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The Plant Cell Feb 2022Plants have evolved sophisticated mechanisms to ensure flowering in favorable conditions for reproductive success. In the model plant Arabidopsis thaliana, FLOWERING...
Plants have evolved sophisticated mechanisms to ensure flowering in favorable conditions for reproductive success. In the model plant Arabidopsis thaliana, FLOWERING LOCUS C (FLC) acts as a central repressor of flowering and the major determinant for winter cold requirement for flowering. FLC is activated in winter annuals by the FRIGIDA (FRI) activator complex containing FRI, FLC EXPRESSOR (FLX), and FLX-LIKE 4 (FLX4), among which FLX and FLX4 are also essential for establishing basal FLC expression in summer annuals. Here we show that a plant RNA polymerase II C-terminal domain phosphatase, C-TERMINAL DOMAIN PHOSPHATASE-LIKE 3 (CPL3), interacts with and dephosphorylates FLX4 through their scaffold protein FLX to inhibit flowering. CPL3-mediated dephosphorylation of FLX4 serves as a key molecular switch that enables binding of dephosphorylated FLX4 to the FLC locus to promote FLC expression, thus repressing flowering in both winter and summer annuals of Arabidopsis. Our findings reveal a molecular switch underlying the activation of FLC for flowering time control.
Topics: Arabidopsis; Arabidopsis Proteins; Flowers; Gene Expression Regulation, Plant; MADS Domain Proteins; Phosphoprotein Phosphatases; Phosphorylation; Plants, Genetically Modified; Nicotiana
PubMed: 34850922
DOI: 10.1093/plcell/koab286 -
The Journal of Biological Chemistry Jun 2009Neurodegenerative tauopathies, including Alzheimer disease, are characterized by abnormal hyperphosphorylation of the microtubule-associated protein Tau. One group of...
Neurodegenerative tauopathies, including Alzheimer disease, are characterized by abnormal hyperphosphorylation of the microtubule-associated protein Tau. One group of tauopathies, known as frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), is directly associated with mutations of the gene tau. However, it is unknown why mutant Tau is highly phosphorylated in the patient brain. In contrast to in vivo high phosphorylation, FTDP-17 Tau is phosphorylated less than wild-type Tau in vitro. Because phosphorylation is a balance between kinase and phosphatase activities, we investigated dephosphorylation of mutant Tau proteins, P301L and R406W. Tau phosphorylated by Cdk5-p25 was dephosphorylated by protein phosphatases in rat brain extracts. Compared with wild-type Tau, R406W was dephosphorylated faster and P301L slower. The two-dimensional phosphopeptide map analysis suggested that faster dephosphorylation of R406W was due to a lack of phosphorylation at Ser-404, which is relatively resistant to dephosphorylation. We studied the effect of the peptidyl-prolyl isomerase Pin1 or microtubule binding on dephosphorylation of wild-type Tau, P301L, and R406W in vitro. Pin1 catalyzes the cis/trans isomerization of phospho-Ser/Thr-Pro sequences in a subset of proteins. Dephosphorylation of wild-type Tau was reduced in brain extracts of Pin1-knockout mice, and this reduction was not observed with P301L and R406W. On the other hand, binding to microtubules almost abolished dephosphorylation of wild-type and mutant Tau proteins. These results demonstrate that mutation of Tau and its association with microtubules may change the conformation of Tau, thereby suppressing dephosphorylation and potentially contributing to the etiology of tauopathies.
Topics: Amino Acid Substitution; Animals; Base Sequence; Brain; DNA Primers; Humans; In Vitro Techniques; Kinetics; Mice; Mice, Knockout; Microtubules; Models, Neurological; Mutation; NIMA-Interacting Peptidylprolyl Isomerase; Peptide Mapping; Peptidylprolyl Isomerase; Phosphorylation; Protein Conformation; Rats; Recombinant Proteins; Tauopathies; tau Proteins
PubMed: 19401603
DOI: 10.1074/jbc.M109.003277 -
American Journal of Physiology.... Oct 2003Ca2+-regulated heat-stable protein of 24 kDa (CRHSP-24) is a serine phosphoprotein originally identified as a physiological substrate for the Ca2+-calmodulin regulated...
Ca2+-regulated heat-stable protein of 24 kDa (CRHSP-24) is a serine phosphoprotein originally identified as a physiological substrate for the Ca2+-calmodulin regulated protein phosphatase calcineurin (PP2B). CRHSP-24 is a paralog of the brain-specific mRNA-binding protein PIPPin and was recently shown to interact with the STYX/dead phosphatase protein in developing spermatids (Wishart MJ and Dixon JE. Proc Natl Acad Sci USA 99: 2112-2117, 2002). Investigation of the effects of phorbol ester (12-o-tetradecanoylphorbol-13-acetate; TPA) and cAMP analogs in 32P-labeled pancreatic acini revealed that these agents acutely dephosphorylated CRHSP-24 by a Ca2+-independent mechanism. Indeed, cAMP- and TPA-mediated dephosphorylation of CRHSP-24 was fully inhibited by the PP1/PP2A inhibitor calyculin A, indicating that the protein is regulated by an additional phosphatase other than PP2B. Supporting this, CRHSP-24 dephosphorylation in response to the Ca2+-mobilizing hormone cholecystokinin was differentially inhibited by calyculin A and the PP2B-selective inhibitor cyclosporin A. Stimulation of acini with secretin, a secretagogue that signals through the cAMP pathway in acini, induced CRHSP-24 dephosphorylation in a concentration-dependent manner. Isoelectric focusing and immunoblotting indicated that elevated cellular Ca2+ dephosphorylated CRHSP-24 on at least three serine sites, whereas cAMP and TPA partially dephosphorylated the protein on at least two sites. The cAMP-mediated dephosphorylation of CRHSP-24 was inhibited by low concentrations of okadaic acid (10 nM) and fostriecin (1 microM), suggesting that CRHSP-24 is regulated by PP2A or PP4. Collectively, these data indicate that CRHSP-24 is regulated by diverse and physiologically relevant signaling pathways in acinar cells, including Ca2+, cAMP, and diacylglycerol.
Topics: Alkenes; Animals; Binding Sites; Calcineurin Inhibitors; Calcium; Cholecystokinin; Cyclic AMP; Cyclosporine; DNA-Binding Proteins; Enzyme Inhibitors; Marine Toxins; Okadaic Acid; Oxazoles; Pancreas; Phosphoprotein Phosphatases; Phosphoproteins; Phosphorylation; Polyenes; Pyrones; Rats; Rats, Sprague-Dawley; Signal Transduction; Tetradecanoylphorbol Acetate; Transcription Factors
PubMed: 12801884
DOI: 10.1152/ajpgi.00111.2003 -
Cells Jul 2023Protocadherin-7 (Pcdh7) is a member of the non-clustered protocadherin δ1 subgroup of the cadherin superfamily. Pcdh7 has been revealed to control osteoclast...
Protocadherin-7 (Pcdh7) is a member of the non-clustered protocadherin δ1 subgroup of the cadherin superfamily. Pcdh7 has been revealed to control osteoclast differentiation by regulating Rho-family small GTPases, RhoA and Rac1, through its intracellular SET binding domain. However, the mechanisms by which small GTPases are regulated downstream of Pcdh7 remain unclear. Here, we demonstrate that protein phosphatase 2A (PP2A)-mediated dephosphorylation of Glycogen synthase kinase-3β (GSK3β) is required for Pcdh7-dependent activation of RhoA during osteoclast differentiation. Pcdh7-deficient (Pcdh7) cells showed impaired PP2A activity, despite their normal expression of PP2A. GSK3β, whose activity is regulated by its inhibitory phosphorylation at Ser9, was dephosphorylated during osteoclast differentiation in a Pcdh7-dependent manner. Inhibition of protein phosphatase by okadaic acid reduced dephosphorylation of GSK3β in Pcdh7 cells, while activation of PP2A by DT-061 rescued impaired dephosphorylation of GSK3β in Pcdh7 cells. Inhibition of GSK3β by AR-A014418 inhibited RANKL-induced RhoA activation and osteoclast differentiation in Pcdh7 cells. On the other hand, DT-061 treatment rescued impaired RhoA activation and RANKL-induced osteoclast differentiation in Pcdh7 cells. Taken together, these results demonstrate that PP2A dephosphorylates GSK3β and thereby activates it in a Pcdh7-dependent manner, which is required for activation of small GTPase RhoA and proper osteoclast differentiation.
Topics: Osteoclasts; Protein Phosphatase 2; Monomeric GTP-Binding Proteins; Protocadherins; Glycogen Synthase Kinase 3 beta; Cadherins
PubMed: 37566044
DOI: 10.3390/cells12151967 -
PloS One 2012Capsules frequently play a key role in bacterial interactions with their environment. Escherichia coli capsules were categorized as groups 1 through 4, each produced by...
Capsules frequently play a key role in bacterial interactions with their environment. Escherichia coli capsules were categorized as groups 1 through 4, each produced by a distinct mechanism. Etk and Etp are members of protein families required for the production of group 1 and group 4 capsules. These members function as a protein tyrosine kinase and protein tyrosine phosphatase, respectively. We show that Etp dephosphorylates Etk in vivo, and mutations rendering Etk or Etp catalytically inactive result in loss of group 4 capsule production, supporting the notion that cyclic phosphorylation and dephosphorylation of Etk is required for capsule formation. Notably, Etp also becomes tyrosine phosphorylated in vivo and catalyzes rapid auto-dephosphorylation. Further analysis identified Tyr121 as the phosphorylated residue of Etp. Etp containing Phe, Glu or Ala in place of Tyr121 retained phosphatase activity and catalyzed dephosphorylation of Etp and Etk. Although EtpY121E and EtpY121A still supported capsule formation, EtpY121F failed to do so. These results suggest that cycles of phosphorylation and dephosphorylation of Etp, as well as Etk, are involved in the formation of group 4 capsule, providing an additional regulatory layer to the complex control of capsule production.
Topics: Amino Acid Sequence; Bacterial Capsules; Escherichia coli; Escherichia coli Proteins; Kinetics; Membrane Proteins; Models, Biological; Molecular Sequence Data; Mutation; Phosphorylation; Phosphotyrosine; Protein-Tyrosine Kinases; Spectrometry, Mass, Electrospray Ionization
PubMed: 22675501
DOI: 10.1371/journal.pone.0037984 -
The Plant Cell Feb 2013The basic Leucine zipper transcription factor ABSCISIC ACID INSENSITIVE5 (ABI5) is a key regulator of abscisic acid (ABA)-mediated seed germination and postgermination...
The basic Leucine zipper transcription factor ABSCISIC ACID INSENSITIVE5 (ABI5) is a key regulator of abscisic acid (ABA)-mediated seed germination and postgermination seedling growth. While a family of SUCROSE NONFERMENTING1-related protein kinase2s (SnRK2s) is responsible for ABA-induced phosphorylation and stabilization of ABI5, the phosphatase(s) responsible for dephosphorylating ABI5 is still unknown. Here, we demonstrate that mutations in FyPP1 (for Phytochrome-associated serine/threonine protein phosphatase1) and FyPP3, two homologous genes encoding the catalytic subunits of Ser/Thr PROTEIN PHOSPHATASE6 (PP6), cause an ABA hypersensitive phenotype in Arabidopsis thaliana, including ABA-mediated inhibition of seed germination and seedling growth. Conversely, overexpression of FyPP causes reduced sensitivity to ABA. The ABA hypersensitive phenotype of FyPP loss-of-function mutants is ABI5 dependent, and the amount of phosphorylated and total ABI5 proteins inversely correlates with the levels of FyPP proteins. Moreover, FyPP proteins physically interact with ABI5 in vitro and in vivo, and the strength of the interaction depends on the ABI5 phosphorylation status. In vitro phosphorylation assays show that FyPP proteins directly dephosphorylate ABI5. Furthermore, genetic and biochemical assays show that FyPP proteins act antagonistically with SnRK2 kinases to regulate ABI5 phosphorylation and ABA responses. Thus, Arabidopsis PP6 phosphatase regulates ABA signaling through dephosphorylation and destabilization of ABI5.
Topics: Abscisic Acid; Arabidopsis; Arabidopsis Proteins; Basic-Leucine Zipper Transcription Factors; Gene Expression Regulation, Plant; Germination; Mutation; Phosphoprotein Phosphatases; Phosphorylation; Plants, Genetically Modified; Protein Interaction Maps; Protein Phosphatase 2; Protein Serine-Threonine Kinases; Protein Stability; Seedlings; Seeds; Signal Transduction
PubMed: 23404889
DOI: 10.1105/tpc.112.105767 -
Molecular and Cellular Biology Dec 1996SHP-1 is an SH2-containing cytoplasmic tyrosine phosphatase that is widely distributed in cells of the hematopoietic system. SHP-1 plays an important role in the signal...
SHP-1 is an SH2-containing cytoplasmic tyrosine phosphatase that is widely distributed in cells of the hematopoietic system. SHP-1 plays an important role in the signal transduction of many cytokine receptors, including the receptor for erythropoietin, by associating via its SH2 domains to the receptors and dephosphorylating key substrates. Recent studies have suggested that SHP-1 regulates the function of Jak family tyrosine kinases, as shown by its constitutive association with the Tyk2 kinase and the hyperphosphorylation of Jak kinases in the motheaten cells that lack functional SHP-1. We have examined the interactions of SHP-1 with two tyrosine kinases activated during engagement of the erythropoietin receptor, the Janus family kinase Jak-2 and the c-fps/fes kinase. Immunoblotting studies with extracts from mouse hematopoietic cells demonstrated that Jak2, but not c-fes, was present in anti-SHP-1 immunoprecipitates, suggesting that SHP-1 selectively associates with Jak2 in vivo. Consistent with this, when SHP-1 was coexpressed with these kinases in Cos-7 cells, it associated with and dephosphorylated Jak2 but not c-fes. Transient cotransfection of truncated forms of SHP-1 with Jak2 demonstrated that the SHP-1-Jak2 interaction is direct and is mediated by a novel binding activity present in the N terminus of SHP-1, independently of SH2 domain-phosphotyrosine interaction. Such SHP-1-Jak2 interaction resulted in induction of the enzymatic activity of the phosphatase in in vitro protein tyrosine phosphatase assays. Interestingly, association of the SH2n domain of SHP-1 with the tyrosine phosphorylated erythropoietin receptor modestly potentiated but was not essential for SHP-1-mediated dephosphorylation of Jak2 and had no effect on c-fes phosphorylation. These data indicate that the main mechanism for regulation of Jak2 phosphorylation by SHP-1 involves a direct, SH2-independent interaction with Jak2 and suggest the existence of similar mechanisms for other members of the Jak family of kinases. They also suggest that such interactions may provide one of the mechanisms that control SHP-1 substrate specificity.
Topics: Animals; COS Cells; Intracellular Signaling Peptides and Proteins; Janus Kinase 2; Mice; Phosphorylation; Protein Tyrosine Phosphatase, Non-Receptor Type 11; Protein Tyrosine Phosphatase, Non-Receptor Type 6; Protein Tyrosine Phosphatases; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; SH2 Domain-Containing Protein Tyrosine Phosphatases
PubMed: 8943354
DOI: 10.1128/MCB.16.12.6985 -
The Journal of Cell Biology May 2021Tight regulation of the APC/C-Cdc20 ubiquitin ligase that targets cyclin B1 for degradation is important for mitotic fidelity. The spindle assembly checkpoint (SAC)...
Tight regulation of the APC/C-Cdc20 ubiquitin ligase that targets cyclin B1 for degradation is important for mitotic fidelity. The spindle assembly checkpoint (SAC) inhibits Cdc20 through the mitotic checkpoint complex (MCC). In addition, phosphorylation of Cdc20 by cyclin B1-Cdk1 independently inhibits APC/C-Cdc20 activation. This creates a conundrum for how Cdc20 is activated before cyclin B1 degradation. Here, we show that the MCC component BubR1 harbors both Cdc20 inhibition and activation activities, allowing for cross-talk between the two Cdc20 inhibition pathways. Specifically, BubR1 acts as a substrate specifier for PP2A-B56 to enable efficient Cdc20 dephosphorylation in the MCC. A mutant Cdc20 mimicking the dephosphorylated state escapes a mitotic checkpoint arrest, arguing that restricting Cdc20 dephosphorylation to the MCC is important. Collectively, our work reveals how Cdc20 can be dephosphorylated in the presence of cyclin B1-Cdk1 activity without causing premature anaphase onset.
Topics: Anaphase; Anaphase-Promoting Complex-Cyclosome; Cdc20 Proteins; Cell Cycle Proteins; Cell Line, Tumor; Cyclin B1; HeLa Cells; Humans; M Phase Cell Cycle Checkpoints; Mitosis; Phosphorylation; Protein Binding; Protein Serine-Threonine Kinases; Spindle Apparatus
PubMed: 33819340
DOI: 10.1083/jcb.202012081 -
Cell Reports Feb 2024Neuronal protein synthesis is required for long-lasting plasticity and long-term memory consolidation. Dephosphorylation of eukaryotic initiation factor 2α is one of...
Neuronal protein synthesis is required for long-lasting plasticity and long-term memory consolidation. Dephosphorylation of eukaryotic initiation factor 2α is one of the key translational control events that is required to increase de novo protein synthesis that underlies long-lasting plasticity and memory consolidation. Here, we interrogate the molecular pathways of translational control that are triggered by neuronal stimulation with brain-derived neurotrophic factor (BDNF), which results in eukaryotic initiation factor 2α (eIF2α) dephosphorylation and increases in de novo protein synthesis. Primary rodent neurons exposed to BDNF display elevated translation of GADD34, which facilitates eIF2α dephosphorylation and subsequent de novo protein synthesis. Furthermore, GADD34 requires G-actin generated by cofilin to dephosphorylate eIF2α and enhance protein synthesis. Finally, GADD34 is required for BDNF-induced translation of synaptic plasticity-related proteins. Overall, we provide evidence that neurons repurpose GADD34, an effector of the integrated stress response, as an orchestrator of rapid increases in eIF2-dependent translation in response to plasticity-inducing stimuli.
Topics: Brain-Derived Neurotrophic Factor; Actin Depolymerizing Factors; Actins; Eukaryotic Initiation Factor-2; Neurons
PubMed: 38219147
DOI: 10.1016/j.celrep.2023.113670 -
Molecular Biology of the Cell Nov 2007Fission yeast mitogen-activated protein kinase (MAPK) Pmk1p is involved in morphogenesis, cytokinesis, and ion homeostasis as part of the cell integrity pathway, and it...
Fission yeast mitogen-activated protein kinase (MAPK) Pmk1p is involved in morphogenesis, cytokinesis, and ion homeostasis as part of the cell integrity pathway, and it becomes activated under multiple stresses, including hyper- or hypotonic conditions, glucose deprivation, cell wall-damaging compounds, and oxidative stress. The only protein phosphatase known to dephosphorylate and inactivate Pmk1p is Pmp1p. We show here that the stress-activated protein kinase (SAPK) pathway and its main effector, Sty1p MAPK, are essential for proper deactivation of Pmk1p under hypertonic stress in a process regulated by Atf1p transcription factor. We demonstrate that tyrosine phosphatases Pyp1p and Pyp2p, and serine/threonine phosphatase Ptc1p, that negatively regulate Sty1p activity and whose expression is dependent on Sty1p-Atf1p function, are involved in Pmk1p dephosphorylation under osmostress. Pyp1p and Ptc1p, in addition to Pmp1p, also control the basal level of MAPK Pmk1p activity in growing cells and associate with, and dephosphorylate Pmk1p both in vitro and in vivo. Our results with Ptc1p provide the first biochemical evidence for a PP2C-type phosphatase acting on more than one MAPK in yeast cells. Importantly, the SAPK-dependent down-regulation of Pmk1p through Pyp1p, Pyp2p, and Ptc1p was not complete, and Pyp1p and Ptc1p phosphatases are able to negatively regulate MAPK Pmk1p activity by an alternative regulatory mechanism. Our data also indicate that Pmk1p phosphorylation oscillates as a function of the cell cycle, peaking at cell separation during cytokinesis, and that Pmp1p phosphatase plays a main role in regulating this process.
Topics: Cell Cycle; Down-Regulation; Enzyme Activation; Gene Deletion; Gene Expression Regulation, Fungal; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Osmotic Pressure; Phosphoprotein Phosphatases; Phosphorylation; Phosphothreonine; Protein Binding; Schizosaccharomyces; Schizosaccharomyces pombe Proteins
PubMed: 17761528
DOI: 10.1091/mbc.e07-05-0484