast, one such signal transduction pathway called the mitotic exit network (MEN) governs the transition from mitosis to the G1 phase of the cell cycle. The MEN is conserved and in metazoans is known as the Hippo tumor-suppressor pathway. We found that signaling through the MEN kinase cascade was mediated by an unusual two-step process. The MEN kinase Cdc15 first phosphorylated the scaffold Nud1. This created a phospho-docking site on Nud1, to which the effector kinase complex Dbf2-Mob1 bound through a phosphoserine-threonine binding domain, in order to be activated by Cdc15. This mechanism of pathway activation has implications for signal transmission through other kinase cascades and might represent a general principle in scaffold-assisted signaling.

Sister-chromatid cohesion is established during S phase when Eco1 acetylates cohesin. In budding yeast, Eco1 activity falls after S phase due to Cdk1-dependent phosphorylation, which triggers ubiquitination by SCF(Cdc4). We show here that Eco1 degradation requires the sequential actions of Cdk1 and two additional kinases, Cdc7-Dbf4 and the GSK-3 homolog Mck1. These kinases recognize motifs primed by previous phosphorylation, resulting in an ordered sequence of three phosphorylation events on Eco1. Only the latter two phosphorylation sites are spaced correctly to bind Cdc4, resulting in strict discrimination between phosphates added by Cdk1 and by Cdc7. Inhibition of Cdc7 by the DNA damage response prevents Eco1 destruction, allowing establishment of cohesion after S phase. This elaborate regulatory system, involving three independent kinases and stringent substrate selection by a ubiquitin ligase, enables robust control of cohesion establishment during normal growth and after stress.

CtIP plays an important role in homologous recombination (HR)-mediated DNA double-stranded break (DSB) repair and interacts with Nbs1 and BRCA1, which are linked to Nijmegen breakage syndrome (NBS) and familial breast cancer, respectively. We identified new CDK phosphorylation sites on CtIP and found that phosphorylation of these newly identified CDK sites induces association of CtIP with the N-terminus FHA and BRCT domains of Nbs1. We further showed that these CDK-dependent phosphorylation events are a prerequisite for ATM to phosphorylate CtIP upon DNA damage, which is important for end resection to activate HR by promoting recruitment of BLM and Exo1 to DSBs. Most notably, this CDK-dependent CtIP and Nbs1 interaction facilitates ATM to phosphorylate CtIP in a substrate-specific manner. These studies reveal one important mechanism to regulate cell-cycle-dependent activation of HR upon DNA damage by coupling CDK- and ATM-mediated phosphorylation of CtIP through modulating the interaction of CtIP with Nbs1, which significantly helps to understand how DSB repair is regulated in mammalian cells to maintain genome stability., Competing Interests: The authors have declared that no competing interests exist.

Here we identify and characterize a cytoskeletal myosin protein required for IRE1α oligomerization, activation, and signaling. Proteomic screening identified nonmuscle myosin heavy chain IIB (NMHCIIB), a subunit of nonmuscle myosin IIB (NMIIB), as an ER stress-dependent interacting protein specific to IRE1α. Loss of NMIIB compromises XBP1s and UPR target gene expression with no effect on the PERK pathway. Mechanistically, NMIIB is required for IRE1α aggregation and foci formation under ER stress. The NMIIB-mediated effect on IRE1α signaling is in part dependent on the phosphorylation of myosin regulatory light chain and the actomyosin contractility of NMIIB. Biologically, the function of NMIIB in ER stress response is conserved as both mammalian cells and C. elegans lacking NMIIB exhibit hypersensitivity to ER stress. Thus, optimal IRE1α activation and signaling require concerted coordination between the ER and cytoskeleton., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The Schizosaccharomyces pombe checkpoint protein Dma1 couples mitotic progression with cytokinesis and is important in delaying mitotic exit and cytokinesis when kinetochores are not properly attached to the mitotic spindle. Dma1 is a ubiquitin ligase and potential functional relative of the human tumor suppressor Chfr. Dma1 delays mitotic exit and cytokinesis by ubiquitinating a scaffold protein (Sid4) of the septation initiation network, which, in turn, antagonizes the ability of the Polo-like kinase Plo1 to promote cell division. Here we identify Dnt1 as a Dma1-binding protein. Several lines of evidence indicate that Dnt1 inhibits Dma1 function during metaphase. First, Dnt1 interacts preferentially with Dma1 during metaphase. Second, Dma1 ubiquitin ligase activity and Sid4 ubiquitination are elevated in dnt1 cells. Third, the enhanced mitotic defects in dnt1Δ plo1 double mutants are partially rescued by deletion of dma1(+), suggesting that the defects in dnt1 plo1 double mutants are attributable to excess Dma1 activity. Taken together, these data show that Dnt1 acts to restrain Dma1 activity in early mitosis to allow normal mitotic progression.

MBF and SBF transcription factors regulate a large family of coordinately expressed G1/S genes required for early cell-cycle functions including DNA replication and repair. SBF is inactivated upon S-phase entry by Clb/CDK whereas MBF targets are repressed by the co-repressor, Nrm1. Using genome-wide expression analysis of cells treated with methyl methane sulfonate (MMS), hydroxyurea (HU) or camptothecin (CPT), we show that genotoxic stress during S phase specifically induces MBF-regulated genes. This occurs via direct phosphorylation of Nrm1 by Rad53, the effector checkpoint kinase, which prevents its binding to MBF target promoters. We conclude that MBF-regulated genes are distinguished from SBF-regulated genes by their sensitivity to activation by the S-phase checkpoint, thereby, providing an effective mechanism for enhancing DNA replication and repair and promoting genome stability.

LATS2 kinase functions as part of the Hippo pathway to promote contact inhibition of growth and tumor suppression by phosphorylating and inhibiting the transcriptional coactivator YAP. LATS2 is activated by the MST2 kinase. How LATS2 is activated by MST2 in response to changes in cell density is unknown. Here we identify the angiomotin-family tight junction protein AMOTL2 as a novel activator of LATS2. Like AMOTL2, the other angiomotin-family proteins AMOT and AMOTL1 also activate LATS2 through a novel conserved domain that binds and activates LATS2. AMOTL2 binds MST2, LATS2, and YAP, suggesting that AMOTL2 might serve as a scaffold protein. We show that LATS2, AMOTL2, and YAP all localize to tight junctions, raising the possibility that clustering of Hippo pathway components at tight junctions might function to trigger LATS2 activation and growth inhibition in response to increased cell density.

Mitotic kinases orchestrate cell cycle processes by phosphorylation of cell cycle regulators. DDA3, a spindle-associated phosphor-protein, is a substrate of mitotic kinases that control chromosome movement and spindle microtubule (MT) dynamics. Through a mass spectrometry analysis, we identified phosphorylation sites on the endogenous mitotic DDA3, which include Ser22, Ser65, Ser70, and Ser223. Phosphorylation of these residues converts interphase form of DDA3 to mitotic form by changing its biochemical activity, as unphosphorylated DDA3 processed both the MT polymerizing and bundling activities, whereas phosphor-mimic mutants lost both activities, only retaining the MT-binding activity. We found that mitotic kinases, such as Cdk1, Aurora A, and Plk1, phosphorylate DDA3 in vitro. Whereas Cdk1 and Aurora A negatively regulate MT-polymerizing and MT-bundling activities, Plk1 does not affect these activities. Interestingly, the phosphorylation of DDA3 by Aurora A and Plk1 inhibits the phosphorylation by other kinases, indicating that sequential phosphorylation is important for the regulation of DDA3 function. We conclude that kinases control the function of DDA3 in the cell cycle by regulating its MT-polymerizing/bundling activities through sequential phosphorylation., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Accurate chromosome segregation requires carefully regulated interactions between kinetochores and microtubules, but how plasticity is achieved to correct diverse attachment defects remains unclear. Here we demonstrate that Aurora B kinase phosphorylates three spatially distinct targets within the conserved outer kinetochore KNL1/Mis12 complex/Ndc80 complex (KMN) network, the key player in kinetochore-microtubule attachments. The combinatorial phosphorylation of the KMN network generates graded levels of microtubule-binding activity, with full phosphorylation severely compromising microtubule binding. Altering the phosphorylation state of each protein causes corresponding chromosome segregation defects. Importantly, the spatial distribution of these targets along the kinetochore axis leads to their differential phosphorylation in response to changes in tension and attachment state. In total, rather than generating exclusively binary changes in microtubule binding, our results suggest a mechanism for the tension-dependent fine-tuning of kinetochore-microtubule interactions., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

In Saccharomyces cerevisiae, the DNA damage response (DDR) is activated by the spatio-temporal colocalization of Mec1-Ddc2 kinase and the 9-1-1 clamp. In the absence of direct means to monitor Mec1 kinase activation in vivo, activation of the checkpoint kinase Rad53 has been taken as a proxy for DDR activation. Here, we identify serine 378 of the Rad55 recombination protein as a direct target site of Mec1. Rad55-S378 phosphorylation leads to an electrophoretic mobility shift of the protein and acts as a sentinel for Mec1 activation in vivo. A single double-stranded break (DSB) in G1-arrested cells causes phosphorylation of Rad55-S378, indicating activation of Mec1 kinase. However, Rad53 kinase is not detectably activated under these conditions. This response required Mec1-Ddc2 and loading of the 9-1-1 clamp by Rad24-RFC, but not Rad9 or Mrc1. In addition to Rad55-S378, two additional direct Mec1 kinase targets are phosphorylated, the middle subunit of the ssDNA-binding protein RPA, RPA2 and histone H2A (H2AX). These data suggest the existence of a truncated signaling pathway in response to a single DSB in G1-arrested cells that activates Mec1 without eliciting a full DDR involving the entire signaling pathway including the effector kinases.

Protein phosphorylation plays an important role in the regulation of centrosome duplication. In budding yeast, numerous lines of evidence suggest a requirement for multiple phosphorylation events on individual components of the centrosome to ensure their proper assembly and function. Here, we report the first example of a single phosphorylation event on a component of the yeast centrosome, or spindle pole body (SPB), that is required for SPB duplication and cell viability. This phosphorylation event is on the essential SPB component Spc29 at a conserved Thr residue, Thr(240). Mutation of Thr(240) to Ala is lethal at normal gene dosage, but an increased copy number of this mutant allele results in a conditional phenotype. Phosphorylation of Thr(240) was found to promote the stability of the protein in vivo and is catalyzed in vitro by the Mps1 kinase. Furthermore, the stability of newly synthesized Spc29 is reduced in a mutant strain with reduced Mps1 kinase activity. These results demonstrate the first evidence for a single phosphorylation event on an SPB component that is absolutely required for SPB duplication and suggest that the Mps1 kinase is responsible for this protein-stabilizing phosphorylation.

The microtubule depolymerase Kif2a controls spindle assembly and dynamics and is essential for chromosome congression and segregation. Through a proteomic analysis, we identified Kif2a as a target for regulation by the Polo-like kinase Plk1. Plk1 interacts with Kif2a, but only in mitosis, in a manner dependent on its kinase activity. Plk1 phosphorylates Kif2a and enhances its depolymerase activity in vitro. Inhibition or depletion of Plk1 decreases microtubule-associated Kif2a signals and increases the spindle microtubule intensity in vivo. Interestingly, Aurora A also interacts with and phosphorylates Kif2a. Phosphorylation of Kif2a by Aurora A suppresses its depolymerase activity in vitro, and inhibition of Aurora A increases the microtubule-associated Kif2a signals and reduces the spindle microtubule intensity in vivo. Thus, Kif2a is regulated positively by Plk1 and negatively by Aurora A. We propose that this antagonistic regulation confers differential stability to microtubules in the spindle versus at the pole versus in the cytosol, and that this spatial differential stability is important for spindle assembly and function.

The Aurora kinase complex, also called the chromosomal passenger complex (CPC), is essential for faithful chromosome segregation and completion of cell division. In Fungi and Animalia, this complex consists of the kinase Aurora B/AIR-2/Ipl1p, INCENP/ICP-1/Sli15p, and Survivin/BIR-1/Bir1p. A fourth subunit, Borealin/Dasra/CSC-1, is required for CPC targeting to centromeres and central spindles and has only been found in Animalia. Here we identified a new core component of the CPC in budding yeast, Nbl1p. NBL1 is essential for viability and nbl1 mutations cause chromosome missegregation and lagging chromosomes. Nbl1p colocalizes and copurifies with the CPC, and it is essential for CPC localization, stability, integrity, and function. Nbl1p is related to the N-terminus of Borealin/Dasra/CSC-1 and is similarly involved in connecting the other CPC subunits. Distant homology searching identified nearly 200, mostly unannotated, Borealin/Dasra/CSC-1-related proteins from nearly 150 species within Fungi and Animalia. Analysis of the sequence of these proteins, combined with comparative protein structure modeling of Bir1p-Nbl1p-Sli15p using the crystal structure of the human Survivin-Borealin-INCENP complex, revealed a striking structural conservation across a broad range of species. Our biological and computational analyses therefore establish that the fundamental design of the CPC is conserved from Fungi to Animalia.

Highly conserved among eukaryotic cells, the AMP-activated kinase (AMPK) is a central regulator of carbon metabolism. To map the complete network of interactions around AMPK in yeast (Snf1) and to evaluate the role of its regulatory subunit Snf4, we measured global mRNA, protein and metabolite levels in wild type, Deltasnf1, Deltasnf4, and Deltasnf1Deltasnf4 knockout strains. Using four newly developed computational tools, including novel DOGMA sub-network analysis, we showed the benefits of three-level ome-data integration to uncover the global Snf1 kinase role in yeast. We for the first time identified Snf1's global regulation on gene and protein expression levels, and showed that yeast Snf1 has a far more extensive function in controlling energy metabolism than reported earlier. Additionally, we identified complementary roles of Snf1 and Snf4. Similar to the function of AMPK in humans, our findings showed that Snf1 is a low-energy checkpoint and that yeast can be used more extensively as a model system for studying the molecular mechanisms underlying the global regulation of AMPK in mammals, failure of which leads to metabolic diseases.

Background: Chromosomal instability (CIN), a feature widely shared by cells from solid tumors, is caused by occasional chromosome missegregations during cell division. Two of the causes of CIN are weakened mitotic checkpoint signaling and persistent merotelic attachments that result in lagging chromosomes during anaphase., Principal Findings: Here we identify an autophosphorylation event on Mps1 that is required to prevent these two causes of CIN. Mps1 is phosphorylated in mitotic cells on at least 7 residues, 4 of which by autophosphorylation. One of these, T676, resides in the activation loop of the kinase domain and a mutant that cannot be phosphorylated on T676 is less active than wild-type Mps1 but is not kinase-dead. Strikingly, cells in which endogenous Mps1 was replaced with this mutant are viable but missegregate chromosomes frequently. Anaphase is initiated in the presence of misaligned and lagging chromosomes, indicative of a weakened checkpoint and persistent merotelic attachments, respectively., Conclusions/significance: We propose that full activity of Mps1 is essential for maintaining chromosomal stability by allowing resolution of merotelic attachments and to ensure that single kinetochores achieve the strength of checkpoint signaling sufficient to prevent premature anaphase onset and chromosomal instability. To our knowledge, phosphorylation of T676 on Mps1 is the first post-translational modification in human cells of which the absence causes checkpoint weakening and CIN without affecting cell viability.

Through a convergence of functional genomic and proteomic studies, we identify Bora as a previously unknown cell cycle protein that interacts with the Plk1 kinase and the SCF-beta-TrCP ubiquitin ligase. We show that the Bora protein peaks in G2 and is degraded by proteasomes in mitosis. Proteolysis of Bora requires the Plk1 kinase activity and is mediated by SCF-beta-TrCP. Plk1 phosphorylates a conserved DSGxxT degron in Bora and promotes its interaction with beta-TrCP. Mutations in this degron stabilize Bora. Expression of a nondegradable Bora variant prolongs the metaphase and delays anaphase onset, indicating a physiological requirement of Bora degradation. Interestingly, the activity of Bora is also required for normal mitotic progression, as knockdown of Bora activates the spindle checkpoint and delays sister chromatid segregation. Mechanistically, Bora regulates spindle stability and microtubule polymerization and promotes tension across sister kinetochores during mitosis. We conclude that tight regulation of the Bora protein by its synthesis and degradation is critical for cell cycle progression.

Activation of the checkpoint kinase Rad53 is a critical response to DNA damage that results in stabilization of stalled replication forks, inhibition of late-origin initiation, up-regulation of dNTP levels, and delayed entry to mitosis. Activation of Rad53 is well understood and involves phosphorylation by the protein kinases Mec1 and Tel1 as well as in trans autophosphorylation by Rad53 itself. However, deactivation of Rad53, which must occur to allow the cell to recover from checkpoint arrest, is not well understood. Here, we present genetic and biochemical evidence that the type 2A-like protein phosphatase Pph3 forms a complex with Psy2 (Pph3-Psy2) that binds and dephosphorylates activated Rad53 during treatment with, and recovery from, methylmethane sulfonate-mediated DNA damage. In the absence of Pph3-Psy2, Rad53 dephosphorylation and the resumption of DNA synthesis are delayed during recovery from DNA damage. This delay in DNA synthesis reflects a failure to restart stalled replication forks, whereas, remarkably, genome replication is eventually completed by initiating late origins of replication despite the presence of hyperphosphorylated Rad53. These findings suggest that Rad53 regulates replication fork restart and initiation of late firing origins independently and that regulation of these processes is mediated by specific Rad53 phosphatases.

Like apoptosis, oncogene-induced senescence is a barrier to tumor development. However, relatively little is known about the signaling pathways mediating the senescence response. p38-regulated/activated protein kinase (PRAK) is a p38 MAPK substrate whose physiological functions are poorly understood. Here we describe a role for PRAK in tumor suppression by demonstrating that PRAK mediates senescence upon activation by p38 in response to oncogenic ras. PRAK deficiency in mice enhances DMBA-induced skin carcinogenesis, coinciding with compromised senescence induction. In primary cells, inactivation of PRAK prevents senescence and promotes oncogenic transformation. Furthermore, we show that PRAK activates p53 by direct phosphorylation. We propose that phosphorylation of p53 by PRAK following activation of p38 MAPK by ras plays an important role in ras-induced senescence and tumor suppression.

Background: Duplicated chromosomes are equally segregated to daughter cells by a bipolar mitotic spindle during cell division. By metaphase, sister chromatids are coupled to microtubule (MT) plus ends from opposite poles of the bipolar spindle via kinetochores. Here we describe a phosphorylation event that promotes the coupling of kinetochores to microtubule plus ends., Results: Dam1 is a kinetochore component that directly binds to microtubules. We identified DAM1-765, a dominant allele of DAM1, in a genetic screen for mutations that increase stress on the spindle pole body (SPB) in Saccharomyces cerevisiae. DAM1-765 contains the single mutation S221F. We show that S221 is one of six Dam1 serines (S13, S49, S217, S218, S221, and S232) phosphorylated by Mps1 in vitro. In cells with single mutations S221F, S218A, or S221A, kinetochores in the metaphase spindle form tight clusters that are closer to the SPBs than in a wild-type cell. Five lines of experimental evidence, including localization of spindle components by fluorescence microscopy, measurement of microtubule dynamics by fluorescence redistribution after photobleaching, and reconstructions of three-dimensional structure by electron tomography, combined with computational modeling of microtubule behavior strongly indicate that, unlike wild-type kinetochores, Dam1-765 kinetochores do not colocalize with an equal number of plus ends. Despite the uncoupling of the kinetochores from the plus ends of MTs, the DAM1-765 cells are viable, complete the cell cycle with the same kinetics as wild-type cells, and biorient their chromosomes as efficiently as wild-type cells., Conclusions: We conclude that phosphorylation of Dam1 residues S218 and S221 by Mps1 is required for efficient coupling of kinetochores to MT plus ends. We find that efficient plus-end coupling is not required for (1) maintenance of chromosome biorientation, (2) maintenance of tension between sister kinetochores, or (3) chromosome segregation.

Elevations in circulating glucose and gut hormones during feeding promote pancreatic islet cell viability in part via the calcium- and cAMP-dependent activation of the transcription factor CREB. Here, we describe a signaling module that mediates the synergistic effects of these pathways on cellular gene expression by stimulating the dephosphorylation and nuclear entry of TORC2, a CREB coactivator. This module consists of the calcium-regulated phosphatase calcineurin and the Ser/Thr kinase SIK2, both of which associate with TORC2. Under resting conditions, TORC2 is sequestered in the cytoplasm via a phosphorylation-dependent interaction with 14-3-3 proteins. Triggering of the calcium and cAMP second messenger pathways by glucose and gut hormones disrupts TORC2:14-3-3 complexes via complementary effects on TORC2 dephosphorylation; calcium influx increases calcineurin activity, whereas cAMP inhibits SIK2 kinase activity. Our results illustrate how a phosphatase/kinase module connects two signaling pathways in response to nutrient and hormonal cues.

The Aurora kinase Ipl1p plays a crucial role in regulating kinetochore-microtubule attachments in budding yeast, but the underlying basis for this regulation is not known. To identify Ipl1p targets, we first purified 28 kinetochore proteins from yeast protein extracts. These studies identified five previously uncharacterized kinetochore proteins and defined two additional kinetochore subcomplexes. We then used mass spectrometry to identify 18 phosphorylation sites in 7 of these 28 proteins. Ten of these phosphorylation sites are targeted directly by Ipl1p, allowing us to identify a consensus phosphorylation site for an Aurora kinase. Our systematic mutational analysis of the Ipl1p phosphorylation sites demonstrated that the essential microtubule binding protein Dam1p is a key Ipl1p target for regulating kinetochore-microtubule attachments in vivo.

The evolutionarily conserved yeast checkpoint protein kinase Rad53 regulates cell cycle progression, transcription, and DNA repair in response to DNA damage. To uncover potential regulatory targets of Rad53, we identified proteins physically associated with it in vivo using protein affinity purification and tandem mass spectrometry. Here we report that Rad53 interacts in a dynamic functional manner with Asf1, a chromatin assembly factor recently shown to mediate deposition of acetylated histones H3 and H4 onto newly replicated DNA. Biochemical and molecular genetic studies suggest that Asf1 is an important target of the Rad53-dependent DNA damage response and that Rad53 may directly regulate chromatin assembly during DNA replication and repair.

We have previously reported that in thrombin-stimulated human platelets, cytosolic phospholipase A(2) (cPLA2) is phosphorylated on Ser-505 by p38 protein kinase and on Ser-727 by an unknown kinase. Pharmacological inhibition of p38 leads to inhibition of cPLA2 phosphorylation at both Ser-505 and Ser-727 suggesting that the kinase responsible for phosphorylation on Ser-727 is activated in a p38-dependent pathway. By using Chinese hamster ovary, HeLa, and HEK293 cells stably transfected with wild type and phosphorylation site mutant forms of cPLA2, we show that phosphorylation of cPLA2 at both Ser-505 and Ser-727 and elevation of Ca(2+) leads to its activation in agonist-stimulated cells. The p38-activated protein kinases MNK1, MSK1, and PRAK1 phosphorylate cPLA2 in vitro uniquely on Ser-727 as shown by mass spectrometry. Furthermore, MNK1 and PRAK1, but not MSK1, is present in platelets and undergo modest activation in response to thrombin. Expression of a dominant negative form of MNK1 in HEK293 cells leads to significant inhibition of cPLA2-mediated arachidonate release. The results suggest that MNK1 or a closely related kinase is responsible for in vivo phosphorylation of cPLA2 on Ser-727.

The SAGA histone acetyltransferase/transcriptional adaptor complex is composed of multiple transcriptional regulators including Ada, Spt, and TAFII proteins. Here we identify an additional novel subunit of the complex, Tra1, an ATM/PI-3-kinase-related homolog of the human TRRAP cofactor, which is essential for c-Myc and E2F-mediated oncogenic transformation. Mass spectrometry, immunoblotting, and immunoprecipitation experiments confirm the stable association of this protein within SAGA. In addition, the Tra1 protein is a component of at least two other histone acetyltransferase protein complexes. These results indicate a role for Tra1 in the regulation of transcriptional activation through the recruitment of HAT activity to an activator-bound promoter.