Rad53 Checkpoint Kinase Phosphorylation Site Preference Identified in the Swi6 Protein of Saccharomyces cerevisiae

ABSTRACT Rad53 of Saccharomyces cerevisiae is a checkpoint kinase whose structure and function are conserved among eukaryotes. When a cell detects damaged DNA, Rad53 activity is dramatically increased, which ultimately leads to changes in DNA replication, repair, and cell division. Despite its central role in checkpoint signaling, little is known about Rad53 substrates or substrate specificity. A number of proteins are implicated as Rad53 substrates; however, the evidence remains indirect. Previously, we have provided evidence that Swi6, a subunit of the Swi4/Swi6 late-G1-specific transcriptional activator, is a substrate of Rad53 in the G1/S DNA damage checkpoint. In the present study we identify Rad53 phosphorylation sites in Swi6 in vitro and demonstrate that at least one of them is targeted by Rad53 in vivo. Mutations in these phosphorylation sites in Swi6 shorten but do not eliminate the Rad53-dependent delay of the G1-to-S transition after DNA damage. We derive a consensus for Rad53 site preference at positions −2 and +2 (−2/+2) and identify its potential substrates in the yeast proteome. Finally, we present evidence that one of these candidates, the cohesin complex subunit Scc1 undergoes DNA damage-dependent phosphorylation, which is in part dependent on Rad53.

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Tethering DNA Damage Checkpoint Mediator Proteins Topoisomerase IIβ-binding Protein 1 (TopBP1) and Claspin to DNA Activates Ataxia-Telangiectasia Mutated and RAD3-related (ATR) Phosphorylation of Checkpoint Kinase 1 (Chk1)

Journal of Biological Chemistry ◽

10.1074/jbc.m111.237958 ◽

2011 ◽

Vol 286 (22) ◽

pp. 19229-19236 ◽

Cited By ~ 29

Author(s):

Laura A. Lindsey-Boltz ◽

Aziz Sancar

Keyword(s):

Dna Damage ◽

Ataxia Telangiectasia ◽

Mammalian Cells ◽

Binding Protein ◽

Effector Proteins ◽

Ataxia Telangiectasia Mutated ◽

Checkpoint Kinase ◽

Atr Kinase

The ataxia-telangiectasia mutated and RAD3-related (ATR) kinase initiates DNA damage signaling pathways in human cells after DNA damage such as that induced upon exposure to ultraviolet light by phosphorylating many effector proteins including the checkpoint kinase Chk1. The conventional view of ATR activation involves a universal signal consisting of genomic regions of replication protein A-covered single-stranded DNA. However, there are some indications that the ATR-mediated checkpoint can be activated by other mechanisms. Here, using the well defined Escherichia coli lac repressor/operator system, we have found that directly tethering the ATR activator topoisomerase IIβ-binding protein 1 (TopBP1) to DNA is sufficient to induce ATR phosphorylation of Chk1 in an in vitro system as well as in vivo in mammalian cells. In addition, we find synergistic activation of ATR phosphorylation of Chk1 when the mediator protein Claspin is also tethered to the DNA with TopBP1. Together, these findings indicate that crowding of checkpoint mediator proteins on DNA is sufficient to activate the ATR kinase.

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Threonine-11, Phosphorylated by Rad3 and ATM In Vitro, Is Required for Activation of Fission Yeast Checkpoint Kinase Cds1

Molecular and Cellular Biology ◽

10.1128/mcb.21.10.3398-3404.2001 ◽

2001 ◽

Vol 21 (10) ◽

pp. 3398-3404 ◽

Cited By ~ 36

Author(s):

Katsunori Tanaka ◽

Michael N. Boddy ◽

Xiao-Bo Chen ◽

Clare H. McGowan ◽

Paul Russell

Keyword(s):

Dna Damage ◽

Dna Replication ◽

Fission Yeast ◽

Ataxia Telangiectasia ◽

Null Mutant ◽

Ataxia Telangiectasia Mutated ◽

Tolerance Mechanisms ◽

Checkpoint Kinase ◽

And Function

ABSTRACT Fission yeast Cds1 is phosphorylated and activated when DNA replication is interrupted by nucleotide starvation or DNA damage. Cds1 enforces the S-M checkpoint that couples mitosis (M) to the completion of DNA synthesis (S). Cds1 also controls replicational stress tolerance mechanisms. Cds1 is regulated by a group of proteins that includes Rad3, a kinase related to human checkpoint kinase ATM (ataxia telangiectasia mutated). ATM phosphorylates serine or threonine followed by glutamine (SQ or TQ). Here we show that in vitro, Rad3 and ATM phosphorylate the N-terminal domain of Cds1 at the motif T11Q12. Substitution of threonine-11 with alanine (T11A) abolished Cds1 activation that occurs when DNA replication is inhibited by hydroxyurea (HU) treatment. Thecds1-T11A mutant was profoundly sensitive to HU, although not quite as sensitive as a cds1− null mutant. Cds1T11A was unable to enforce the S-M checkpoint. These results strongly suggest that Rad3-dependent phosphorylation of Cds1 at threonine-11 is required for Cds1 activation and function.

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A proline-rich sequence unique to MEK1 and MEK2 is required for raf binding and regulates MEK function.

Molecular and Cellular Biology ◽

10.1128/mcb.15.10.5214 ◽

1995 ◽

Vol 15 (10) ◽

pp. 5214-5225 ◽

Cited By ~ 137

Author(s):

A D Catling ◽

H J Schaeffer ◽

C W Reuter ◽

G R Reddy ◽

M J Weber

Keyword(s):

Protein Interactions ◽

Critical Role ◽

Phosphorylation Site ◽

Specific Protein ◽

Protein Protein Interactions ◽

Serum Stimulation ◽

And Function

Mammalian MEK1 and MEK2 contain a proline-rich (PR) sequence that is absent both from the yeast homologs Ste7 and Byr1 and from a recently cloned activator of the JNK/stress-activated protein kinases, SEK1/MKK4. Since this PR sequence occurs in MEKs that are regulated by Raf family enzymes but is missing from MEKs and SEKs activated independently of Raf, we sought to investigate the role of this sequence in MEK1 and MEK2 regulation and function. Deletion of the PR sequence from MEK1 blocked the ability of MEK1 to associate with members of the Raf family and markedly attenuated activation of the protein in vivo following growth factor stimulation. In addition, this sequence was necessary for efficient activation of MEK1 in vitro by B-Raf but dispensable for activation by a novel MEK1 activator which we have previously detected in fractionated fibroblast extracts. Furthermore, we found that a phosphorylation site within the PR sequence of MEK1 was required for sustained MEK1 activity in response to serum stimulation of quiescent fibroblasts. Consistent with this observation, we observed that MEK2, which lacks a phosphorylation site at the corresponding position, was activated only transiently following serum stimulation. Finally, we found that deletion of the PR sequence from a constitutively activated MEK1 mutant rendered the protein nontransforming in Rat1 fibroblasts. These observations indicate a critical role for the PR sequence in directing specific protein-protein interactions important for the activation, inactivation, and downstream functioning of the MEKs.

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The Arabidopsis Rho of Plants GTPase ROP1 Is a Potential Calcium-Dependent Protein Kinase (CDPK) Substrate

Plants ◽

10.3390/plants10102053 ◽

2021 ◽

Vol 10 (10) ◽

pp. 2053

Author(s):

Dalma Ménesi ◽

Éva Klement ◽

Györgyi Ferenc ◽

Attila Fehér

Keyword(s):

Protein Kinases ◽

Phosphorylation Site ◽

Interaction Strength ◽

Yeast Cells ◽

Phosphorylation Sites ◽

Mutant Proteins ◽

In Vivo Test ◽

Calcium Dependent

Plant Rho-type GTPases (ROPs) are versatile molecular switches involved in a number of signal transduction pathways. Although it is well known that they are indirectly linked to protein kinases, our knowledge about their direct functional interaction with upstream or downstream protein kinases is scarce. It is reasonable to suppose that similarly to their animal counterparts, ROPs might also be regulated by phosphorylation. There is only, however, very limited experimental evidence to support this view. Here, we present the analysis of two potential phosphorylation sites of AtROP1 and two types of potential ROP-kinases. The S74 site of AtROP1 has been previously shown to potentially regulate AtROP1 activation dependent on its phosphorylation state. However, the kinase phosphorylating this evolutionarily conserved site could not be identified: we show here that despite of the appropriate phosphorylation site consensus sequences around S74 neither the selected AGC nor CPK kinases phosphorylate S74 of AtROP1 in vitro. However, we identified several phosphorylation sites other than S74 for the CPK17 and 34 kinases in AtROP1. One of these sites, S97, was tested for biological relevance. Although the mutation of S97 to alanine (which cannot be phosphorylated) or glutamic acid (which mimics phosphorylation) somewhat altered the protein interaction strength of AtROP1 in yeast cells, the mutant proteins did not modify pollen tube growth in an in vivo test.

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Cdc25A Serine 123 Phosphorylation Couples Centrosome Duplication with DNA Replication and Regulates Tumorigenesis

Molecular and Cellular Biology ◽

10.1128/mcb.00138-08 ◽

2008 ◽

Vol 28 (24) ◽

pp. 7442-7450 ◽

Cited By ~ 10

Author(s):

Sathyavageeswaran Shreeram ◽

Weng Kee Hee ◽

Dmitry V. Bulavin

Keyword(s):

Cell Cycle ◽

Dna Damage ◽

Dna Replication ◽

Cell Cycle Progression ◽

Phosphorylation Site ◽

Centrosome Duplication ◽

Cycle Progression ◽

Cdc25a Phosphatase

ABSTRACT The cell division cycle 25A (Cdc25A) phosphatase is a critical regulator of cell cycle progression under normal conditions and after stress. Stress-induced degradation of Cdc25A has been proposed as a major way of delaying cell cycle progression. In vitro studies pointed toward serine 123 as a key site in regulation of Cdc25A stability after exposure to ionizing radiation (IR). To address the role of this phosphorylation site in vivo, we generated a knock-in mouse in which alanine was substituted for serine 123. The Cdc25 S123A knock-in mice appeared normal, and, unexpectedly, cells derived from them exhibited unperturbed cell cycle and DNA damage responses. In turn, we found that Cdc25A was present in centrosomes and that Cdc25A levels were not reduced after IR in knock-in cells. This resulted in centrosome amplification due to lack of induction of Cdk2 inhibitory phosphorylation after IR specifically in centrosomes. Further, Cdc25A knock-in animals appeared sensitive to IR-induced carcinogenesis. Our findings indicate that Cdc25A S123 phosphorylation is crucial for coupling centrosome duplication to DNA replication cycles after DNA damage and therefore is likely to play a role in the regulation of tumorigenesis.

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Regulation of p53 Function and Stability by Phosphorylation

Molecular and Cellular Biology ◽

10.1128/mcb.19.3.1751 ◽

1999 ◽

Vol 19 (3) ◽

pp. 1751-1758 ◽

Cited By ~ 304

Author(s):

Margaret Ashcroft ◽

Michael H. G. Kubbutat ◽

Karen H. Vousden

Keyword(s):

Dna Damage ◽

Protein Kinases ◽

Phosphorylation Site ◽

Tumor Suppressor Protein ◽

Phosphorylation Sites ◽

Wild Type ◽

Mutant Proteins ◽

Encoding Gene ◽

P53 Tumor Suppressor Protein

ABSTRACT The p53 tumor suppressor protein can be phosphorylated at several sites within the N- and C-terminal domains, and several protein kinases have been shown to phosphorylate p53 in vitro. In this study, we examined the activity of p53 proteins with combined mutations at all of the reported N-terminal phosphorylation sites (p53N-term), all of the C-terminal phosphorylation sites (p53C-term), or all of the phosphorylation sites together (p53N/C-term). Each of these mutant proteins retained transcriptional transactivation functions, indicating that phosphorylation is not essential for this activity of p53, although a subtle contribution of the C-terminal phosphorylation sites to the activation of expression of the endogenous p21Waf1/Cip1-encoding gene was detected. Mutation of the phosphorylation sites to alanine did not affect the sensitivity of p53 to binding to or degradation by Mdm2, although alteration of residues 15 and 37 to aspartic acid, which could mimic phosphorylation, resulted in a slight resistance to Mdm2-mediated degradation, consistent with recent reports that phosphorylation at these sites inhibits the p53-Mdm2 interaction. However, expression of the phosphorylation site mutant proteins in both wild-type p53-expressing and p53-null lines showed that all of the mutant proteins retained the ability to be stabilized following DNA damage. This indicates that phosphorylation is not essential for DNA damage-induced stabilization of p53, although phosphorylation could clearly contribute to p53 stabilization under some conditions.

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Phosphorylation of GAP-43 T172 is a molecular marker of growing axons in a wide range of mammals including primates

Molecular Brain ◽

10.1186/s13041-021-00755-0 ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Masayasu Okada ◽

Yosuke Kawagoe ◽

Yuta Sato ◽

Motohiro Nozumi ◽

Yuya Ishikawa ◽

...

Keyword(s):

Molecular Marker ◽

Phosphorylation Site ◽

Axon Growth ◽

Growth Cones ◽

Specific Protein ◽

Phosphorylation Sites ◽

Quarter Century ◽

Wide Range

AbstractGAP-43 is a vertebrate neuron-specific protein and that is strongly related to axon growth and regeneration; thus, this protein has been utilized as a classical molecular marker of these events and growth cones. Although GAP-43 was biochemically characterized more than a quarter century ago, how this protein is related to these events is still not clear. Recently, we identified many phosphorylation sites in the growth cone membrane proteins of rodent brains. Two phosphorylation sites of GAP-43, S96 and T172, were found within the top 10 hit sites among all proteins. S96 has already been characterized (Kawasaki et al., 2018), and here, phosphorylation of T172 was characterized. In vitro (cultured neurons) and in vivo, an antibody specific to phosphorylated T172 (pT172 antibody) specifically recognized cultured growth cones and growing axons in developing mouse neurons, respectively. Immunoblotting showed that pT172 antigens were more rapidly downregulated throughout development than those of pS96 antibody. From the primary structure, this phosphorylation site was predicted to be conserved in a wide range of animals including primates. In the developing marmoset brainstem and in differentiated neurons derived from human induced pluripotent stem cells, immunoreactivity with pT172 antibody revealed patterns similar to those in mice. pT172 antibody also labeled regenerating axons following sciatic nerve injury. Taken together, the T172 residue is widely conserved in a wide range of mammals including primates, and pT172 is a new candidate molecular marker for growing axons.

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A RAD9-dependent cell cycle arrest in response to unresolved recombination intermediates in Saccharomyces cerevisiae

10.1101/736850 ◽

2019 ◽

Author(s):

Hardeep Kaur ◽

GN Krishnaprasad ◽

Michael Lichten

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Cycle ◽

Dna Damage ◽

Cell Division ◽

Cell Cycle Arrest ◽

Mitotic Cell ◽

Cycle Arrest ◽

Associated Lesions

AbstractIn Saccharomyces cerevisiae, the conserved Sgs1-Top3-Rmi1 helicase-decatenase regulates homologous recombination by limiting accumulation of recombination intermediates that are precursors of crossovers. In vitro studies have suggested that the dissolution of double-Holliday junction joint molecules by Sgs1-driven convergent junction migration and Top3-Rmi1 mediated strand decatenation could be responsible for this. To ask if dissolution occurs in vivo, we conditionally depleted Sgs1 and/or Rmi1 during return to growth, a procedure where recombination intermediates formed during meiosis are resolved when cells resume the mitotic cell cycle. Sgs1 depletion during return to growth delayed joint molecule resolution, but ultimately most were resolved and cells divided normally. In contrast, Rmi1 depletion resulted in delayed and incomplete joint molecule resolution, and most cells did not divide. rad9Δ mutation restored cell division in Rmi1-depleted cells, indicating that the DNA damage checkpoint caused this cell cycle arrest. Restored cell division in rad9Δ, Rmi1-depleted cells frequently produced anucleate cells, consistent with the suggestion that persistent recombination intermediates prevented chromosome segregation. Our findings indicate that Sgs1-Top3-Rmi1 acts in vivo, as it does in vitro, to promote recombination intermediate resolution by dissolution. They also indicate that, in the absence of Top3-Rmi1 activity, unresolved recombination intermediates persist and activate the DNA damage response, which is usually thought to be activated by much earlier DNA damage-associated lesions.

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Non-Canonical G-quadruplexes cause the hCEB1 minisatellite instability in Saccharomyces cerevisiae

eLife ◽

10.7554/elife.26884 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 19

Author(s):

Aurèle Piazza ◽

Xiaojie Cui ◽

Michael Adrian ◽

Frédéric Samazan ◽

Brahim Heddi ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Structure Function ◽

Genomic Instability ◽

Function Analysis ◽

Structural Features ◽

Detectable Effect ◽

Definition Of ◽

And Function

G-quadruplexes (G4) are polymorphic four-stranded structures formed by certain G-rich nucleic acids in vitro, but the sequence and structural features dictating their formation and function in vivo remains uncertain. Here we report a structure-function analysis of the complex hCEB1 G4-forming sequence. We isolated four G4 conformations in vitro, all of which bear unusual structural features: Form 1 bears a V-shaped loop and a snapback guanine; Form 2 contains a terminal G-triad; Form 3 bears a zero-nucleotide loop; and Form 4 is a zero-nucleotide loop monomer or an interlocked dimer. In vivo, Form 1 and Form 2 differently account for 2/3rd of the genomic instability of hCEB1 in two G4-stabilizing conditions. Form 3 and an unidentified form contribute to the remaining instability, while Form 4 has no detectable effect. This work underscores the structural polymorphisms originated from a single highly G-rich sequence and demonstrates the existence of non-canonical G4s in cells, thus broadening the definition of G4-forming sequences.

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The biogenesis and function of nucleosome arrays

Nature Communications ◽

10.1038/s41467-021-27285-6 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Ashish Kumar Singh ◽

Tamás Schauer ◽

Lena Pfaller ◽

Tobias Straub ◽

Felix Mueller-Planitz

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Damage ◽

Dna Sequence ◽

Chromatin Remodeling ◽

Genome Engineering ◽

Genotoxic Stress ◽

Eukaryotic Cells ◽

Nucleosome Density ◽

And Function

AbstractNumerous chromatin remodeling enzymes position nucleosomes in eukaryotic cells. Aside from these factors, transcription, DNA sequence, and statistical positioning of nucleosomes also shape the nucleosome landscape. The precise contributions of these processes remain unclear due to their functional redundancy in vivo. By incisive genome engineering, we radically decreased their redundancy in Saccharomyces cerevisiae. The transcriptional machinery strongly disrupts evenly spaced nucleosomes. Proper nucleosome density and DNA sequence are critical for their biogenesis. The INO80 remodeling complex helps space nucleosomes in vivo and positions the first nucleosome over genes in an H2A.Z-independent fashion. INO80 requires its Arp8 subunit but unexpectedly not the Nhp10 module for spacing. Cells with irregularly spaced nucleosomes suffer from genotoxic stress including DNA damage, recombination and transpositions. We derive a model of the biogenesis of the nucleosome landscape and suggest that it evolved not only to regulate but also to protect the genome.

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