Evolutionary radiation pattern of novel protein phosphatases revealed by analysis of protein data from the completely sequenced genomes of humans, green algae, and higher plants

In addition to the major serine/threonine-specific phosphoprotein phosphatase, Mg(2+)-dependent phosphoprotein phosphatase, and protein tyrosine phosphatase families, there are novel protein phosphatases, including enzymes with aspartic acid-based catalysis and subfamilies of protein tyrosine phosphatases, whose evolutionary history and representation in plants is poorly characterized. We have searched the protein data sets encoded by the well-finished nuclear genomes of the higher plants Arabidopsis (Arabidopsis thaliana) and Oryza sativa, and the latest draft data sets from the tree Populus trichocarpa and the green algae Chlamydomonas reinhardtii and Ostreococcus tauri, for homologs to several classes of novel protein phosphatases. The Arabidopsis proteins, in combination with previously published data, provide a complete inventory of known types of protein phosphatases in this organism. Phylogenetic analysis of these proteins reveals a pattern of evolution where a diverse set of protein phosphatases was present early in the history of eukaryotes, and the division of plant and animal evolution resulted in two distinct sets of protein phosphatases. The green algae occupy an intermediate position, and show similarity to both plants and animals, depending on the protein. Of specific interest are the lack of cell division cycle (CDC) phosphatases CDC25 and CDC14, and the seeming adaptation of CDC14 as a protein interaction domain in higher plants. In addition, there is a dramatic increase in proteins containing RNA polymerase C-terminal domain phosphatase-like catalytic domains in the higher plants. Expression analysis of Arabidopsis phosphatase genes differentially amplified in plants (specifically the C-terminal domain phosphatase-like phosphatases) shows patterns of tissue-specific expression with a statistically significant number of correlated genes encoding putative signal transduction proteins.     

Regulation of neuronal plasticity in the central nervous system by phosphorylation and dephosphorylation

Neuronal plasticity can be defined as adaptive changes in structure and function of the nervous system, an obvious example of which is the capacity to remember and learn. Long-term potentiation and long-term depression are the experimental models of memory in the central nervous system (CNS), and have been frequently utilized for the analysis of the molecular mechanisms of memory formation. Extensive studies have demonstrated that various kinases and phosphatases regulate neuronal plasticity by phosphorylating and dephosphorylating proteins essential to the basic processes of adaptive changes in the CNS. These proteins include receptors, ion channels, synaptic vesicle proteins, and nuclear proteins. Multifunctional kinases (cAMP-dependent protein kinase, Ca²⁺/phospholipid-dependent protein kinase, and Ca²⁺/calmodulin-dependent protein kinases) and phosphatases (calcineurin, protein phosphatases 1, and 2A) that specifically modulate the phosphorylation status of neuronal-signaling proteins have been shown to be required for neuronal plasticity. In general, kinases are involved in upregulation of the activity of target substrates, and phosphatases downregulate them. Although this rule is applicable in most of the cases studied, there are also a number of exceptions. A variety of regulation mechanisms via phosphorylation and dephosphorylation mediated by multiple kinases and phosphatases are discussed.     

Purinergic Receptor-mediated Rapid Depletion of Nuclear Phosphorylated Akt Depends on Pleckstrin Homology Domain Leucine-rich Repeat Phosphatase,Calcineurin, Protein Phosphatase 2A,and PTEN Phosphatases

Akt is an important oncoprotein, and data suggest a critical role for nuclear Akt in cancer development. We have previously described a rapid (3–5 min) and P2X7-dependent depletion of nuclear phosphorylated Akt (pAkt) and effects on downstream targets, and here we studied mechanisms behind the pAkt depletion. We show that cholesterol-lowering drugs, statins, or extracellular ATP, induced a complex and coordinated response in insulin-stimulated A549 cells leading to depletion of nuclear pAkt. It involved protein/lipid phosphatases PTEN, pleckstrin homology domain leucine-rich repeat phosphatase (PHLPP1 and -2), protein phosphatase 2A (PP2A), and calcineurin. We employed immunocytology, immunoprecipitation, and proximity ligation assay techniques and show that PHLPP and calcineurin translocated to the nucleus and formed complexes with Akt within 3 min. Also PTEN translocated to the nucleus and then co-localized with pAkt close to the nuclear membrane. An inhibitor of the scaffolding immunophilin FK506-binding protein 51 (FKBP51) and calcineurin, FK506, prevented depletion of nuclear pAkt. Furthermore, okadaic acid, an inhibitor of PP2A, prevented the nuclear pAkt depletion. Chemical inhibition and siRNA indicated that PHLPP, PP2A, and PTEN were required for a robust depletion of nuclear pAkt, and in prostate cancer cells lacking PTEN, transfection of PTEN restored the statin-induced pAkt depletion. The activation of protein and lipid phosphatases was paralleled by a rapid proliferating cell nuclear antigen (PCNA) translocation to the nucleus, a PCNA-p21^cip1 complex formation, and cyclin D1 degradation. We conclude that these effects reflect a signaling pathway for rapid depletion of pAkt that may stop the cell cycle.     

Characterization of MPF activation by okadaic acid in Xenopus oocyte

Okadaic acid (OA), a specific inhibitor of protein phosphatases, induces a rapid activation (30 min) of MPF when microinjected into the Xenopus oocyte. Neither protein synthesis inhibitors nor cAMP counteract the action of OA. These results indicate that the inhibition of protein phosphatase(s) is sufficient for the in vivo activation of MPF even after the full activation of cAMP-dependent protein kinase. In all experimental conditions (plus or minus inhibitors of protein synthesis; normal or elevated cAMP levels) OA induces a burst of protein phosphorylation together with the activation of MPF. Cytological analysis shows that OA provokes the breakdown of the nuclear envelope, the depolymerization of lamin and the condensation of the chromosomes. However, no metaphase spindles are organized, indicating that inhibition of protein phosphatases strongly affects the function of the microtubule organizing center.     

Proteomic characterization of protein phosphatase complexes of the mammalian nucleus

Our knowledge of the serine/threonine protein phosphatases of the mammalian nucleus is limited compared with their cytosolic counterparts. Microcystin-Sepharose chromatography and mass spectrometry were utilized to affinity purify and identify protein phosphatase-associated proteins from isolated rat liver nuclei. Far Western analysis with labeled protein phosphatase 1 (PP1) showed that many more PP1 binding proteins exist in the nucleus than were previously demonstrated. Mass spectrometry confirmed the presence in the nucleus of the mammalian PP1 isoforms alpha1, alpha2, beta, and gamma1, plus the Aalpha and several of the B and B' subunits that are complexed to PP2A. Other proteins enriched on the microcystin matrix include the spliceosomal proteins known as the U2 snRNPs SAP145 and SAP155 and the U5 snRNPs p116 and p200, myosin heavy chain, and a nuclear PP1 myosin-targeting subunit related to M110. The putative RNA binding protein ZAP was also established as a nuclear PP1 binding protein using the criteria of co-purification with PP1 on microcystin-Sepharose, co-immunoprecipation, binding PP1 in an overlay assay, and presence of a putative PP1 binding site (KKRVRWAD). These results further support a key role for protein phosphatases in several nuclear functions, including the regulation of pre-mRNA splicing.     

MKP-2: out of the DUSP-bin and back into the limelight

The MKPs (mitogen-activated protein kinase phosphatases) are a family of at least ten DUSPs (dual-specificity phosphatases) which function to terminate the activity of the MAPKs (mitogen-activated protein kinases). Several members have already been demonstrated to have distinct roles in immune function, cancer, fetal development and metabolic disorders. One DUSP of renewed interest is the inducible nuclear phosphatase MKP-2, which dephosphorylates both ERK (extracellular-signal-regulated kinase) and JNK (c-Jun N-terminal kinase) in vitro. Recently, the understanding of MKP-2 function has been advanced due to the development of mouse knockout models, which has resulted in the discovery of novel roles for MKP-2 in the regulation of sepsis, infection and cell-cycle progression that are distinct from those of other DUSPs. However, many functions for MKP-2 still await to be characterized.     

Okadaic acid and microcystin insensitive PPP-family phosphatases may represent novel biotechnology targets

Reversible protein phosphorylation is of central importance to the proper cellular functioning of all living organisms. Catalyzed by the opposing reactions of protein kinases and phosphatases, dysfunction in reversible protein phosphorylation can result in a wide variety of cellular aberrations. In eukaryotic organisms there exists four classes of protein phosphatases, of which the PPP-family protein phosphatases have documented susceptibility to a range of protein and small molecule inhibitors. These inhibitors have been of great importance to the biochemical characterization of PPP-family protein phosphatases since their discovery, but also maintain in natura biological significance with their endogenous regulatory properties (protein inhibitors) and toxicity (small molecule inhibitors). Recently, two unique PPP-family protein phosphatases, named the Shewanella-like protein phosphatases (SLP phosphatases), from Arabidopsis thaliana were characterized and found to be phylogenetically similar to the PPP-family protein phosphatases protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A), while completely lacking sensitivity to the classic PPP-family phosphatase small molecule inhibitors okadaic acid and microcystin-LR. SLP phosphatases were also found to be absent in metazoans, but present in a wide range of bacteria, fungi and protozoa responsible for human disease. The unique biochemical properties and evolutionary heritage of SLP phosphatases suggests they could not only be potential biotechnology targets for agriculture, but may also prove to be of interest for future therapeutic drug development.     

细胞核CaMK和Calcineurin 对大鼠心肌肥厚发生的作用

目的:研究大鼠心肌肥厚时,钙依赖的蛋白激酶和蛋白磷酸酶在心肌细胞膜、细胞浆和细胞核的分布规律,以探讨核钙信号与核反应在心肌肥厚发生过程中的病理生理意义.方法:制备腹主动脉缩窄大鼠心肌肥厚模型,同位素32P掺入法分别测定心肌细胞核、细胞浆和细胞膜的蛋白激酶活性及用无机磷生成显色法测定其蛋白磷酸酶活性.结果:腹主动脉缩窄术后4周大鼠心肌显著肥厚,伴有明显的血液动力学异常.与正常对照组相比较,腹主动脉缩窄心肌肥厚组心肌细胞核钙调素蛋白激酶(CaMK)活性增加101.1%(P＜0.01),其膜的酶活性升高40.2%(P＜0.01),而胞浆的酶活性不变(P＞0.05);心肌细胞核钙调神经磷酸酶(Calcineurin)活性增加43.6%(P＜0.05),膜和胞浆中其活性增加无显著性(P＞0.05).正常组和腹主动脉缩窄心肌肥厚组心肌细胞CaMK和Calcineurin活性分布为核＞膜＞胞浆(P＜0.01).结论:腹主动脉缩窄心肌肥厚时核内钙依赖的CaMK和Calcineurin活性增加,提示压力超负荷时细胞核内钙调节的蛋白磷酸化和去磷酸化水平增高,可能在介导心肌肥厚的发生中起重要作用.     

Stimulatory effect of okadaic acid, an inhibitor of protein phosphatases, on nuclear envelope breakdown and protein phosphorylation in mouse oocytes and one-cell embryos.

Treatment of one-cell mouse embryos with okadaic acid (OA), which is an inhibitor of protein phosphatases 1 and 2A, induces a concentration-dependent precocious nuclear envelope breakdown (NEBD) of the pronuclei; at 10 microM okadaic acid, NEBD starts to occur after 1 hr and the embryos become committed to NEBD after about 45 min. Correlated with NEBD is the conversion of a protein of Mr 32,000 (p32) to more highly phosphorylated forms. One-cell embryos cultured continuously in OA-containing medium do not cleave, whereas one-cell embryos incubated for 15-60 min prior to transfer to OA-free medium reveal a time-dependent inhibition in their ability to cleave. OA treatment of oocytes that are arrested from resuming spontaneous maturation by either a phosphodiesterase inhibitor or biologically active phorbol diester results in germinal vesicle breakdown and the maturation-associated changes in the pattern of protein phosphorylation, which include the apparent phosphorylation of p32. Results of these experiments implicate protein phosphatases in the G2 to M transition of the cell cycle in both meiotic and mitotic cells.     

Protein phosphorylation and oocyte maturation : II. Inhibition of starfish oocyte maturation by intracellular microinjection of protein phosphatases 1 and 2A and alkaline phosphatase

Oocyte maturation (meiosis re-initiation) in starfish is induced by the natural hormone 1-methyladenine (1-MeAde). Following hormonal stimulation of the oocyte, an intracellular Maturation Promoting Factor (MPF) appears in the cytoplasm which triggers nuclear envelope breakdown and maturation divisions. Microinjection of pure preparations of the catalytic subunits of protein phosphatases 1 and 2A inhibits 1-MeAde-induced maturation in a dose-dependent manner. Calmodulin-dependent protein phosphatase 2B is inefficient. Maturation induced by mimetics of 1-MeAde, such as dithiothreitol (DTT), methylglyoxal-bis(guanylhydrazone) (MGBG), 8-hydroxyeicosatetraenoic acid (8 HETE) and arachidonic acid (AA) is also inhibited by these protein phosphatases. In all cases inhibition can be reversed by increasing the concentration of 1-Me-Ade or of mimetic. Alkaline phosphatase also inhibits maturation in a dose-dependent way and in a reversible manner. Microinjection of protein phosphatase is still effective when preformed long after the end of the hormone-dependent period, and can even be effective a few minutes before the breakdown of the nuclear envelope. No detectable MPF activity is found in 1-MeAde-treated phosphatase-injected oocytes. However, microinjection of phosphatase 2A simultaneously with MPF (obtained from 1-MeAde-treated donors) does not result in inhibition. These results constitute direct evidence for the necessity of an elevated level of phosphorylated proteins for MPF activity and maturation. The mode of action of 1-MeAde in inducing starfish oocyte maturation is discussed in relation to protein phosphorylation.     

Visualization of phosphatase activity in living cells with a FRET-based calcineurin activity sensor

Protein kinases and phosphatases are organized into complex intracellular signaling networks designed to coordinate their activities in both space and time. In order to better understand the molecular mechanisms underlying the regulation of signal transduction networks, it is important to define the spatiotemporal dynamics of both protein kinases and phosphatases within their endogenous environment. Herein, we report the development of a genetically-encoded protein biosensor designed to specifically probe the activity of the Ca2+/calmodulin-dependent protein phosphatase, calcineurin. Our reporter design utilizes a phosphatase activity-dependent molecular switch based on the N-terminal regulatory domain of the nuclear factor of activated T-cells as a specific substrate of calcineurin, sandwiched between cyan fluorescent protein and yellow fluorescent protein. Using this reporter, calcineurin activity can be monitored as dephosphorylation-induced increases in fluorescence resonance energy transfer and can be simultaneously imaged with intracellular calcium dynamics. The successful design of a prototype phosphatase activity sensor lays a foundation for studying targeting and compartmentation of phosphatases.     

The complement of protein phosphatase catalytic subunits encoded in the genome of Arabidopsis

Reversible protein phosphorylation is critically important in the modulation of a wide variety of cellular functions. Several families of protein phosphatases remove phosphate groups placed on key cellular proteins by protein kinases. The complete genomic sequence of the model plant Arabidopsis permits a comprehensive survey of the phosphatases encoded by this organism. Several errors in the sequencing project gene models were found via analysis of predicted phosphatase coding sequences. Structural sequence probes from aligned and unaligned sequence models, and all-against-all BLAST searches, were used to identify 112 phosphatase catalytic subunit sequences, distributed among the serine (Ser)/threonine (Thr) phosphatases (STs) of the protein phosphatase P (PPP) family, STs of the protein phosphatase M (PPM) family (protein phosphatases 2C [PP2Cs] subfamily), protein tyrosine (Tyr) phosphatases (PTPs), low-M(r) protein Tyr phosphatases, and dual-specificity (Tyr and Ser/Thr) phosphatases (DSPs). The Arabidopsis genome contains an abundance of PP2Cs (69) and a dearth of PTPs (one). Eight sequences were identified as new protein phosphatase candidates: five dual-specificity phosphatases and three PP2Cs. We used phylogenetic analyses to infer clustering patterns reflecting sequence similarity and evolutionary ancestry. These clusters, particularly for the largely unexplored PP2C set, will be a rich source of material for plant biologists, allowing the systematic sampling of protein function by genetic and biochemical means.     

Protein phosphatases are involved in the in vivo activation of histone H1 kinase in mouse oocyte

Histone H1 kinase and protein phosphorylation have been studied in mouse oocyte. Histone H1 kinase activity increases when the oocyte enters M-phase at the time of GVBD and is paralleled with a burst of protein phosphorylation. This activity dramatically drops after parthenogenetic activation induced by puromycin. Okadic acid (OA), a potent inhibitor of protein phosphatases, induces GVBD when oocytes are arrested in the first meiotic prophase by dbc-AMP; the continuous presence of the phosphatase inhibitor, however, inhibits the polymerization of metaphase microtubules. Following activation of metaphase II-arrested mouse eggs by puromycin, OA can induce the breakdown of the nuclear envelope and the activation of histone H1 kinase. This indicates that in the absence of protein synthesis, and therefore of cyclin synthesis, inhibition of protein phosphatases may be sufficient to induce the entry into M-phase during the first cell cycle of the mouse parthenogenetic activated oocyte.     

The specificity of extracellular signal-regulated kinase 2 dephosphorylation by protein phosphatases

The extracellular signal-regulated protein kinase 2 (ERK2) is the founding member of a family of mitogen-activated protein kinases (MAPKs) that are central components of signal transduction pathways for cell proliferation, stress responses, and differentiation. The MAPKs are unique among the Ser/Thr protein kinases in that they require both Thr and Tyr phosphorylation for full activation. The dual phosphorylation of Thr-183 and Tyr-185 in ERK2 is catalyzed by MAPK/ERK kinase 1 (MEK1). However, the identity and relative activity of protein phosphatases that inactivate ERK2 are less well established. In this study, we performed a kinetic analysis of ERK2 dephosphorylation by protein phosphatases using a continuous spectrophotometric enzyme-coupled assay that measures the inorganic phosphate produced in the reaction. Eleven different protein phosphatases, many previously suggested to be involved in ERK2 regulation, were compared, including tyrosine-specific phosphatases (PTP1B, CD45, and HePTP), dual specificity MAPK phosphatases (VHR, MKP3, and MKP5), and Ser/Thr protein phosphatases (PP1, PP2A, PP2B, PP2C alpha, and lambda PP). The results provide biochemical evidence that protein phosphatases display exquisite specificity in their substrate recognition and implicate HePTP, MKP3, and PP2A as ERK2 phosphatases. The fact that ERK2 inactivation could be carried out by multiple specific phosphatases shows that signals can be integrated into the pathway at the phosphatase level to determine the cellular response to external stimuli. Important insights into the roles of various protein phosphatases in ERK2 kinase signaling are obtained, and further analysis of the mechanism by which different protein phosphatases recognize and inactivate MAPKs will increase our understanding of how this kinase family is regulated.     

Characterization of microcystin-LR, a potent inhibitor of type 1 and type 2A protein phosphatases

The level of protein phosphorylation is dependent on the relative activities of both protein kinases and protein phosphatases. By comparison with protein kinases, however, there have been considerably fewer studies on the functions of serine/threonine protein phosphatases. This is partly due to a lack of specific protein phosphatase inhibitors that can be used as probes. In the present study we characterize the inhibitory effects of microcystin-LR, a hepatotoxic cyclic peptide associated with most strains of the blue-green algae Microcystis aeruginosa found in the Northern hemisphere, that proves to be a potent inhibitor of type 1 (IC50 = 1.7 nM) and type 2A (IC50 = 0.04 nM) protein phosphatases. Microcystin-LR inhibited the activity of both type 1 and type 2A phosphatases greater than 10-fold more potently than okadaic acid under the same conditions. Type 2A protein phosphatases in dilute mammalian cell extracts were found to be completely inhibited by 0.5 nM microcystin-LR while type 1 protein phosphatases were only slightly affected at this concentration. Thus, microcystin-LR may prove to be a useful probe for the study and identification cellular processes which are mediated by protein phosphatases.