-
The Journal of Biological Chemistry Aug 2013Protein translation initiation is a tightly controlled process responding to nutrient availability and mitogen stimulation. Serving as one of the most important negative...
Protein translation initiation is a tightly controlled process responding to nutrient availability and mitogen stimulation. Serving as one of the most important negative regulators of protein translation, 4E binding protein 1 (4E-BP1) binds to translation initiation factor 4E and inhibits cap-dependent translation in a phosphorylation-dependent manner. Although it has been demonstrated previously that the phosphorylation of 4E-BP1 is controlled by mammalian target of rapamycin in the mammalian target of rapamycin complex 1, the mechanism underlying the dephosphorylation of 4E-BP1 remains elusive. Here, we report the identification of PPM1G as the phosphatase of 4E-BP1. A coimmunoprecipitation experiment reveals that PPM1G binds to 4E-BP1 in cells and that purified PPM1G dephosphorylates 4E-BP1 in vitro. Knockdown of PPM1G in 293E and colon cancer HCT116 cells results in an increase in the phosphorylation of 4E-BP1 at both the Thr-37/46 and Ser-65 sites. Furthermore, the time course of 4E-BP1 dephosphorylation induced by amino acid starvation or mammalian target of rapamycin inhibition is slowed down significantly in PPM1G knockdown cells. Functionally, the amount of 4E-BP1 bound to the cap-dependent translation initiation complex is decreased when the expression of PPM1G is depleted. As a result, the rate of cap-dependent translation, cell size, and protein content are increased in PPM1G knockdown cells. Taken together, our study has identified protein phosphatase PPM1G as a novel regulator of cap-dependent protein translation by negatively controlling the phosphorylation of 4E-BP1.
Topics: Adaptor Proteins, Signal Transducing; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Humans; Phosphoprotein Phosphatases; Phosphoproteins; Phosphorylation; Protein Biosynthesis; Protein Phosphatase 2C; TOR Serine-Threonine Kinases
PubMed: 23814053
DOI: 10.1074/jbc.M113.492371 -
Molecular and Cellular Biology Apr 1991Simian virus 40 (SV40) large-T antigen and the cellular protein p53 were phosphorylated in vivo by growing cells in the presence of 32Pi. The large-T/p53 complex was...
Simian virus 40 (SV40) large-T antigen and the cellular protein p53 were phosphorylated in vivo by growing cells in the presence of 32Pi. The large-T/p53 complex was isolated by immunoprecipitation and used as a substrate for protein phosphatase 2A (PP2A) consisting of the catalytic subunit (C) and the two regulatory subunits, A and B. Three different purified forms of PP2A, including free C, the AC form, and the ABC form, could readily dephosphorylate both proteins. With both large-T and p53, the C subunit was most active, followed by the AC form, which was more active than the ABC form. The activity of all three forms of PP2A toward these proteins was strongly stimulated by manganese ions and to a lesser extent by magnesium ions. The presence of complexed p53 did not affect the dephosphorylation of large-T antigen by PP2A. The dephosphorylation of individual phosphorylation sites of large-T and p53 were determined by two-dimensional peptide mapping. Individual sites within large-T and p53 were dephosphorylated at different rates by all three forms of PP2A. The phosphates at Ser-120 and Ser-123 of large-T, which affect binding to the origin of SV40 DNA, were removed most rapidly. Three of the six major phosphopeptides of p53 were readily dephosphorylated, while the remaining three were relatively resistant to PP2A. Dephosphorylation of most of the sites in large-T and p53 by the AC form was inhibited by SV40 small-t antigen. The inhibition was most apparent for those sites which were preferentially dephosphorylated. Inhibition was specific for the AC form; no effect was observed on the dephosphorylation of either protein by the free C subunit or the ABC form. The inhibitory effect of small-t on dephosphorylation by PP2A could explain its role in transformation.
Topics: Antigens, Polyomavirus Transforming; Peptide Mapping; Phosphoprotein Phosphatases; Phosphorylation; Precipitin Tests; Protein Phosphatase 2; Simian virus 40; Substrate Specificity; Tumor Suppressor Protein p53
PubMed: 1848668
DOI: 10.1128/mcb.11.4.1996-2003.1991 -
Journal of Lipid Research Nov 2022In the yeast Saccharomyces cerevisiae, the PAH1-encoded Mg-dependent phosphatidate (PA) phosphatase Pah1 regulates the bifurcation of PA to diacylglycerol (DAG) for...
In the yeast Saccharomyces cerevisiae, the PAH1-encoded Mg-dependent phosphatidate (PA) phosphatase Pah1 regulates the bifurcation of PA to diacylglycerol (DAG) for triacylglycerol (TAG) synthesis and to CDP-DAG for phospholipid synthesis. Pah1 function is mainly regulated via control of its cellular location by phosphorylation and dephosphorylation. Pah1 phosphorylated by multiple protein kinases is sequestered in the cytosol apart from its substrate PA in the membrane. The phosphorylated Pah1 is then recruited and dephosphorylated by the protein phosphatase complex Nem1 (catalytic subunit)-Spo7 (regulatory subunit) in the endoplasmic reticulum. The dephosphorylated Pah1 hops onto and scoots along the membrane to recognize PA for its dephosphorylation to DAG. Here, we developed a proteoliposome model system that mimics the Nem1-Spo7/Pah1 phosphatase cascade to provide a tool for studying Pah1 regulation. Purified Nem1-Spo7 was reconstituted into phospholipid vesicles prepared in accordance with the phospholipid composition of the nuclear/endoplasmic reticulum membrane. The Nem1-Spo7 phosphatase reconstituted in the proteoliposomes, which were measured 60 nm in an average diameter, was catalytically active on Pah1 phosphorylated by Pho85-Pho80, and its active site was located at the external side of the phospholipid bilayer. Moreover, we determined that PA stimulated the Nem1-Spo7 activity, and the regulatory effect was governed by the nature of the phosphate headgroup but not by the fatty acyl moiety of PA. The reconstitution system for the Nem1-Spo7/Pah1 phosphatase cascade, which starts with the phosphorylation of Pah1 by Pho85-Pho80 and ends with the production of DAG, is a significant advance to understand a regulatory cascade in yeast lipid synthesis.
Topics: Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Phosphatidic Acids; Phosphoric Monoester Hydrolases; Phosphatidate Phosphatase; Membrane Proteins; Nuclear Proteins
PubMed: 36314526
DOI: 10.1016/j.jlr.2022.100282 -
Proceedings of the National Academy of... Aug 1988Five protein kinases are shown to serve as specific phosphatases in the absence of ADP. Although the rates of hydrolysis are very slow compared to the forward...
Five protein kinases are shown to serve as specific phosphatases in the absence of ADP. Although the rates of hydrolysis are very slow compared to the forward phosphorylation rates under optimal conditions, they are of the same order as the reverse reaction in the presence of ADP. Because cells contain approximately equal to 3 mM ATP, neither the reverse reaction nor the phosphatase is likely to play a physiological role. beta-casein B phosphorylated by the catalytic subunit of cAMP-dependent protein kinase (protein kinase A) is specifically dephosphorylated by protein kinase A but not by polypeptide-dependent protein kinase (protein kinase P). beta-casein B phosphorylated by protein kinase P is specifically dephosphorylated by protein kinase P but not by protein kinase A. Histone H1 phosphorylated by protein kinase C is dephosphorylated by the same enzyme in the absence of ADP. In all cases tested addition of ADP and F1-ATPase accelerates moderately the rate of dephosphorylation. Native H+-ATPase from yeast plasma membranes is isolated mainly in the phosphorylated form. It is dephosphorylated and rephosphorylated by protein kinase P but not by protein kinase A. Protein-tyrosine kinase of the epidermal growth factor receptor phosphorylates the random synthetic polypeptide poly(Glu80Tyr20). The phosphorylated polymer is specifically dephosphorylated in the absence of ADP by epidermal growth factor receptor preparations but not by insulin receptor preparations. The same polymer phosphorylated by insulin receptor is dephosphorylated by insulin receptor but not by epidermal growth factor receptor preparations. By using a cycle of dephosphorylation-rephosphorylation, it is possible to identify proteins that are phosphorylated by these protein kinases in vivo. Should this method be applicable to additional protein kinases, it should be possible to estimate the quantitative contribution of each protein kinase to a single phosphoprotein.
Topics: ErbB Receptors; Phosphoproteins; Phosphorylation; Protein Kinases; Protein Serine-Threonine Kinases; Protein-Tyrosine Kinases; Proton-Translocating ATPases; Receptor, Insulin
PubMed: 2901092
DOI: 10.1073/pnas.85.16.5849 -
FEBS Letters Mar 1994Microtubule-associated protein tau is abnormally hyperphosphorylated and forms the major protein subunit of paired helical filaments (PHF) in Alzheimer disease brains....
Microtubule-associated protein tau is abnormally hyperphosphorylated and forms the major protein subunit of paired helical filaments (PHF) in Alzheimer disease brains. The abnormally phosphorylated sites Ser-199, Ser-202, Ser-396 and Ser-404 but not Ser-46 and Ser-235 of Alzheimer tau were found to be dephosphorylated by protein phosphatase-1 and this dephosphorylation was activated by Mn2+. In contrast, protein phosphatase-2C did not dephosphorylate any of these sites. Both protein phosphatase-1 and -2C had high activities towards [32P]tau phosphorylated by cAMP-dependent protein kinase. These results suggest that both protein phosphatase-1 and -2C might be associated with normal phosphorylation state of tau, but only the former and not the latter phosphatase is involved in its abnormal phosphorylation in Alzheimer disease.
Topics: Aged; Alzheimer Disease; Animals; Cattle; Cyclic AMP-Dependent Protein Kinases; Humans; Male; Middle Aged; Phosphoprotein Phosphatases; Phosphorylation; Protein Phosphatase 1; Rabbits; Serine; tau Proteins
PubMed: 8137929
DOI: 10.1016/0014-5793(94)80247-5 -
Journal of Biochemistry Mar 1981When a plasma membrane preparation isolated from rat liver was incubated with [gamma-32P]ATP and Mg2+, protein-bound 32P increased rapidly, followed by a gradual...
When a plasma membrane preparation isolated from rat liver was incubated with [gamma-32P]ATP and Mg2+, protein-bound 32P increased rapidly, followed by a gradual decrease. The time course suggested the existence of membrane-bound kinase(s) and phosphatase(s) phosphorylating and dephosphorylating endogenous proteins. The extent of phosphorylation was not affected by inclusion of cyclic AMP in the reaction mixture. The extent of the maximum phosphorylation was dependent on membrane concentration, owing to rapid hydrolysis of ATP by the membrane-bound ATPase activity. Thus, phosphorylation proceeded further on repeated addition of ATP. Both phosphorylation and dephosphorylation were stimulated by Mg2+, an effective rate of phosphorylation being obtained at 15 mM. Pi up to 20 mM stimulated phosphorylation with little effect on the rate of dephosphorylation. At higher phosphate concentrations, the maximum 32P-incorporation decreased again, and at 100 mM, dephosphorylation was prevented significantly. Autoradiography after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and urea revealed six main phosphorylated bands, two of which (Band 3 and 5) were partly extractable with 1 M NaCl. In the presence of 100 mM Pi, very strong phosphorylation of Band 5 (about 23,000 daltons) was noted, and a new strongly labeled band (Band P, about 20,000 daltons) was observed. It was concluded that the phosphoproteins in the membrane may be turned over at different rates and high concentrations of Pi may affect the turnover rate of some phosphoproteins, probably through interference with the phosphatase.
Topics: Adenosine Triphosphate; Animals; Cell Membrane; Electrophoresis, Polyacrylamide Gel; In Vitro Techniques; Liver; Male; Membrane Proteins; Phosphates; Phosphoprotein Phosphatases; Phosphoproteins; Phosphorylation; Protein Kinases; Rats; Sodium-Potassium-Exchanging ATPase
PubMed: 6270066
DOI: 10.1093/oxfordjournals.jbchem.a133252 -
Science Signaling Feb 2021, an important human parasite, has a flagellum that controls cell motility, morphogenesis, proliferation, and cell-cell communication. Inheritance of the newly assembled...
, an important human parasite, has a flagellum that controls cell motility, morphogenesis, proliferation, and cell-cell communication. Inheritance of the newly assembled flagellum during the cell cycle requires the Polo-like kinase homolog TbPLK and the kinetoplastid-specific protein phosphatase KPP1, although whether TbPLK acts on KPP1 or vice versa has been unclear. Here, we showed that dephosphorylation of TbPLK on Thr by KPP1 maintained low TbPLK activity in the flagellum-associated hook complex structure, thereby ensuring proper flagellum positioning and attachment. This dephosphorylation event required the recognition of phosphorylated Thr in the activation loop of TbPLK by the N-terminal Plus3 domain of KPP1 and the dephosphorylation of phosphorylated Thr in TbPLK by the C-terminal catalytic domain of KPP1. Dephosphorylation of TbPLK by KPP1 prevented hyperphosphorylation of the hook complex protein TbCentrin2, thereby allowing timely dephosphorylation of phosphorylated TbCentrin2 for hook complex duplication and flagellum positioning and attachment. Thus, KPP1 attenuates TbPLK activity by dephosphorylating TbPLK to facilitate flagellum inheritance.
Topics: Cell Cycle; Flagella; Phosphoprotein Phosphatases; Protozoan Proteins; Trypanosoma brucei brucei
PubMed: 33563698
DOI: 10.1126/scisignal.abc6435 -
The Journal of Cell Biology May 2022Autophagy is a conserved eukaryotic lysosomal degradation pathway that responds to environmental and cellular cues. Autophagy is essential for the meiotic exit and...
Autophagy is a conserved eukaryotic lysosomal degradation pathway that responds to environmental and cellular cues. Autophagy is essential for the meiotic exit and sporulation in budding yeast, but the underlying molecular mechanisms remain unknown. Here, we show that autophagy is maintained during meiosis and stimulated in anaphase I and II. Cells with higher levels of autophagy complete meiosis faster, and genetically enhanced autophagy increases meiotic kinetics and sporulation efficiency. Strikingly, our data reveal that the conserved phosphatase Cdc14 regulates meiosis-specific autophagy. Cdc14 is activated in anaphase I and II, accompanying its subcellular relocation from the nucleolus to the cytoplasm, where it dephosphorylates Atg13 to stimulate Atg1 kinase activity and thus autophagy. Together, our findings reveal a meiosis-tailored mechanism that spatiotemporally controls meiotic autophagy activity to ensure meiosis progression, exit, and sporulation.
Topics: Adaptor Proteins, Signal Transducing; Anaphase; Autophagy; Autophagy-Related Proteins; Cell Cycle Proteins; Meiosis; Protein Tyrosine Phosphatases; Saccharomyces cerevisiae Proteins
PubMed: 35238874
DOI: 10.1083/jcb.202107151 -
Biochimica Et Biophysica Acta.... Jun 2021PGAM5 is a protein phosphatase located in the inner mitochondrial membrane through its transmembrane (TM) domain and is cleaved within the TM domain upon mitochondrial...
PGAM5 is a protein phosphatase located in the inner mitochondrial membrane through its transmembrane (TM) domain and is cleaved within the TM domain upon mitochondrial dysfunction. We found previously that cleaved PGAM5 is released from mitochondria, following proteasome-mediated rupture of the outer mitochondrial membrane during mitophagy, a selective form of autophagy specific to mitochondria. Here, we examined the role of cleaved PGAM5 outside mitochondria. Deletion mutants that mimic cleaved PGAM5 existed not only in the cytosol but also in the nucleus, and a fraction of cleaved PGAM5 translocated to the nucleus during mitophagy induced by the uncoupler CCCP. We identified serine/arginine-related nuclear matrix protein of 160 kDa (SRm160)/SRRM1, which contains a highly phosphorylated domain rich in arginine/serine dipeptides, called the RS domain, as a nuclear protein that interacts with PGAM5. PGAM5 dephosphorylated SRm160, and incubation of lysates from WT cells, but not of those from PGAM5-deficient cells, induced dephosphorylation of SRm160 and another RS domain-containing protein SRSF1, one of the most characterized serine/arginine-rich (SR) proteins. Moreover, phosphorylation of these proteins and other SR proteins, which are commonly reactive toward the 1H4 monoclonal antibody that detects phosphorylated SR proteins, decreased during mitophagy, largely because of PGAM5 activity. These results suggest that PGAM5 regulates phosphorylation of these nuclear proteins during mitophagy. Because SRm160 and SR proteins play critical roles in mRNA metabolism, PGAM5 may coordinate cellular responses to mitochondrial stress at least in part through post-transcriptional and pre-translational events.
Topics: Antigens, Nuclear; Cell Nucleus; Cytosol; HeLa Cells; Humans; Membrane Proteins; Mitochondria; Mitochondrial Membrane Transport Proteins; Mitochondrial Membranes; Mitochondrial Proteins; Mitophagy; Nuclear Matrix-Associated Proteins; Phosphoprotein Phosphatases; Phosphorylation; RNA-Binding Proteins; Serine-Arginine Splicing Factors; Ubiquitin-Protein Ligases
PubMed: 33872670
DOI: 10.1016/j.bbamcr.2021.119045 -
MBio May 2021The circadian clock controls the phosphorylation and activity of eukaryotic translation initiation factor 2α (eIF2α). In , the clock drives a daytime peak in the...
The circadian clock controls the phosphorylation and activity of eukaryotic translation initiation factor 2α (eIF2α). In , the clock drives a daytime peak in the activity of the eIF2α kinase CPC-3, the homolog of yeast and mammalian GCN2 kinase. This leads to increased levels of phosphorylated eIF2α (P-eIF2α) and reduced mRNA translation initiation during the day. We hypothesized that rhythmic eIF2α activity also requires dephosphorylation of P-eIF2α at night by phosphatases. In support of this hypothesis, we show that mutation of PPP-1, a homolog of the yeast eIF2α phosphatase GLC7, leads to high and arrhythmic P-eIF2α levels, while maintaining core circadian oscillator function. PPP-1 levels are clock-controlled, peaking in the early evening, and rhythmic PPP-1 levels are necessary for rhythmic P-eIF2α accumulation. Deletion of the N terminus of eIF2γ, the region necessary for eIF2γ interaction with GLC7 in yeast, led to high and arrhythmic P-eIF2α levels. These data supported that eIF2γ functions to recruit PPP-1 to dephosphorylate eIF2α at night. Thus, in addition to the activity of CPC-3 kinase, circadian clock regulation of eIF2α activity requires dephosphorylation by PPP-1 phosphatase at night. These data show how the circadian clock controls the activity a central regulator of translation, critical for cellular metabolism and growth control, through the temporal coordination of phosphorylation and dephosphorylation events. Circadian clock control of mRNA translation contributes to the daily cycling of a significant proportion of the cellular protein synthesis, but how this is accomplished is not understood. We discovered that the clock in the model fungus regulates rhythms in protein synthesis by controlling the phosphorylation and dephosphorylation of a conserved translation initiation factor eIF2α. During the day, eIF2α is phosphorylated and inactivated by CPC-3 kinase. At night, a clock-controlled phosphatase, PPP-1, dephosphorylates and activates eIF2α, leading to increased nighttime protein synthesis. Translation requires significant cellular energy; thus, partitioning translation to the night by the clock provides a mechanism to coordinate energy metabolism with protein synthesis and cellular growth.
Topics: Circadian Clocks; Eukaryotic Initiation Factor-2; Fungal Proteins; Neurospora crassa; Phosphorylation; Protein Biosynthesis; Protein Phosphatase 1
PubMed: 34006661
DOI: 10.1128/mBio.00871-21