-
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 -
Molecular and Cellular Biology Jan 2020yclase-ssociated rotein (CAP1) is a conserved actin-regulating protein that enhances actin filament dynamics and also regulates adhesion in mammalian cells. We...
Dynamic Phosphorylation and Dephosphorylation of Cyclase-Associated Protein 1 by Antagonistic Signaling through Cyclin-Dependent Kinase 5 and cAMP Are Critical for the Protein Functions in Actin Filament Disassembly and Cell Adhesion.
yclase-ssociated rotein (CAP1) is a conserved actin-regulating protein that enhances actin filament dynamics and also regulates adhesion in mammalian cells. We previously found that phosphorylation at the Ser307/Ser309 tandem site controls its association with cofilin and actin and is important for CAP1 to regulate the actin cytoskeleton. Here, we report that transient Ser307/Ser309 phosphorylation is required for CAP1 function in both actin filament disassembly and cell adhesion. Both the phosphomimetic and the nonphosphorylatable CAP1 mutant, which resist transition between phosphorylated and dephosphorylated forms, had defects in rescuing the reduced rate of actin filament disassembly in the CAP1 knockdown HeLa cells. The phosphorylation mutants also had defects in alleviating the elevated ocal dhesion inase (FAK) activity and the enhanced focal adhesions in the knockdown cells. In dissecting further phosphoregulatory cell signals for CAP1, we found that yclin-ependent inase (CDK5) phosphorylates both Ser307 and Ser309 residues, whereas cAMP signaling induces dephosphorylation at the tandem site, through its effectors rotein inase (PKA) and xchange roteins directly ctivated by AMP (Epac). No evidence supports an involvement of activated protein phosphatase in executing the dephosphorylation downstream from cAMP, whereas preventing CAP1 from accessing its kinase CDK5 appears to underlie CAP1 dephosphorylation induced by cAMP. Therefore, this study provides direct cellular evidence that transient phosphorylation is required for CAP1 functions in both actin filament turnover and adhesion, and the novel mechanistic insights substantially extend our knowledge of the cell signals that function in concert to regulate CAP1 by facilitating its transient phosphorylation.
Topics: Actin Cytoskeleton; Actin Depolymerizing Factors; Actins; Bridged Bicyclo Compounds, Heterocyclic; Cell Adhesion; Cell Cycle Proteins; Cell Line, Tumor; Cyclic AMP; Cyclin-Dependent Kinase 5; Cytoskeletal Proteins; Focal Adhesion Protein-Tyrosine Kinases; HEK293 Cells; HeLa Cells; Humans; Microfilament Proteins; Phosphorylation; Signal Transduction; Thiazolidines
PubMed: 31791978
DOI: 10.1128/MCB.00282-19 -
The Journal of Experimental Medicine Aug 2022Rap1 GTPase drives assembly of the Mig-10/RIAM/Lamellipodin (MRL protein)-integrin-talin (MIT) complex that enables integrin-dependent lymphocyte functions. Here we used...
Rap1 GTPase drives assembly of the Mig-10/RIAM/Lamellipodin (MRL protein)-integrin-talin (MIT) complex that enables integrin-dependent lymphocyte functions. Here we used tandem affinity tag-based proteomics to isolate and analyze the MIT complex and reveal that Phostensin (Ptsn), a regulatory subunit of protein phosphatase 1, is a component of the complex. Ptsn mediates dephosphorylation of Rap1, thereby preserving the activity and membrane localization of Rap1 to stabilize the MIT complex. CRISPR/Cas9-induced deletion of PPP1R18, which encodes Ptsn, markedly suppresses integrin activation in Jurkat human T cells. We generated apparently healthy Ppp1r18-/- mice that manifest lymphocytosis and reduced population of peripheral lymphoid tissues ascribable, in part, to defective activation of integrins αLβ2 and α4β7. Ppp1r18-/- T cells exhibit reduced capacity to induce colitis in a murine adoptive transfer model. Thus, Ptsn enables lymphocyte integrin-mediated functions by dephosphorylating Rap1 to stabilize the MIT complex. As a consequence, loss of Ptsn ameliorates T cell-mediated colitis.
Topics: Adaptor Proteins, Signal Transducing; Animals; Cell Adhesion; Colitis; Integrins; Lymphoid Tissue; Membrane Proteins; Mice; Protein Phosphatase 1; T-Lymphocytes; Talin; rap1 GTP-Binding Proteins
PubMed: 35766979
DOI: 10.1084/jem.20211637 -
Molecular Systems Biology Jul 2015Concentration gradients provide spatial information for tissue patterning and cell organization, and their robustness under natural fluctuations is an evolutionary...
Concentration gradients provide spatial information for tissue patterning and cell organization, and their robustness under natural fluctuations is an evolutionary advantage. In rod-shaped Schizosaccharomyces pombe cells, the DYRK-family kinase Pom1 gradients control cell division timing and placement. Upon dephosphorylation by a Tea4-phosphatase complex, Pom1 associates with the plasma membrane at cell poles, where it diffuses and detaches upon auto-phosphorylation. Here, we demonstrate that Pom1 auto-phosphorylates intermolecularly, both in vitro and in vivo, which confers robustness to the gradient. Quantitative imaging reveals this robustness through two system's properties: The Pom1 gradient amplitude is inversely correlated with its decay length and is buffered against fluctuations in Tea4 levels. A theoretical model of Pom1 gradient formation through intermolecular auto-phosphorylation predicts both properties qualitatively and quantitatively. This provides a telling example where gradient robustness through super-linear decay, a principle hypothesized a decade ago, is achieved through autocatalysis. Concentration-dependent autocatalysis may be a widely used simple feedback to buffer biological activities.
Topics: Algorithms; Cell Division; Cell Membrane; Microtubule-Associated Proteins; Phosphorylation; Protein Kinases; Schizosaccharomyces; Schizosaccharomyces pombe Proteins
PubMed: 26150232
DOI: 10.15252/msb.20145996 -
Molecular Systems Biology Dec 2023Phosphoprotein phosphatases (PPPs) regulate major signaling pathways, but the determinants of phosphatase specificity are poorly understood. This is because methods to...
Phosphoprotein phosphatases (PPPs) regulate major signaling pathways, but the determinants of phosphatase specificity are poorly understood. This is because methods to investigate this at scale are lacking. Here, we develop a novel in vitro assay, MRBLE:Dephos, that allows multiplexing of dephosphorylation reactions to determine phosphatase preferences. Using MRBLE:Dephos, we establish amino acid preferences of the residues surrounding the dephosphorylation site for PP1 and PP2A-B55, which reveals common and unique preferences. To compare the MRBLE:Dephos results to cellular substrates, we focused on mitotic exit that requires extensive dephosphorylation by PP1 and PP2A-B55. We use specific inhibition of PP1 and PP2A-B55 in mitotic exit lysates coupled with phosphoproteomics to identify more than 2,000 regulated sites. Importantly, the sites dephosphorylated during mitotic exit reveal key signatures that are consistent with MRBLE:Dephos. Furthermore, integration of our phosphoproteomic data with mitotic interactomes of PP1 and PP2A-B55 provides insight into how binding of phosphatases to substrates shapes dephosphorylation. Collectively, we develop novel approaches to investigate protein phosphatases that provide insight into mitotic exit regulation.
Topics: Phosphorylation; Protein Phosphatase 2; Mitosis; Signal Transduction; Substrate Specificity
PubMed: 37916966
DOI: 10.15252/msb.202311782 -
EMBO Reports May 2020The tumor suppressor Smad4, a key mediator of the TGF-β/BMP pathways, is essential for development and tissue homeostasis. Phosphorylation of Smad4 in its linker region...
The tumor suppressor Smad4, a key mediator of the TGF-β/BMP pathways, is essential for development and tissue homeostasis. Phosphorylation of Smad4 in its linker region catalyzed by the mitogen-activated protein kinase (MAPK) plays a pivotal role in regulating its transcriptional activity and stability. In contrast, roles of Smad4 dephosphorylation as a control mechanism of TGF-β/BMP signaling and the phosphatases responsible for its dephosphorylation remain so far elusive. Here, we identify Wip1 as a Smad4 phosphatase. Wip1 selectively binds and dephosphorylates Smad4 at Thr277, a key MAPK phosphorylation site, thereby regulating its nuclear accumulation and half-life. In Xenopus embryos, Wip1 limits mesoderm formation and favors neural induction by inhibiting TGF-β/BMP signals. Wip1 restrains TGF-β-induced growth arrest, migration, and invasion in human cells and enhances the tumorigenicity of cancer cells by repressing the antimitogenic activity of Smad4. We propose that Wip1-dependent dephosphorylation of Smad4 is critical for the regulation of TGF-β signaling.
Topics: Animals; Humans; Mitogen-Activated Protein Kinases; Phosphorylation; Protein Phosphatase 2C; Signal Transduction; Smad4 Protein; Transforming Growth Factor beta; Xenopus Proteins; Xenopus laevis
PubMed: 32103600
DOI: 10.15252/embr.201948693 -
ELife Dec 2020Mitophagy plays an important role in mitochondrial homeostasis. In yeast, the phosphorylation of the mitophagy receptor Atg32 by casein kinase 2 is essential for... (Comparative Study)
Comparative Study
Mitophagy plays an important role in mitochondrial homeostasis. In yeast, the phosphorylation of the mitophagy receptor Atg32 by casein kinase 2 is essential for mitophagy. This phosphorylation is counteracted by the yeast equivalent of the STRIPAK complex consisting of the PP2A-like protein phosphatase Ppg1 and Far3-7-8-9-10-11 (Far complex), but the underlying mechanism remains elusive. Here we show that two subpopulations of the Far complex reside in the mitochondria and endoplasmic reticulum, respectively, and play distinct roles; the former inhibits mitophagy via Atg32 dephosphorylation, and the latter regulates TORC2 signaling. Ppg1 and Far11 form a subcomplex, and Ppg1 activity is required for the assembling integrity of Ppg1-Far11-Far8. The Far complex preferentially interacts with phosphorylated Atg32, and this interaction is weakened by mitophagy induction. Furthermore, the artificial tethering of Far8 to Atg32 prevents mitophagy. Taken together, the Ppg1-mediated Far complex formation and its dissociation from Atg32 are crucial for mitophagy regulation.
Topics: Autophagy-Related Proteins; Endoplasmic Reticulum; Gene Expression Regulation, Fungal; Mitochondria; Mitophagy; Multiprotein Complexes; Phosphoprotein Phosphatases; Phosphorylation; Receptors, Cytoplasmic and Nuclear; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Signal Transduction
PubMed: 33317697
DOI: 10.7554/eLife.63694 -
Communications Biology Jun 2023Protein Tyrosine Phosphatase 1B (PTP1B) is the prototypical protein tyrosine phosphatase and plays an essential role in the regulation of several kinase-driven...
Protein Tyrosine Phosphatase 1B (PTP1B) is the prototypical protein tyrosine phosphatase and plays an essential role in the regulation of several kinase-driven signalling pathways. PTP1B displays a preference for bisphosphorylated substrates. Here we identify PTP1B as an inhibitor of IL-6 and show that, in vitro, it can dephosphorylate all four members of the JAK family. In order to gain a detailed understanding of the molecular mechanism of JAK dephosphorylation, we undertook a structural and biochemical analysis of the dephosphorylation reaction. We identified a product-trapping PTP1B mutant that allowed visualisation of the tyrosine and phosphate products of the reaction and a substrate-trapping mutant with a vastly decreased off-rate compared to those previously described. The latter mutant was used to determine the structure of bisphosphorylated JAK peptides bound to the enzyme active site. These structures revealed that the downstream phosphotyrosine preferentially engaged the active site, in contrast to the analogous region of IRK. Biochemical analysis confirmed this preference. In this binding mode, the previously identified second aryl binding site remains unoccupied and the non-substrate phosphotyrosine engages Arg47. Mutation of this arginine disrupts the preference for the downstream phosphotyrosine. This study reveals a previously unappreciated plasticity in how PTP1B interacts with different substrates.
Topics: Phosphoric Monoester Hydrolases; Phosphotyrosine; Janus Kinases; Arginine; Binding Sites
PubMed: 37316570
DOI: 10.1038/s42003-023-05020-9 -
The Journal of Biological Chemistry Apr 2023To cope with an increased external osmolarity, the budding yeast Saccharomyces cerevisiae activates the Hog1 mitogen-activated protein kinase (MAPK) through the...
To cope with an increased external osmolarity, the budding yeast Saccharomyces cerevisiae activates the Hog1 mitogen-activated protein kinase (MAPK) through the high-osmolarity glycerol (HOG) pathway, which governs adaptive responses to osmostress. In the HOG pathway, two apparently redundant upstream branches, termed SLN1 and SHO1, activate cognate MAP3Ks (MAPKK kinase) Ssk2/22 and Ste11, respectively. These MAP3Ks, when activated, phosphorylate and thus activate the Pbs2 MAP2K (MAPK kinase), which in turn phosphorylates and activates Hog1. Previous studies have shown that protein tyrosine phosphatases and the serine/threonine protein phosphatases type 2C negatively regulate the HOG pathway to prevent its excessive and inappropriate activation, which is detrimental to cell growth. The tyrosine phosphatases Ptp2 and Ptp3 dephosphorylate Hog1 at Tyr-176, whereas the protein phosphatase type 2Cs Ptc1 and Ptc2 dephosphorylate Hog1 at Thr-174. In contrast, the identities of phosphatases that dephosphorylate Pbs2 remained less clear. Here, we examined the phosphorylation status of Pbs2 at the activating phosphorylation sites Ser-514 and Thr-518 (S514 and T518) in various mutants, both in the unstimulated and osmostressed conditions. Thus, we found that Ptc1-Ptc4 collectively regulate Pbs2 negatively, but each Ptc acts differently to the two phosphorylation sites in Pbs2. T518 is predominantly dephosphorylated by Ptc1, while S514 can be dephosphorylated by any of Ptc1-4 to an appreciable extent. We also show that Pbs2 dephosphorylation by Ptc1 requires the adaptor protein Nbp2 that recruits Ptc1 to Pbs2, thus highlighting the complex processes involved in regulating adaptive responses to osmostress.
Topics: Glycerol; Intracellular Signaling Peptides and Proteins; MAP Kinase Kinase Kinases; Mitogen-Activated Protein Kinase Kinases; Osmolar Concentration; Phosphoprotein Phosphatases; Phosphorylation; Protein Kinases; Protein Phosphatase 2C; Protein Tyrosine Phosphatases; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Signal Transduction
PubMed: 36870684
DOI: 10.1016/j.jbc.2023.104569