Phosphoproteomic Analysis of Thermomorphogenic Responses in Arabidopsis

Plants rapidly adapt to elevated ambient temperature by adjusting their growth and developmental programs. To date, a number of experiments have been carried out to understand how plants sense and respond to warm temperatures. However, how warm temperature signals are relayed from thermosensors to transcriptional regulators is largely unknown. To identify new early regulators of plant thermo-responsiveness, we performed phosphoproteomic analysis using TMT (Tandem Mass Tags) labeling and phosphopeptide enrichment with Arabidopsis etiolated seedlings treated with or without 3h of warm temperatures (29°C). In total, we identified 13,160 phosphopeptides in 5,125 proteins with 10,700 quantifiable phosphorylation sites. Among them, 200 sites (180 proteins) were upregulated, while 120 sites (87 proteins) were downregulated by elevated temperature. GO (Gene Ontology) analysis indicated that phosphorelay-related molecular function was enriched among the differentially phosphorylated proteins. We selected ATL6 (ARABIDOPSIS TOXICOS EN LEVADURA 6) from them and expressed its native and phosphorylation-site mutated (S343A S357A) forms in Arabidopsis and found that the mutated form of ATL6 was less stable than that of the native form both in vivo and in cell-free degradation assays. Taken together, our data revealed extensive protein phosphorylation during thermo-responsiveness, providing new candidate proteins/genes for studying plant thermomorphogenesis in the future.

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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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Differential Hormone-Dependent Phosphorylation of Progesterone Receptor A and B Forms Revealed by a Phosphoserine Site-Specific Monoclonal Antibody

Molecular Endocrinology ◽

10.1210/mend.14.1.0413 ◽

2000 ◽

Vol 14 (1) ◽

pp. 52-65 ◽

Cited By ~ 56

Author(s):

David L. Clemm ◽

Lori Sherman ◽

Viroj Boonyaratanakornkit ◽

William T. Schrader ◽

Nancy L. Weigel ◽

...

Keyword(s):

Progesterone Receptor ◽

Phosphorylation Site ◽

Phosphorylation Sites ◽

Experimental Conditions ◽

Western Blots ◽

Amino Terminal ◽

Differential Reactivity ◽

Flanking Sequences ◽

Human Progesterone Receptor

Abstract Human progesterone receptor (PR) is phosphorylated on multiple serine residues (at least seven sites) in a manner that involves distinct groups of sites coordinately regulated by hormone and different kinases. Progress on defining a functional role for PR phosphorylation has been hampered both by the complexity of phosphorylation and the lack of simple, nonradioactive methods to detect the influence of ligands and other signaling pathways on specific PR phosphorylation sites in vivo. Toward this end, we have produced monoclonal antibodies (MAbs) that recognize specific phosphorylation sites within human PR including a basal site at Ser 190 (MAb P190) and a hormone-induced site at Ser 294 (MAb P294). Biochemical experiments showed the differential reactivity of the P190 and P294 MAbs for phosphorylated and unphosphorylated forms of PR. Both MAbs recognize specific phosphorylated forms of PR under different experimental conditions including denatured PR protein by Western blots and PR in its native conformation in solution or complexed to specific target DNA. As detected by Western blot of T47D cells treated with hormone for different times, hormone-dependent down-regulation of total PR and the Ser 190 phosphorylation site occurred in parallel, whereas the Ser 294 phosphorylation site was down-regulated more rapidly. This difference in kinetics suggests that the Ser 294 site is more labile than basal sites and is acted upon by distinct phosphatases. A strong preferential hormone-dependent phosphorylation of Ser 294 was observed on PR-B as compared with the amino-terminal truncated A form of PR. This was unexpected because Ser 294 and flanking sequences are identical on both proteins, suggesting that a distinct conformation of the N-terminal domain of PR-A inhibits phosphorylation of this site. That Ser 294 lies within an inhibitory domain that mediates the unique repressive functions of PR-A raises the possibility that differential phosphorylation of Ser 294 is involved in the distinct functional properties of PR-A and PR-B.

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

Molecular and Cellular Biology ◽

10.1128/mcb.23.10.3405-3416.2003 ◽

2003 ◽

Vol 23 (10) ◽

pp. 3405-3416 ◽

Cited By ~ 32

Author(s):

Julia M. Sidorova ◽

Linda L. Breeden

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Damage ◽

Phosphorylation Site ◽

Site Preference ◽

Phosphorylation Sites ◽

Checkpoint Kinase ◽

Checkpoint Signaling ◽

And Function

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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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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The relative roles of specific N- and C-terminal phosphorylation sites in the disassembly of intermediate filament in mitotic BHK-21 cells

Journal of Cell Science ◽

10.1242/jcs.109.4.817 ◽

1996 ◽

Vol 109 (4) ◽

pp. 817-826 ◽

Cited By ~ 1

Author(s):

Y.H. Chou ◽

P. Opal ◽

R.A. Quinlan ◽

R.D. Goldman

Keyword(s):

Protein Kinase ◽

Phosphorylation Site ◽

Supramolecular Organization ◽

Phosphorylation Sites ◽

Bhk Cells ◽

Terminal Domain ◽

Kinase Phosphorylation ◽

Mitotic Cells

Previously we identified p34cdc2 as one of two protein kinases mediating the hyperphosphorylation and disassembly of vimentin in mitotic BHK-21 cells. In this paper, we identify the second kinase as a 37 kDa protein. This p37 protein kinase phosphorylates vimentin on two adjacent residues (thr-457 and ser-458) which are located in the C-terminal non-alpha-helical domain. Contrary to the p34cdc2 mediated N-terminal phosphorylation (at ser-55) which can disassemble vimentin intermediate filaments (IF) in vitro, p37 protein kinase phosphorylates vimentin-IF without obviously affecting its structure in vitro. We have further examined the in vivo role(s) of vimentin phosphorylation in the disassembly of the IF network in mitotic BHK cells by transient transfection assays. In untransfected BHK cells, the interphase vimentin IF networks are disassembled into non-filamentous aggregates when cells enter mitosis. Transfection of cells with vimentin cDNA lacking the p34cdc2 phosphorylation site (ser55:ala) effectively prevents mitotic cells from disassembling their IF. In contrast, apparently normal disassembly takes place in cells transfected with cDNA containing mutated p37 kinase phosphorylation sites (thr457:ala/ser458:ala). Transfection of cells with vimentin cDNAs lacking both the N- and C-terminal phosphorylation sites yields a phenotype indistinguishable from that obtained with the single N-terminal mutant. Taken together, our results demonstrate that the site-specific phosphorylation of the N-terminal domain, but not the C-terminal domain of vimentin plays an important role in determining the state of IF polymerization and supramolecular organization in mitotic cells.

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Multisite phosphorylation of doublecortin by cyclin-dependent kinase 5

Biochemical Journal ◽

10.1042/bj20040324 ◽

2004 ◽

Vol 381 (2) ◽

pp. 471-481 ◽

Cited By ~ 28

Author(s):

Mark E. GRAHAM ◽

Patricia RUMA-HAYNES ◽

Amanda G. CAPES-DAVIS ◽

Joanne M. DUNN ◽

Timothy C. TAN ◽

...

Keyword(s):

Phosphorylation Site ◽

Site Directed Mutagenesis ◽

Phosphorylation Sites ◽

Single Mutation ◽

Cyclin Dependent Kinase ◽

Major Site ◽

Multisite Phosphorylation ◽

Cyclin Dependent Kinase 5

Doublecortin (DCX) is a 40 kDa microtubule-associated protein required for normal neural migration and cortical layering during development. Mutations in the human DCX gene cause a disruption of cortical neuronal migration. Defects in cdk5 (cyclin-dependent kinase 5) also cause defects in neural migration and cortical layering. DCX is a substrate for cdk5 in vitro and in vivo and the major site of in vitro phosphorylation is Ser-297. We used a highly developed MS strategy to identify the cdk5 phosphorylation sites and determine the major and minor sites. Several phosphopeptides were identified from a tryptic digest of 32P-labelled, cdk5-phosphorylated DCX using a combination of off-line HPLC and matrix-assisted laser-desorption ionization-MS with alkaline phosphatase treatment. Tandem MS/MS enabled the identification of seven phosphorylation sites for cdk5. Monitoring of 32P label indicated that there was one major site, Ser-28, at the N-terminus, and a major site, Ser-339, in the serine/proline-rich domain at the C-terminus. Five other sites, Ser-287, Thr-289, Ser-297, Thr-326 and Ser-332, were also found in the tail. Site-directed mutagenesis largely supported these findings. Single mutation of Ser-28 reduced but did not abolish phosphorylation. Double, rather than single, mutation for Ser-332 and Ser-339 was required to reduce overall phosphorylation, suggesting an interaction between these sites. Truncations of the tail produced a significant reduction in cdk5 phosphorylation of DCX. These results do not support Ser-297 as the major cdk5 phosphorylation site in DCX, but indicate that DCX is subject to complex multisite phosphorylation. This illustrates the importance of a well-developed MS strategy to identify phosphorylation sites.

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ATR Dependent Phosphorylation of FANCM Is Required for the Fanconi Anemia Pathway.

Blood ◽

10.1182/blood.v110.11.837.837 ◽

2007 ◽

Vol 110 (11) ◽

pp. 837-837

Author(s):

Thiyam R. Singh ◽

Abdullah M. Ali ◽

Chang-hu Du ◽

Ruhikanta A. Meetei

Keyword(s):

Dna Damage ◽

Fanconi Anemia ◽

Bone Marrow Failure ◽

Phosphorylation Site ◽

Cell Cycle Checkpoint ◽

Phosphorylation Sites ◽

Core Complex ◽

Knock Out ◽

Hct116 Cells

Abstract Fanconi anemia (FA) is a rare, recessive disorder characterized by progressive bone marrow failure, developmental abnormalities, chromosome instability, cellular hypersensitivity to DNA cross-linking agents, and predisposition to cancer, mainly leukemias and squamous cell carcinomas of the head and neck. We have shown that FANCM which is one of the FA core complex proteins is hyperphosphorylated in response to DNA damage suggesting that it may serve as a signal transducer through which the activity of the FA-core complex is regulated. The cell cycle checkpoint kinase, ATR has been shown to act upstream of the FA pathway, however, its substrate within the FA-core complex has not been identified yet. FANCM contains multiple predicted ATR phosphorylation sites suggesting that FANCM could be a direct ATR target. In this study, we examined the roles of ATR in regulating FANCM phosphorylation in response to DNA damage: by kinetics study we found that phosphorylation of FANCM is concurrent with FANCD2 monoubiquitination; siRNA mediated suppression of ATR activity abrogates both phosphorylation of FANCM and monoubiquitination of FANCD2; and ATR knock out HCT116 cells display defective phosphorylation of FANCM as well as defective monoubiquitination of FANCD2 indicating that DNA damage induced phosphorylation of FANCM is ATR dependant. Furthermore, we used mass spectrometry to identify the in vivo phosphorylation sites of FANCM and found a novel DNA damage-inducible phosphorylation site (S-1045; one of the potential ATR phosphorylation sites) within FANCM protein. Using ATR knock out HCT116 cells and the anti-p-S1045 antibody, we show that phosphorylation of FANCM at S-1045 is ATR dependant. The biological relevance of phosphorylation of FANCM at S1045 in FA pathway will be investigated by functional complementation analysis with non phosphorylatable FANCM mutants in FANCM deficient cells.

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Identification of Raf-1 S471 as a Novel Phosphorylation Site Critical for Raf-1 and B-Raf Kinase Activities and for MEK Binding

Molecular Biology of the Cell ◽

10.1091/mbc.e05-02-0090 ◽

2005 ◽

Vol 16 (10) ◽

pp. 4733-4744 ◽

Cited By ~ 27

Author(s):

Jun Zhu ◽

Vitaly Balan ◽

Agnieszka Bronisz ◽

Karina Balan ◽

Hengrui Sun ◽

...

Keyword(s):

Kinase Activity ◽

Cell Transformation ◽

Signal Transmission ◽

Phosphorylation Site ◽

Kinase Domain ◽

Mitogen Activated Protein Kinase ◽

Phosphorylation Sites ◽

Raf Kinase ◽

Mitogen Activated Protein

The Ras-Raf-MAPK cascade is a key growth-signaling pathway and its uncontrolled activation results in cell transformation. Although the general features of the signal transmission along the cascade are reasonably defined, the mechanisms underlying Raf activation remain incompletely understood. Here, we show that Raf-1 dephosphorylation, primarily at epidermal growth factor (EGF)-induced sites, abolishes Raf-1 kinase activity. Using mass spectrometry, we identified five novel in vivo Raf-1 phosphorylation sites, one of which, S471, is located in subdomain VIB of Raf-1 kinase domain. Mutational analyses demonstrated that Raf-1 S471 is critical for Raf-1 kinase activity and for its interaction with mitogen-activated protein kinase kinase (MEK). Similarly, mutation of the corresponding B-Raf site, S578, resulted in an inactive kinase, suggesting that the same Raf-1 and B-Raf phosphorylation is needed for Raf kinase activation. Importantly, the naturally occurring, cancer-associated B-Raf activating mutation V599E suppressed the S578A mutation, suggesting that introducing a charged residue at this region eliminates the need for an activating phosphorylation. Our results demonstrate an essential role of specific EGF-induced Raf-1 phosphorylation sites in Raf-1 activation, identify Raf-1 S471 as a novel phosphorylation site critical for Raf-1 and B-Raf kinase activities, and point to the possibility that the V599E mutation activates B-Raf by mimicking a phosphorylation at the S578 site.

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Structure and function of C-CAM1: effects of the cytoplasmic domain on cell aggregation

Biochemical Journal ◽

10.1042/bj3110239 ◽

1995 ◽

Vol 311 (1) ◽

pp. 239-245 ◽

Cited By ~ 18

Author(s):

S H Lin ◽

W Luo ◽

K Earley ◽

P Cheung ◽

D C Hixson

Keyword(s):

Signal Transduction ◽

Cell Adhesion ◽

Amino Acid ◽

Cyclic Amp ◽

Phosphorylation Site ◽

Cytoplasmic Domain ◽

Tumour Suppressor ◽

Phosphorylation Sites ◽

Cytoplasmic Domains

C-CAMs are epithelial cell-adhesion molecules of the immunoglobulin supergene family with sequences highly homologous to carcinoembryonic antigen (CEA). C-CAMs and their human homologues, biliary glycoproteins, are unique among the CEA-family proteins in that they have cytoplasmic domains. Furthermore, alternative splicing generates C-CAM isoforms with different cytoplasmic domains, suggesting that the cytoplasmic domains of C-CAM may play important roles in regulating the function or functions of C-CAM. By using both sense and antisense approaches, we have shown that C-CAM1 is a tumour suppressor in prostate carcinogenesis. This observation raises the possibility that the cytoplasmic domain of C-CAM1 may be involved in signal transduction or interaction with cytoskeletal elements to elicit the tumour suppressor function. The cytoplasmic domain of C-CAM1 contains several potential phosphorylation sites, including putative consensus sequences for cyclic AMP-dependent kinase and tyrosine kinase. One of the potential tyrosine phosphorylation sites is located within the antigen-receptor homology (ARH) domain. The ARH domain of the membrane-bound IgM molecule is necessary for signal transduction in B-cells. These structural features suggest that the cytoplasmic domain of C-CAM1 may be important for signal transduction. To test this possibility, we generated several site-directed C-CAM1 mutants and tested their ability to support adhesion and their abilities to be phosphorylated in vivo. Results from these studies revealed that Tyr-488 is phosphorylated in vivo. However, replacing this tyrosine with phenylalanine did not significantly compromise its adhesion function. Similarly, Ser and Thr residues are phosphorylated in vivo, but deletion of the potential cyclic AMP-dependent kinase site did not significantly reduce the adhesion function. These results suggest that the kinase phosphorylation sites in the cytoplasmic domain of C-CAM1 are not required for the adhesion function. However, these phosphorylation sites are probably involved in the regulation of C-CAM-mediated signal transduction. Thus, there are probably distinct structural requirements for the adhesion and the signal transduction functions of C-CAM. Incidentally, a C-CAM1 deletion mutant containing a 10-amino-acid cytoplasmic domain was able to support adhesion activity. This is in contrast to our previous finding that a C-CAM isoform, C-CAM3, with a 6-amino-acid cytoplasmic domain could not support cell adhesion. This result indicates that the extra four amino acids, which are absent in C-CAM3 and contain a potential Ser/Thr phosphorylation site, are important for the adhesion function.

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Recent Development of Machine Learning Methods in Microbial Phosphorylation Sites

Current Genomics ◽

10.2174/1389202921666200427210833 ◽

2020 ◽

Vol 21 (3) ◽

pp. 194-203 ◽

Cited By ~ 2

Author(s):

Md. Mamunur Rashid ◽

Swakkhar Shatabda ◽

Md. Mehedi Hasan ◽

Hiroyuki Kurata

Keyword(s):

Machine Learning ◽

Phosphorylation Site ◽

Window Size ◽

Proteome Analysis ◽

Phosphorylation Sites ◽

Cellular Functions ◽

Phosphorylated Proteins ◽

Technical Advances ◽

Complete Survey ◽

Software Utility

A variety of protein post-translational modifications has been identified that control many cellular functions. Phosphorylation studies in mycobacterial organisms have shown critical importance in diverse biological processes, such as intercellular communication and cell division. Recent technical advances in high-precision mass spectrometry have determined a large number of microbial phosphorylated proteins and phosphorylation sites throughout the proteome analysis. Identification of phosphorylated proteins with specific modified residues through experimentation is often laborintensive, costly and time-consuming. All these limitations could be overcome through the application of machine learning (ML) approaches. However, only a limited number of computational phosphorylation site prediction tools have been developed so far. This work aims to present a complete survey of the existing ML-predictors for microbial phosphorylation. We cover a variety of important aspects for developing a successful predictor, including operating ML algorithms, feature selection methods, window size, and software utility. Initially, we review the currently available phosphorylation site databases of the microbiome, the state-of-the-art ML approaches, working principles, and their performances. Lastly, we discuss the limitations and future directions of the computational ML methods for the prediction of phosphorylation.

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