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Proceedings of the National Academy of... Jul 2015Transient protein synthesis inhibition, mediated by phosphorylation of the α subunit of eukaryotic translation initiation factor 2 (eIF2α), is an important protective...
Transient protein synthesis inhibition, mediated by phosphorylation of the α subunit of eukaryotic translation initiation factor 2 (eIF2α), is an important protective mechanism cells use during stress conditions. Following relief of the stress, the growth arrest and DNA damage-inducible protein GADD34 associates with the broadly acting serine/threonine protein phosphatase 1 (PP1) to dephosphorylate eIF2α. Whereas the PP1-binding motif on GADD34 has been defined, it remains to be determined how GADD34 directs PP1 to specifically dephosphorylate eIF2α. In this report, we map a novel eIF2α-binding motif to the C terminus of GADD34 in a region distinct from where PP1 binds to GADD34. This motif is characterized by the consensus sequence Rx[Gnl]x(1-2)Wxxx[Arlv]x[Dn][Rg]xRFxx[Rlvk][Ivc], where capital letters are preferred and x is any residue. Point mutations altering the eIF2α-binding motif impair the ability of GADD34 to interact with eIF2α, promote eIF2α dephosphorylation, and suppress PKR toxicity in yeast. Interestingly, this eIF2α-docking motif is conserved among viral orthologs of GADD34, and is necessary for the proteins produced by African swine fever virus, Canarypox virus, and Herpes simplex virus to promote eIF2α dephosphorylation. Taken together, these data indicate that GADD34 and its viral orthologs direct specific dephosphorylation of eIF2α by interacting with both PP1 and eIF2α through independent binding motifs.
Topics: Amino Acid Motifs; Amino Acid Sequence; Binding Sites; Eukaryotic Initiation Factor-2; Humans; Immunoblotting; Molecular Sequence Data; Phosphorylation; Point Mutation; Protein Binding; Protein Phosphatase 1; Saccharomyces cerevisiae; Sequence Homology, Amino Acid
PubMed: 26100893
DOI: 10.1073/pnas.1501557112 -
PloS One 2011The Hippo/MST1 signaling pathway plays an important role in the regulation of cell proliferation and apoptosis. As a major downstream target of the Hippo/MST1 pathway,...
BACKGROUND
The Hippo/MST1 signaling pathway plays an important role in the regulation of cell proliferation and apoptosis. As a major downstream target of the Hippo/MST1 pathway, YAP2 (Yes-associated protein 2) functions as a transcriptional cofactor that has been implicated in many biological processes, including organ size control and cancer development. MST1/Lats kinase inhibits YAP2's nuclear accumulation and transcriptional activity through inducing the phosphorylation at serine 127 and the sequential association with 14-3-3 proteins. However, the dephosphorylation of YAP2 is not fully appreciated.
METHODOLOGY/PRINCIPAL FINDINGS
In the present study, we demonstrate that PP1A (catalytic subunit of protein phosphatase-1) interacts with and dephosphorylates YAP2 in vitro and in vivo, and PP1A-mediated dephosphorylation induces the nuclear accumulation and transcriptional activation of YAP2. Inhibition of PP1 by okadiac acid (OA) increases the phosphorylation at serine 127 and cytoplasmic translocation of YAP2 proteins, thereby mitigating its transcription activity. PP1A expression enhances YAP2's pro-survival capability and YAP2 knockdown sensitizes ovarian cancer cells to cisplatin treatment.
CONCLUSIONS/SIGNIFICANCE
Our findings define a novel molecular mechanism that YAP2 is positively regulated by PP1-mediated dephosphorylation in the cell survival.
Topics: Adaptor Proteins, Signal Transducing; Cell Line, Tumor; Cell Nucleus; Cell Survival; Humans; Phosphoproteins; Phosphorylation; Protein Binding; Protein Phosphatase 1; Transcription Factors; Transcription, Genetic; Transcriptional Activation; YAP-Signaling Proteins
PubMed: 21909427
DOI: 10.1371/journal.pone.0024288 -
ELife Mar 2019Cell-cell communication in multicellular organisms depends on the dynamic and reversible phosphorylation of protein tyrosine residues. The receptor-linked protein...
Cell-cell communication in multicellular organisms depends on the dynamic and reversible phosphorylation of protein tyrosine residues. The receptor-linked protein tyrosine phosphatases (RPTPs) receive cues from the extracellular environment and are well placed to influence cell signaling. However, the direct events downstream of these receptors have been challenging to resolve. We report here that the homophilic receptor PTPRK is stabilized at cell-cell contacts in epithelial cells. By combining interaction studies, quantitative tyrosine phosphoproteomics, proximity labeling and dephosphorylation assays we identify high confidence PTPRK substrates. PTPRK directly and selectively dephosphorylates at least five substrates, including Afadin, PARD3 and δ-catenin family members, which are all important cell-cell adhesion regulators. In line with this, loss of PTPRK phosphatase activity leads to disrupted cell junctions and increased invasive characteristics. Thus, identifying PTPRK substrates provides insight into its downstream signaling and a potential molecular explanation for its proposed tumor suppressor function.
Topics: Adaptor Proteins, Signal Transducing; Catenins; Cell Adhesion; Cell Cycle Proteins; Cell Line; Epithelial Cells; Humans; Microfilament Proteins; Phosphorylation; Protein Processing, Post-Translational; Receptor-Like Protein Tyrosine Phosphatases, Class 2; Delta Catenin
PubMed: 30924770
DOI: 10.7554/eLife.44597 -
Oncotarget Jan 2017Aberrant phosphorylation and overexpression of BCR-ABL fusion protein are responsible for the main pathogenesis in chronic myeloid leukemia (CML). Phosphorylated BCR-ABL...
Aberrant phosphorylation and overexpression of BCR-ABL fusion protein are responsible for the main pathogenesis in chronic myeloid leukemia (CML). Phosphorylated BCR-ABL Y177 recruits GRB2 adaptor and triggers leukemic RAS-MAPK and PI3K-AKT signals. In this study, we engineered a SPOA system to dephosphorylate and degrade BCR-ABL by targeting BCR-ABL Y177. We tested its effect on BCR-ABL phosphorylation and expression, as well as cell proliferation and apoptosis in CML cells. We found that SPOA remarkably dephosphorylated BCR-ABL Y177, prevented GRB2 recruitment, and uncoupled RAS-MAPK and PI3K-AKT signals. Meanwhile, SPOA degraded BCR-ABL oncoprotein in ubiquitin-independent manner and depressed the signal transduction of STAT5 and CRKL by BCR-ABL. Furthermore, SPOA inhibited proliferation and induced apoptosis in CML cells and depressed the oncogenecity of K562 cells in mice. These results provide evidence that dephosphorylating and degrading oncogenic BCR-ABL offer an alternative CML therapy.
Topics: Animals; Apoptosis; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; Disease Models, Animal; Fusion Proteins, bcr-abl; Gene Expression Regulation; Humans; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Mice; Phosphorylation; Proteasome Endopeptidase Complex; Protein Binding; Proteolysis; Signal Transduction; Ubiquitin
PubMed: 27926512
DOI: 10.18632/oncotarget.13754 -
Cell Adhesion & Migration 2015Altered phosphorylation status of the C-terminal Thr residues of Ezrin/Radixin/Moesin (ERM) is often linked to cell shape change. To determine the role of phophorylated...
Altered phosphorylation status of the C-terminal Thr residues of Ezrin/Radixin/Moesin (ERM) is often linked to cell shape change. To determine the role of phophorylated ERM, we modified phosphorylation status of ERM and investigated changes in cell adhesion and morphology. Treatment with Calyculin-A (Cal-A), a protein phosphatase inhibitor, dramatically augmented phosphorylated ERM (phospho-ERM). Cal-A-treatment or expression of phospho-mimetic Moesin mutant (Moesin-TD) induced cell rounding in adherent cells. Moreover, reattachment of detached cells to substrate was inhibited by either treatment. Phospho-ERM, Moesin-TD and actin cytoskeleton were observed at the plasma membrane of such round cells. Augmented cell surface rigidity was also observed in both cases. Meanwhile, non-adherent KG-1 cells were rather rich in phospho-ERM. Treatment with Staurosporine, a protein kinase inhibitor that dephosphorylates phospho-ERM, up-regulated the integrin-dependent adhesion of KG-1 cells to substrate. These findings strongly suggest the followings: (1) Phospho-ERM inhibit cell adhesion, and therefore, dephosphorylation of ERM proteins is essential for cell adhesion. (2) Phospho-ERM induce formation and/or maintenance of spherical cell shape. (3) ERM are constitutively both phosphorylated and dephosphorylated in cultured adherent and non-adherent cells.
Topics: Cell Adhesion; Cell Line; Cell Membrane; Cell Shape; Cytoskeletal Proteins; Cytoskeleton; Humans; Marine Toxins; Membrane Proteins; Microfilament Proteins; Oxazoles; Phosphorylation
PubMed: 26555866
DOI: 10.1080/19336918.2015.1113366 -
Proteomics Apr 2012Protein phosphorylation is reversibly regulated by the interplay between kinases and phosphatases. Recent developments within the field of proteomics have revealed the...
Protein phosphorylation is reversibly regulated by the interplay between kinases and phosphatases. Recent developments within the field of proteomics have revealed the extent of this modification in nature. To date there is still a lack of information about phosphatase specificity for different proteomes and their conditions to achieve maximum enzyme activity. This information is important per se, and in addition often requested in functional and biochemical in vitro studies, where a dephosphorylated sample is needed as a negative control to define baseline conditions. In this study, we have addressed the effectiveness of two phosphatases endogenously present in the heart (protein phosphatases 1 and 2A) and two generic phosphatases (alkaline phosphatase and lambda protein phosphatase) on three cardiac subproteomes known to be regulated by phosphorylation. We optimized the dephoshorylating conditions on a cardiac tissue fraction comprising cytosolic and myofilament proteins using 2DE and MS. The two most efficient conditions were further investigated on a mitochondrial-enriched fraction. Dephosphorylation of specific proteins depends on the phosphatase, its concentration, as well as sample preparation including buffer composition. Finally, we analyzed the efficiency of alkaline phosphatase, the phosphatase with the broadest substrate specificity, using TiO(2) peptide enrichment and 2DLC-MS/MS. Under these conditions, 95% of the detected cardiac cytoplasmic-enriched phospho-proteome was dephosphorylated. In summary, targeting dephosphorylation of the cardiac muscle subproteomes or a specific protein will drive the selection of the specific phosphatase, and each requires different conditions for optimal performance.
Topics: Animals; Electrophoresis, Gel, Two-Dimensional; Mice; Muscle Proteins; Myocardium; Phosphorylation; Protein Phosphatase 1; Protein Phosphatase 2; Proteomics
PubMed: 22522803
DOI: 10.1002/pmic.201100116 -
Scientific Reports Sep 2020Control of the protein phosphorylation status is a major mechanism for regulation of cellular processes, and its alteration often lead to functional disorders. Ppz1, a...
Control of the protein phosphorylation status is a major mechanism for regulation of cellular processes, and its alteration often lead to functional disorders. Ppz1, a protein phosphatase only found in fungi, is the most toxic protein when overexpressed in Saccharomyces cerevisiae. To investigate the molecular basis of this phenomenon, we carried out combined genome-wide transcriptomic and phosphoproteomic analyses. We have found that Ppz1 overexpression causes major changes in gene expression, affecting ~ 20% of the genome, together with oxidative stress and increase in total adenylate pools. Concurrently, we observe changes in the phosphorylation pattern of near 400 proteins (mainly dephosphorylated), including many proteins involved in mitotic cell cycle and bud emergence, rapid dephosphorylation of Snf1 and its downstream transcription factor Mig1, and phosphorylation of Hog1 and its downstream transcription factor Sko1. Deletion of HOG1 attenuates the growth defect of Ppz1-overexpressing cells, while that of SKO1 aggravates it. Our results demonstrate that Ppz1 overexpression has a widespread impact in the yeast cells and reveals new aspects of the regulation of the cell cycle.
Topics: Cell Cycle; DNA Damage; Gene Expression Regulation, Fungal; Metabolome; Phosphoprotein Phosphatases; Phosphorylation; Reactive Oxygen Species; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Transcriptome
PubMed: 32973189
DOI: 10.1038/s41598-020-72391-y -
The Journal of Biological Chemistry Jan 1998A synthetic peptide corresponding to the autophosphorylation site of Ca2+/calmodulin-dependent protein kinase II (CaMKII) (residues 281-289) was conjugated to...
A synthetic peptide corresponding to the autophosphorylation site of Ca2+/calmodulin-dependent protein kinase II (CaMKII) (residues 281-289) was conjugated to paramagnetic particles, and phosphorylated by a constitutively active CaMKII fragment. Using this phosphopeptide conjugate as a substrate, a calyculin A-insensitive, Mn(2+)-dependent, and poly-L-lysine-stimulated protein phosphatase activity was detected in the crude extract of rat brain. The protein phosphatase (designated as CaMKII phosphatase) (CaMKIIPase) was purified to near homogeneity from rat brain. CaMKIIPase showed apparent molecular weights of 54,000 and 65,000, on SDS-polyacrylamide gel electrophoresis and gel-filtration analysis, respectively. It was not inhibited by 100 nM calyculin A or 10 microM okadaic acid. Mn2+, but not Mg2+, was absolutely required for activity. CaMKIIPase was potently activated by polycations. Autophosphorylated CaMKII was dephosphorylated by CaMKIIPase, whereas phosphorylase kinase, mixed histones, myelin basic protein, and alpha-casein (which had been phosphorylated by cAMP-dependent protein kinase) and phosphorylase a (phosphorylated by phosphorylase kinase) were not significantly dephosphorylated. No other proteins than CaMKII in rat brain extract which had been phosphorylated by CaMKII were dephosphorylated. The stimulated Ca(2+)-independent activity of autophosphorylated CaMKII was reversed by the action of CaMKIIPase. Thus, CaMKIIPase appears to be a specialized protein phosphatase for the regulation of CaMKII.
Topics: Animals; Brain; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calcium-Calmodulin-Dependent Protein Kinases; Cations; Enzyme Activation; Molecular Weight; Phosphoprotein Phosphatases; Phosphorylation; Polyamines; Polyelectrolytes; Rats; Substrate Specificity
PubMed: 9442023
DOI: 10.1074/jbc.273.4.1904 -
The Journal of Biological Chemistry Dec 2012Nucleocytoplasmic shuttling of Hxk2 induced by glucose levels has been reported recently. Here we present evidence that indicates that Hxk2 nucleocytoplasmic traffic is...
Nucleocytoplasmic shuttling of Hxk2 induced by glucose levels has been reported recently. Here we present evidence that indicates that Hxk2 nucleocytoplasmic traffic is regulated by phosphorylation and dephosphorylation at serine 14. Moreover, we identified the protein kinase Snf1 and the protein phosphatase Glc7-Reg1 as novel regulatory partners for the nucleocytoplasmic shuttling of Hxk2. Functional studies revealed that, in contrast to the wild-type protein, the dephosphorylation-mimicking mutant of Hxk2 retains its nuclear localization in low glucose conditions, and the phosphomimetic mutant of Hxk2 retains its cytoplasmic localization in high glucose conditions. Interaction experiments of Hxk2 with Kap60 and Xpo1 indicated that nuclear import of the S14D mutant of Hxk2 is severely decreased but that the export is significantly enhanced. Conversely, nuclear import of the S14A mutant of Hxk2 was significantly enhanced, although the export was severely decreased. The interaction of Hxk2 with Kap60 and Xpo1 was found to occur in the dephosphorylated and phosphorylated states of the protein, respectively. In addition, we found that Hxk2 is a substrate for Snf1. Mutational analysis indicated that serine 14 is a major in vitro and in vivo phosphorylation site for Snf1. We also provide evidence that dephosphorylation of Hxk2 at serine 14 is a protein phosphatase Glc7-Reg1-dependent process. Taken together, this study establishes a functional link between Hxk2, Reg1, and Snf1 signaling, which involves the regulation of Hxk2 nucleocytoplasmic shuttling by phosphorylation-dephosphorylation of serine 14.
Topics: Active Transport, Cell Nucleus; Amino Acid Substitution; Cell Nucleus; Cytoplasm; Hexokinase; Karyopherins; Mutation, Missense; Phosphorylation; Protein Phosphatase 1; Protein Serine-Threonine Kinases; Receptors, Cytoplasmic and Nuclear; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Signal Transduction; Exportin 1 Protein
PubMed: 23066030
DOI: 10.1074/jbc.M112.401679 -
Investigative Ophthalmology & Visual... Feb 2013CaBP4 is a neuronal Ca(2+)-binding protein that is expressed in the retina and in the cochlea, and is essential for normal photoreceptor synaptic function. CaBP4 is...
PURPOSE
CaBP4 is a neuronal Ca(2+)-binding protein that is expressed in the retina and in the cochlea, and is essential for normal photoreceptor synaptic function. CaBP4 is phosphorylated by protein kinase C zeta (PKCζ) in the retina at serine 37, which affects its interaction with and modulation of voltage-gated Ca(v)1 Ca(2+) channels. In this study, we investigated the potential role and functional significance of protein phosphatase 2A (PP2A) in CaBP4 dephosphorylation.
METHODS
The effect of protein phosphatase inhibitors, light, and overexpression of PP2A subunits on CaBP4 dephosphorylation was measured in in vitro assays. Pull-down experiments using retinal or transfected HEK293 cell lysates were used to investigate the association between CaBP4 and PP2A subunits. Electrophysiologic recordings of cotransfected HEK293 cells were performed to analyze the effect of CaBP4 dephosphorylation in modulating Ca(v)1.3 currents.
RESULTS
PP2A inhibitors, okadaic acid (OA), and fostriecin, but not PP1 selective inhibitors, NIPP-1, and inhibitor 2, block CaBP4 dephosphorylation in retinal lysates. Increased phosphatase activity in light-dependent conditions reverses phosphorylation of CaBP4 by PKCζ. In HEK293 cells, overexpression of PP2A enhances the rate of dephosphorylation of CaBP4. In addition, inhibition of protein phosphatase activity by OA increases CaBP4 phosphorylation and potentiates the modulatory effect of CaBP4 on Ca(v)1.3 Ca(2+) channels in HEK293T cells.
CONCLUSIONS
This study provides evidence that CaBP4 is dephosphorylated by PP2A in the retina. Our findings reveal a novel role for protein phosphatases in regulating CaBP4 function in the retina, which may fine tune presynaptic Ca(2+) signals at the photoreceptor synapse.
Topics: Adaptation, Ocular; Animals; Calcium Channels, L-Type; Calcium-Binding Proteins; Dark Adaptation; Enzyme Inhibitors; HEK293 Cells; Humans; Intracellular Signaling Peptides and Proteins; Mice; Mice, Inbred C57BL; Models, Biological; Nerve Tissue Proteins; Okadaic Acid; Patch-Clamp Techniques; Phosphoprotein Phosphatases; Phosphorylation; Polyenes; Protein Kinase C; Protein Phosphatase 2; Protein Phosphatase 2C; Pyrones; Retina; Signal Transduction
PubMed: 23341017
DOI: 10.1167/iovs.12-11319