-
ELife Oct 2019Mutations that activate LRRK2 protein kinase cause Parkinson's disease. LRRK2 phosphorylates a subset of Rab GTPases within their Switch-II motif controlling interaction...
Mutations that activate LRRK2 protein kinase cause Parkinson's disease. LRRK2 phosphorylates a subset of Rab GTPases within their Switch-II motif controlling interaction with effectors. An siRNA screen of all human protein phosphatases revealed that a poorly studied protein phosphatase, PPM1H, counteracts LRRK2 signaling by specifically dephosphorylating Rab proteins. PPM1H knockout increased endogenous Rab phosphorylation and inhibited Rab dephosphorylation in human A549 cells. Overexpression of PPM1H suppressed LRRK2-mediated Rab phosphorylation. PPM1H also efficiently and directly dephosphorylated Rab8A in biochemical studies. A "substrate-trapping" PPM1H mutant (Asp288Ala) binds with high affinity to endogenous, LRRK2-phosphorylated Rab proteins, thereby blocking dephosphorylation seen upon addition of LRRK2 inhibitors. PPM1H is localized to the Golgi and its knockdown suppresses primary cilia formation, similar to pathogenic LRRK2. Thus, PPM1H acts as a key modulator of LRRK2 signaling by controlling dephosphorylation of Rab proteins. PPM1H activity enhancers could offer a new therapeutic approach to prevent or treat Parkinson's disease.
Topics: Humans; Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; Phosphoprotein Phosphatases; Phosphorylation; RNA, Small Interfering; Signal Transduction; rab GTP-Binding Proteins
PubMed: 31663853
DOI: 10.7554/eLife.50416 -
The Plant Cell Oct 2022Autophagy, a conserved pathway that carries out the bulk degradation of cytoplasmic material in eukaryotic cells, is critical for plant physiology and development. This...
Autophagy, a conserved pathway that carries out the bulk degradation of cytoplasmic material in eukaryotic cells, is critical for plant physiology and development. This process is tightly regulated by ATG13, a core component of the ATG1 kinase complex, which initiates autophagy. Although ATG13 is known to be dephosphorylated immediately after nutrient starvation, the phosphatase regulating this process is poorly understood. Here, we determined that the Arabidopsis (Arabidopsis thaliana) septuple mutant (topp-7m) and octuple mutant (topp-8m) of TYPE ONE PROTEIN PHOSPHATASE (TOPP) exhibited significantly reduced tolerance to fixed-carbon (C) starvation due to compromised autophagy activity. Genetic analysis placed TOPP upstream of autophagy. Interestingly, ATG13a was found to be an interactor of TOPP. TOPP directly dephosphorylated ATG13a in vitro and in vivo. We identified 18 phosphorylation sites in ATG13a by LC-MS. Phospho-dead ATG13a at these 18 sites significantly promoted autophagy and increased the tolerance of the atg13ab mutant to fixed-C starvation. The dephosphorylation of ATG13a facilitated ATG1a-ATG13a complex formation. Consistently, the recruitment of ATG13a for ATG1a was markedly inhibited in topp-7m-1. Finally, TOPP-controlled dephosphorylation of ATG13a boosted ATG1a phosphorylation. Taken together, our study reveals the crucial role of TOPP in regulating autophagy by stimulating the formation of the ATG1a-ATG13a complex by dephosphorylating ATG13a in Arabidopsis.
Topics: Arabidopsis; Carbon; Protein Kinases; Autophagy; Autophagy-Related Proteins; Phosphorylation; Phosphoprotein Phosphatases
PubMed: 35961047
DOI: 10.1093/plcell/koac251 -
The Journal of Cell Biology Dec 2018As a dividing cell exits mitosis and daughter cells enter interphase, many proteins must be dephosphorylated. The protein phosphatase 2A (PP2A) with its B55 regulatory...
As a dividing cell exits mitosis and daughter cells enter interphase, many proteins must be dephosphorylated. The protein phosphatase 2A (PP2A) with its B55 regulatory subunit plays a crucial role in this transition, but the identity of its substrates and how their dephosphorylation promotes mitotic exit are largely unknown. We conducted a maternal-effect screen in to identify genes that function with PP2A-B55/Tws in the cell cycle. We found that eggs that receive reduced levels of Tws and of components of the nuclear envelope (NE) often fail development, concomitant with NE defects following meiosis and in syncytial mitoses. Our mechanistic studies using cells indicate that PP2A-Tws promotes nuclear envelope reformation (NER) during mitotic exit by dephosphorylating BAF and suggests that PP2A-Tws targets additional NE components, including Lamin and Nup107. This work establishes as a powerful model to further dissect the molecular mechanisms of NER and suggests additional roles of PP2A-Tws in the completion of meiosis and mitosis.
Topics: Animals; Aquaporins; DNA-Binding Proteins; Drosophila Proteins; Drosophila melanogaster; Lamins; Mitosis; Models, Biological; Nuclear Envelope; Nuclear Proteins; Phosphoprotein Phosphatases
PubMed: 30309980
DOI: 10.1083/jcb.201804018 -
Journal of Neurochemistry Jan 2004Protein phosphatase 5 (PP5) is a 58-kDa novel phosphoseryl/phosphothreonyl protein phosphatase. It is ubiquitously expressed in all mammalian tissues examined, with a...
Protein phosphatase 5 (PP5) is a 58-kDa novel phosphoseryl/phosphothreonyl protein phosphatase. It is ubiquitously expressed in all mammalian tissues examined, with a high level in the brain, but little is known about its physiological substrates. We found that this phosphatase dephosphorylated recombinant tau phosphorylated with cAMP-dependent protein kinase and glycogen synthase kinase-3beta, as well as abnormally hyperphosphorylated tau isolated from brains of patients with Alzheimer's disease. The specific activity of PP5 toward tau was comparable to those reported with other protein substrates examined to date. The PP5 activity toward tau was stimulated by arachidonic acid by 30- to 45-fold. Immunostaining demonstrated that PP5 was primarily cytoplasmic in PC12 cells and in neurons of postmortem human brain tissue. A small pool of PP5 associated with microtubules. Expression of active PP5 in PC12 cells resulted in reduced phosphorylation of tau, suggesting that PP5 can also dephosphorylate tau in cells. These results suggest that PP5 plays a role in the dephosphorylation of tau and might be involved in the molecular pathogenesis of Alzheimer's disease.
Topics: Animals; Binding Sites; Cytoplasm; Humans; Male; Microtubule-Associated Proteins; Nuclear Proteins; PC12 Cells; Phosphoprotein Phosphatases; Phosphorylation; Rats; Rats, Wistar; tau Proteins
PubMed: 14690518
DOI: 10.1111/j.1471-4159.2004.02147.x -
The Journal of Biological Chemistry Dec 2000Prolactin (PRL) plays a central and crucial role in the regulation of milk protein gene expression in mammary epithelial cells. PRL binding to its cognate receptor leads...
Prolactin (PRL) plays a central and crucial role in the regulation of milk protein gene expression in mammary epithelial cells. PRL binding to its cognate receptor leads to receptor dimerization and activation of the tyrosine kinase Janus kinase 2 (JAK2), associated with the membrane-proximal, intracellular domain of the receptor. In turn, JAK2 phosphorylates and activates STAT5, a member of the signal transducers and activators of transcription (STAT) family. We have recently reported that 16 different protein-tyrosine phosphatases (PTP) were expressed in lactating mouse mammary gland and mammary epithelial cells (Aoki, N., Kawamura, M., Yamaguchi-Aoki, Y., Ohira, S., and Matsuda, T. (1999) J. Biochem. (Tokyo) 125, 669-675). We investigated the involvement of each PTP in PRL signaling. Among the 12 phosphatases including SHP-2 examined, a cytosolic phosphatase PTP1B was found to specifically dephosphorylate STAT5a and STAT5b in transfected COS7 and in vitro. Nuclear translocation of STAT5a and STAT5b was largely inhibited upon overexpression of PTP1B. The PRL-dependent transcriptional activation of the beta-casein gene promoter was also inhibited by PTP1B. Furthermore, retrovirus-mediated overexpression of PTP1B resulted in dephosphorylation of endogenous STAT5 and down-regulation of beta-casein gene expression in mammary epithelial COMMA-1D cells when the cells were treated with lactogenic hormones. Endogenous tyrosine-phosphorylated STAT5 proteins in mammary epithelial COMMA-1D cells as well as tyrosine-phosphorylated STAT5a and STAT5b expressed in COS7 cells were co-precipitated by substrate-trapping mutants of recombinant PTP1B. These results strongly suggest that PTP1B dephosphorylates PRL-activated STAT5a and STAT5b, thereby negatively regulating PRL-mediated signaling pathway.
Topics: Animals; Blotting, Western; Breast; COS Cells; Caseins; Cell Nucleus; Cytosol; DNA, Complementary; DNA-Binding Proteins; Down-Regulation; Epithelial Cells; Immunoblotting; Intracellular Signaling Peptides and Proteins; Janus Kinase 2; Mice; Milk Proteins; Phosphorylation; Prolactin; Protein Structure, Tertiary; Protein Tyrosine Phosphatase, Non-Receptor Type 1; Protein Tyrosine Phosphatase, Non-Receptor Type 11; Protein Tyrosine Phosphatase, Non-Receptor Type 6; Protein Tyrosine Phosphatases; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Recombinant Fusion Proteins; Recombinant Proteins; Retroviridae; STAT5 Transcription Factor; Signal Transduction; Substrate Specificity; Time Factors; Trans-Activators; Transcriptional Activation; Transfection
PubMed: 10993888
DOI: 10.1074/jbc.M005615200 -
PloS One Apr 2011The cyclin-dependent kinase CDK9/cyclin T1 induces HIV-1 transcription by phosphorylating the carboxyterminal domain (CTD) of RNA polymerase II (RNAPII). CDK9 activity...
The cyclin-dependent kinase CDK9/cyclin T1 induces HIV-1 transcription by phosphorylating the carboxyterminal domain (CTD) of RNA polymerase II (RNAPII). CDK9 activity is regulated by protein phosphatase-1 (PP1) which was previously shown to dephosphorylate CDK9 Thr186. Here, we analyzed the effect of PP1 on RNAPII phosphorylation and CDK9 activity. The selective inhibition of PP1 by okadaic acid and by NIPP1 inhibited phosphorylation of RNAPII CTD in vitro and in vivo. Expression of the central domain of NIPP1 in cultured cells inhibited the enzymatic activity of CDK9 suggesting its activation by PP1. Comparison of dephosphorylation of CDK9 phosphorylated by ((32)P) in vivo and dephosphorylation of CDK9's Thr186 analyzed by Thr186 phospho-specific antibodies, indicated that a residue other than Thr186 might be dephosphorylated by PP1. Analysis of dephosphorylation of phosphorylated peptides derived from CDK9's T-loop suggested that PP1 dephosphorylates CDK9 Ser175. In cultured cells, CDK9 was found to be phosphorylated on Ser175 as determined by combination of Hunter 2D peptide mapping and LC-MS analysis. CDK9 S175A mutant was active and S175D--inactive, and dephosphorylation of CDK9's Ser175 upregulated HIV-1 transcription in PP1-dependent manner. Collectively, our results point to CDK9 Ser175 as novel PP1-regulatory site which dephosphorylation upregulates CDK9 activity and contribute to the activation of HIV-1 transcription.
Topics: Amino Acid Sequence; Cell Line; Cyclin-Dependent Kinase 9; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; HIV-1; Humans; Mass Spectrometry; Molecular Sequence Data; Mutation; Peptide Mapping; Phosphorylation; Protein Phosphatase 1; Serine; Transcription, Genetic
PubMed: 21533037
DOI: 10.1371/journal.pone.0018985 -
The Journal of Biological Chemistry Dec 2006Smad proteins transduce bone morphogenetic protein (BMP) and transforming growth factor-beta (TGFbeta) signals upon phosphorylation of their C-terminal SXS motif by...
Dephosphorylation of the linker regions of Smad1 and Smad2/3 by small C-terminal domain phosphatases has distinct outcomes for bone morphogenetic protein and transforming growth factor-beta pathways.
Smad proteins transduce bone morphogenetic protein (BMP) and transforming growth factor-beta (TGFbeta) signals upon phosphorylation of their C-terminal SXS motif by receptor kinases. The activity of Smad1 in the BMP pathway and Smad2/3 in the TGFbeta pathway is restricted by pathway cross-talk and feedback through protein kinases, including MAPK, CDK2/4, p38MAPK, JNK, and others. These kinases phosphorylate Smads 1-3 at the region that links the N-terminal DNA-binding domain and the C-terminal transcriptional domain. Phosphatases that dephosphorylate the linker region are therefore likely to play an integral part in the regulation of Smad activity. We reported previously that small C-terminal domain phosphatases 1, 2, and 3 (SCP1-3) dephosphorylate Smad1 C-terminal tail, thereby attenuating BMP signaling. Here we provide evidence that SCP1-3 also dephosphorylate the linker regions of Smad1 and Smad2/3 in vitro, in mammalian cells and in Xenopus embryos. Overexpression of SCP 1, 2, or 3 decreased linker phosphorylation of Smads 1, 2 and 3. Moreover, RNA interference-mediated knockdown of SCP1/2 increased the BMP-dependent phosphorylation of the Smad1 linker region as well as the C terminus. In contrast, SCP1/2 knockdown increased the TGFbeta-dependent linker phosphorylation of Smad2/3 but not the C-terminal phosphorylation. Consequently, SCP1/2 knockdown inhibited TGFbeta transcriptional responses, but it enhanced BMP transcriptional responses. Thus, by dephosphorylating Smad2/3 at the linker (inhibitory) but not the C-terminal (activating) site, the SCPs enhance TGFbeta signaling, and by dephosphorylating Smad1 at both sites, the SCPs reset Smad1 to the basal unphosphorylated state.
Topics: Animals; Bone Morphogenetic Proteins; Cell Line; Cell Line, Tumor; Humans; Nuclear Proteins; Peptide Fragments; Phosphoprotein Phosphatases; Phosphorylation; Protein Structure, Tertiary; Signal Transduction; Smad1 Protein; Smad2 Protein; Smad3 Protein; Transforming Growth Factor beta1; Tumor Suppressor Proteins; Xenopus laevis
PubMed: 17085434
DOI: 10.1074/jbc.M610172200 -
The Journal of Biological Chemistry Aug 1993The activation of extracellular signal-regulated kinase (ERK) or mitogen-activated protein kinase (MAPK) by a dual specific kinase, MEK (MAPK or ERK kinase), is a...
The activation of extracellular signal-regulated kinase (ERK) or mitogen-activated protein kinase (MAPK) by a dual specific kinase, MEK (MAPK or ERK kinase), is a critical event in the mitogenic signal transduction pathway. However, little is known about the mechanism of ERK inactivation, which occurs after stimulation. In this report, we demonstrated that a dual specific protein phosphatase, HVH1 (human VH1 phosphatase homolog) whose expression is induced by mitogenic growth factors, specifically inactivates ERK. When several phosphoproteins were tested for recombinant HVH1, only MEK-activated ERK1 was dephosphorylated. HVH1 selectively dephosphorylated threonine and tyrosine residues but not serine residues of the activated ERK1. Inactivation of ERK1 by HVH1 could be reversed by MEK, suggesting that HVH1 dephosphorylates the same residues that are recognized and phosphorylated by MEK. Our results suggest that mitogenic growth factors transiently activate ERK (peak at 5 min followed by a rapid decline) by temporally activating MEK (the on signal) and inducing the expression of HVH phosphatase (the off signal).
Topics: Base Sequence; DNA; Dual-Specificity Phosphatases; Enzyme Induction; Humans; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Molecular Sequence Data; Phosphorylation; Polymerase Chain Reaction; Protein Serine-Threonine Kinases; Protein Tyrosine Phosphatases; Protein-Tyrosine Kinases; Signal Transduction; Substrate Specificity
PubMed: 8344896
DOI: No ID Found -
Blood Jan 2009Bcl2 is associated with chemoresistance and poor prognosis in patients with various hematologic malignancies. DNA damage-induced p53/Bcl2 interaction at the outer...
Bcl2 is associated with chemoresistance and poor prognosis in patients with various hematologic malignancies. DNA damage-induced p53/Bcl2 interaction at the outer mitochondrial membrane results in a Bcl2 conformational change with loss of its antiapoptotic activity in interleukin-3-dependent myeloid H7 cells. Here we find that specific disruption of protein phosphatase 2A (PP2A) activity by either expression of small t antigen or depletion of PP2A/C by RNA interference enhances Bcl2 phosphorylation and suppresses cisplatin-stimulated p53/Bcl2 binding in association with prolonged cell survival. By contrast, treatment of cells with C2-ceramide (a potent PP2A activator) or expression of the PP2A catalytic subunit (PP2A/C) inhibits Bcl2 phosphorylation, leading to increased p53/Bcl2 binding and apoptotic cell death. Mechanistically, PP2A-mediated dephosphorylation of Bcl2 in vitro promotes its direct interaction with p53 as well as a conformational change in Bcl2. PP2A directly interacts with the BH4 domain of Bcl2 as a docking site to potentially "bridge" PP2A to Bcl2's flexible loop domain containing the target serine 70 phosphorylation site. Thus, PP2A may provide a dual inhibitory effect on Bcl2's survival function by both dephosphorylating Bcl2 and enhancing p53-Bcl2 binding. Activating PP2A to dephosphorylate Bcl2 and/or increase Bcl2/p53 binding may represent an efficient and novel approach for treatment of hematologic malignancies.
Topics: Animals; Antigens, Viral, Tumor; Apoptosis; Cell Line, Tumor; Cell Survival; Ceramides; DNA Damage; Enzyme Activators; Hematologic Neoplasms; Interleukin-3; Mice; Mitochondrial Membranes; Phosphoprotein Phosphatases; Phosphorylation; Protein Binding; Protein Phosphatase 2C; Protein Structure, Tertiary; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; RNA Interference; Tumor Suppressor Protein p53
PubMed: 18845789
DOI: 10.1182/blood-2008-06-165134 -
Nature Communications Aug 2019Very little is known about how Ser/Thr protein phosphatases specifically recruit and dephosphorylate substrates. Here, we identify how the Na/H-exchanger 1 (NHE1), a key...
Very little is known about how Ser/Thr protein phosphatases specifically recruit and dephosphorylate substrates. Here, we identify how the Na/H-exchanger 1 (NHE1), a key regulator of cellular pH homeostasis, is regulated by the Ser/Thr phosphatase calcineurin (CN). NHE1 activity is increased by phosphorylation of NHE1 residue T779, which is specifically dephosphorylated by CN. While it is known that Ser/Thr protein phosphatases prefer pThr over pSer, we show that this preference is not key to this exquisite CN selectivity. Rather a combination of molecular mechanisms, including recognition motifs, dynamic charge-charge interactions and a substrate interaction pocket lead to selective dephosphorylation of pT779. Our data identify T779 as a site regulating NHE1-mediated cellular acid extrusion and provides a molecular understanding of NHE1 substrate selection by CN, specifically, and how phosphatases recruit specific substrates, generally.
Topics: Acids; Calcineurin; Cell Membrane; Crystallography, X-Ray; Homeostasis; Humans; Hydrogen-Ion Concentration; MCF-7 Cells; Molecular Docking Simulation; Nuclear Magnetic Resonance, Biomolecular; Phosphorylation; Protein Binding; Recombinant Proteins; Sodium-Hydrogen Exchanger 1; Substrate Specificity
PubMed: 31375679
DOI: 10.1038/s41467-019-11391-7