Identification and characterization of an alternatively spliced isoform of the human protein phosphatase 2Aα catalytic subunit

PP2A is the main serine/threonine-specific phosphatase in animal cells. The active phosphatase has been described as a holoenzyme consisting of a catalytic, a scaffolding, and a variable regulatory subunit, all encoded by multiple genes, allowing for the assembly of more than 70 different holoenzymes. The catalytic subunit can also interact with α4, TIPRL (TIP41, TOR signaling pathway regulator-like), the methyl-transferase LCMT-1, and the methyl-esterase PME-1. Here, we report that the gene encoding the catalytic subunit PP2Acα can generate two mRNA types, the standard mRNA and a shorter isoform, lacking exon 5, which we termed PP2Acα2. Higher levels of the PP2Acα2 mRNA, equivalent to the level of the longer PP2Acα mRNA, were detected in peripheral blood mononuclear cells that were left to rest for 24 h. After this time, the peripheral blood mononuclear cells are still viable and the PP2Acα2 mRNA decreases soon after they are transferred to culture medium, showing that generation of the shorter isoform depends on the incubation conditions. FLAG-tagged PP2Acα2 expressed in HEK293 is catalytically inactive. It displays a specific interaction profile with enhanced binding to the α4 regulatory subunit, but no binding to the scaffolding subunit and PME-1. Consistently, α4 out-competes PME-1 and LCMT-1 for binding to both PP2Acα isoforms in pulldown assays. Together with molecular modeling studies, this suggests that all three regulators share a common binding surface on the catalytic subunit. Our findings add important new insights into the complex mechanisms of PP2A regulation.     

Identification and characterization of the ATP·Mg-dependent protein phosphatase activator (F A) as a microtubule protein kinase in the brain

The activating factor of ATP·Mg-dependent protein phosphatase (F _A) has been identified in brain microtubules. When using purified MAP-2 (microtubule associated protein 2) and tau proteins as substrates,F _A could phosphorylate MAP-2 to 16 moles of phosphates per mole of protein with aK _m value of 0.4 µM, and tau proteins to 4 moles of phosphates per mole of proteins with aK _m value of about 3 µM. When using microtubules as substrates,F _A could enhance many-fold the endogenous phosphorylation of many microtubule-associated proteins including MAP-2, tau proteins, and several low-molecular-weight MAPs. In contrast to other reported MAP kinases, such as cAMP-dependent protein kinase and Ca⁺²/phospholipid-dependent protein kinase, theF _A-catalyzed phosphorylation of tau proteins could cause an electrophoretic mobility shift on sodium dodecyl sulfate polyacrylamide gel electrophoresis, suggesting that a dramatic conformational change of tau proteins was produced byF _A. Peptide mapping analysis of the phosphopeptides derived from SV8 protease digestion revealed thatF _A could phosphorylate MAP-2 and tau proteins on at least four specific sites distinctly different from those phosphorylated by cAMP-dependent and Ca⁺²/phospholipid-dependent MAP kinases. Quantitative analysis further indicated that approximately 19% of the total endogenous kinase activity in brain microtubules was due toF _A. Taken together, the results provide initial evidence that the ATP·Mg-dependent protein phosphatase activating factor (F _A) is a potent and unique MAP kinase, and may represent one of the major factors involved in phosphorylation of brain microtubules.     

Knock-down of protein phosphatase 2A subunit B’γ promotes phosphorylation of CALRETICULIN 1 in Arabidopsis thaliana

Different types of plant pathogens may cause enormous losses in agriculture and also have an ecological impact in the nature. On molecular level, disease resistance is acquired through the action of tightly interconnected signaling pathways that may induce highly specific immune reactions in plant cells. Controlled protein dephosphorylation through protein phosphatase 2A activity is emerging as a crucial mechanism that regulates diverse signaling events in plants. PP2A is predominantly trimeric, and consists of a catalytic subunit, a scaffold subunit A, and a variable regulatory subunit B, which determines the target specificity of the PP2A holoenzyme.¹ Recently, we uncovered a specific role for a regulatory subunit B’γ of PP2A as a negative regulator of immune reactions in Arabidopsis thaliana (hereafter Arabidopsis).² Knock-down pp2a-b’γ mutants show constitutive activation of defense related genes, imbalanced antioxidant metabolism and premature disintegration of chloroplasts upon ageing. Proteomic analysis of soluble leaf extracts further revealed that the constitutive defense response in pp2a-b’γ leaves associates with increased levels of Cu/Zn superoxide dismutase, aconitase as well as components of the methionine-salvage pathway, suggesting PP2A-B’γ modulates methionine metabolism in leaves.     

Identification of a DNA-binding factor that recognizes an α-coixin promoter and interacts with a Coix Opaque-2 like protein

Transient expression and electrophoretic mobility shift assay were used to investigate the cis elements and the DNA-binding proteins involved in the regulation of expression of a 22 kDa zein-like -coixin gene. A set of unidirectional deletions was generated in a 962 bp fragment of the -coixin promoter that had been previously fused to the reporter gene GUS. The constructs were assayed by transient expression in immature maize endosperm. There was no significant decrease in GUS activity as deletions progressed from –1084 to –238. However, deletion from –238 to –158, which partially deleted the O2c box, resulted in a dramatic decrease in GUS activity emphasizing the importance of the O2 box in the quantitative expression of the gene. The –238 promoter fragment interacted with Coix endosperm nuclear proteins to form 5 DNA-protein complexes, C1–C5, as detected by EMSA. The same retarded complexes were observed when the –158 promoter fragment was used in the binding reactions. Reactions with nuclear extracts isolated from Coix endosperms harvested from 6 to 35 days after pollination revealed that the 5 DNA-protein complexes that interact with the -coixin promoter are differentially assembled during seed development. Deletion analysis carried out on the –238/ATG promoter fragment showed that a 35 bp region from –86 to –51 is essential for the formation of the complexes observed. When nuclear extracts were incubated with an antiserum raised against the maize Opaque-2 protein, the formation of 4 complexes, C1, C3, C4 and C5, was prevented indicating that an Opaque-2 like protein participates in the formation of those complexes. Complex C2 was not affected by the addition of the O2 antibody, suggesting the existence of a novel nuclear factor, CBF1, that binds to the promoter and makes protein-protein associations with other proteins present in Coix endosperm nuclei.     

Identification of protein kinase disruptions as suppressors of the calcium sensitivity of S. cerevisiae Δptp2 Δmsg5 protein phosphatase double disruptant

The double disruptant of the S. cerevisiae protein phosphatase (PPase) genes, PTP2 (phosphotyrosine-specific PPase) and MSG5 (phosphotyrosine and phosphothreonine/serine-PPase) causes calcium-sensitive growth (Ca^s). Previous study using Fluorescent-activated cell sorting (FACS) analysis showed that this growth defect with calcium occurs at G1–S transition in the cell cycle. We discovered that six non-essential protein kinase (PKase) disruptions (Δbck1, Δmkk1, Δslt2/Δmpk1, Δmck1, Δssk2 and Δyak1) suppressed the Ca^s-phenotype of the Δptp2 Δmsg5 double disruptant. Bck1p, Mkk1p and Slt2p are components of the mitogen-activated protein kinase (MAPK) cascade of cell wall integrity pathway (Slt2 pathway), and Mck1p is its down regulator. Ssk2p is the MAPK kinase kinase of the high-osmolarity glycerol (HOG) pathway, while Yak1p is a negative regulator for the cAMP-dependent PKA pathway. FACS analysis revealed that only the disruption of Δssk2 and Δyak1 but not Δbck1, Δmkk1, Δslt2 and Δmck1 was able to suppress the delayed G1–S transition, suggesting that suppression of the growth defect is not always accompanied by suppression of the G1–S transition delay. The discovery of these PKases as suppressors revealed that in addition to the previously anticipated Slt2 pathway, HOG, Yak1p and Mck1p regulatory pathways may also be involved in the calcium sensitivity of the Δptp2 Δmsg5 double disruptant.     

Protein tyrosine phosphatase interacting protein 51—a jack-of-all-trades protein

Protein tyrosine phosphatase interacting protein 51 (PTPIP51) interacts both in vitro and in vivo with PTP1B, a protein tyrosine phosphatase involved in cellular regulation. PTPIP51 is known to be expressed in many different types of tissues. It is involved in cellular processes such as proliferation, differentiation and apoptosis. Nevertheless, the exact cellular function of PTPIP51 is still unknown. The present review summarizes our current knowledge of the PTPIP51 gene and its mRNA and protein structure.     

Identification of the ATP·Mg-dependent protein phosphatase activator (Fa) as a synapsin I kinase that inhibits cross-linking of synapsin I with brain microtubules

The ATP·Mg-dependent protein phosphatase activating factor (Fa) has been identified and purified to near homogeneity from brain. In this report, as evidenced on SDS-polyacrylamide gel electrophoresis followed by autoradiography, factorFa has further been identified as a cAMP and Ca²⁺-independent brain kinase that could phosphorylate synapsin I, a neuronal protein that coats synaptic vesicles, binds to cytoskeleton, and is believed to be involved in the modulation of neurotransmission. Kinetic study further indicated that factorFa could phosphorylate synapsin I with a lowK _m value of about 2 µM and with a molar ratio of 1 mol of phosphate per mole of protein. Peptide mapping analysis revealed that factorFa specifically phosphorylated the tail region of synapsin I but on a unique site distinct from those phosphorylated by Ca²⁺/calmodulin-dependent protein kinase II and cAMP-dependent protein kinase, the two well-established synapsin I kinases. Functional study further revealed that factorFa could phosphorylate this unique specific site on the tail region of synapsin I and thereby inhibit cross-linking of synapsin I with microtubules. The results further suggest the possible involvement of factorFa as a synapsin I kinase in the regulation of axonal transport process of synaptic vesicles via the promotion of vesicles motility during neurotransmission.     

Identification of a combination of SNPs associated with Graves’ disease using swarm intelligence

Graves’ disease, the production of thyroid-stimulating hormone receptor-stimulating antibodies leading to hyperthyroidism, is one of the most common forms of human autoimmune disease. It is widely agreed that complex diseases are not controlled simply by an individual gene or DNA variation but by their combination. Single nucleotide polymorphisms (SNPs), which are the most common form of DNA variation, have great potential as a medical diagnostic tool. In this paper, the P-value is used as a SNP pre-selection criterion, and a wrapper algorithm with binary particle swarm optimization is used to find the rule for discriminating between affected and control subjects. We analyzed the association between combinations of SNPs and Graves’ disease by investigating 108 SNPs in 384 cases and 652 controls. We evaluated our method by differentiating between cases and controls in a five-fold cross validation test, and it achieved a 72.9% prediction accuracy with a combination of 17 SNPs. The experimental results showed that SNPs, even those with a high P-value, have a greater effect on Graves’ disease when acting in a combination.     

Cyclin G-associated kinase regulates protein phosphatase 2A by phosphorylation of its B'γ subunit

Protein phosphatase 2A (PP2A) bearing the B’γ (= B’α/B56γ1/PR61γ) subunit is recruited to dephosphorylation targets by cyclin G. We demonstrate here that cyclin G-associated kinase (GAK), a component of the GAK/B’γ/cyclin G complex, directly phosphorylates the B’γ-Thr104 residue and regulates PP2A activity. Indeed, an anti-B’γ-pT104 antibody detected immunofluorescence signals at the chromosome and centrosome during mitosis; these signals were reduced by siRNA-mediated GAK knockdown. After DNA damage by γ-irradiation, the chromosome signals formed foci that colocalized with a DNA double-strand break (DSB) marker H2AX-pS139 (γH2AX) and CHK2-pT68. Moreover, B’γ-pT104 enhanced PP2A holoenzyme assembly and PP2A activity, as shown by the results of an in vitro phosphatase assay. These results suggest a novel role for GAK as a regulator of dephosphorylation events under the control of the PP2A B’γ subunit.     

CK2α - protein phosphatase 2A molecular complex: Possible interaction with the MAP kinase pathway

Despite its wide range of known substrates, the signaling function of protein kinase CK2 is still enigmatic. Mounting evidence suggests that CK2, the catalytic subunit of holoenzymic CK2, may exist free of its usual regulatory partner CK2, raising the possibility that free CK2 has regulation and function distinct from those of the holoenzyme. We previously reported that CK2 could bind to the core dimer of protein phosphatase 2A, and indirectly cause down-regulation of the PP2A substrate MEK1, possibly via activation of PP2A and/or targeting of PP2A to some element of the Ras/Raf/MEK pathway. Here, these results are confirmed and extended. By using transfection experiments and immune kinase assays, we show that endogenous PP2Ac and CK2 are the only major substrates associating with epitope-tagged CK2, and that expression of activated Raf results in disruption of the CK2- PP2A association. Such disruption might be a necessary step for maximal activation of the MAP kinase pathway by Raf. In keeping with this idea, overexpression of CK2 dose-dependently inhibits the mitogen-induced activation of cotransfected, epitope-tagged MAP kinase. We suggest that the CK2 free form of CK2 is both a target and a regulator of Raf/MAPK signaling.     

Angiotensin IV upregulates the activity of protein phosphatase 1α in Neura-2A cells

The peptide angiotensin IV (Ang IV) is a derivative of angiotensin II. While insulin regulated amino peptidase (IRAP) has been proposed as a potential receptor for Ang IV, the signalling pathways of Ang IV through IRAP remain elusive. We applied high-resolution mass spectrometry to perform a systemic quantitative phosphoproteome of Neura-2A (N2A) cells treated with and without Ang IV using sta ble-isotope labeling by amino acids in cell culture (SILAC), and identified a reduction in the phosphorylation of a major Ser/Thr protein phosphorylase 1 (PP1) upon Ang IV treatment. In addition, spinophilin (spn), a PP1 regulatory protein that plays important functions in the neural system, was expressed at higher levels. Immunoblotting revealed decreased phosphorylation of p70S6 kinase (p70^S6K) and the major cell cycle modulator retinoblastoma protein (pRB). These changes are consistent with an observed decrease in cell proliferation. Taken together, our study suggests that Ang IV functions via regulating the activity of PP1.     

Identification of nucleolin as a lipid-raft-dependent β1-integrin-interacting protein in A375 cell migration

Lipid rafts are related to cell surface receptor function. Integrin is a major surface receptor protein in cell adhesion and migration on the extracellular matrix (ECM). Here, we showed that lipid rafts played a critical role in human melanoma A375 cell spreading and migration on fibronectin; an important component of the ECM that interacts with β1 integrin. We found that the disruption of lipid rafts did not markedly inhibit the expression and activation of β1 integrin. By coimmunoprecipitation and mass spectrometry, we investigated the influence of lipid rafts on the β1 integrin complex and identified nucleolin as a potential lipid-raft-dependent β1-integrin-interacting protein. Upon confirmation of the interaction between β1 integrin and nucleolin, further studies revealed that nucleolin colocalized with β1 integrin in lipid rafts and raft disruption interrupted their association. In addition, knockdown of nucleolin markedly attenuated A375 cell spreading and migration on fibronectin. Taken together, we demonstrated that nucleolin is a critical lipid-raft-dependent β1-integrin-interacting protein in A375 cell spreading and migration on fibronectin.     

Identification of a disruptor of the MDM2-p53 protein–protein interaction facilitated by high-throughput in silico docking

NSC 333003 has been identified from the NCI Diversity Set as an inhibitor of the MDM2-p53 protein–protein interaction by in silico docking (virtual screening). Its potency and chemical characteristics render it well suited for lead optimization studies that can result in more potent analogs with improved drug-like properties. Its synthesis was achieved using an acid catalyzed condensation reaction from commercially available benzothiazole hydrazine and pyridyl phenyl ketone in refluxing methanol. Stereochemical implications for this compound are described.     

Identification of N-Glycosylation in Hepatocellular Carcinoma Patients’ Serum with a Comparative Proteomic Approach

Aim

This study is to explore the different expressions of serum N-glycoproteins and glycosylation sites between hepatocellular carcinoma (HCC) patients and healthy controls.

Method

We combined high abundant proteins depletion and hydrophilic affinity method to enrich the glycoproteins. Through liquid chromatography-tandem mass spectrometry (LC-MS/MS), we extensively surveyed different expressions of glycosylation sites and glycoproteins between the two groups.

Result

This approach identified 152 glycosylation sites and 54 glycoproteins expressed differently between HCC patients and healthy controls. With the absolute values of Pearson coefficients of at least 0.8, eight proteins were identified significantly up or down regulated in HCC serum. Those proteins are supposed to be involved in several biological processes, cellular components and molecular functions of hepatocarcinogenesis. Several of them had been reported abnormally regulated in several kinds of malignant tumors, and may be promising biomarkers of HCC.

Conclusion

Our work provides a systematic and quantitative method of glycoproteomics and demonstrates some key changes in clinical HCC serum. These proteomic signatures may help to unveil the underlying mechanisms of hepatocarcinogenesis and may be useful for the exploration of candidate biomarkers.     

Monoubiquitination promotes calpain cleavage of the protein phosphatase 2A (PP2A) regulatory subunit α4, altering PP2A stability and microtubule-associated protein phosphorylation

Multiple neurodegenerative disorders are linked to aberrant phosphorylation of microtubule-associated proteins (MAPs). Protein phosphatase 2A (PP2A) is the major MAP phosphatase; however, little is known about its regulation at microtubules. α4 binds the PP2A catalytic subunit (PP2Ac) and the microtubule-associated E3 ubiquitin ligase MID1, and through unknown mechanisms can both reduce and enhance PP2Ac stability. We show MID1-dependent monoubiquitination of α4 triggers calpain-mediated cleavage and switches α4's activity from protective to destructive, resulting in increased Tau phosphorylation. This regulatory mechanism appears important in MAP-dependent pathologies as levels of cleaved α4 are decreased in Opitz syndrome and increased in Alzheimer disease, disorders characterized by MAP hypophosphorylation and hyperphosphorylation, respectively. These findings indicate that regulated inter-domain cleavage controls the dual functions of α4, and dysregulation of α4 cleavage may contribute to Opitz syndrome and Alzheimer disease.     

A critical role for the protein phosphatase 2A B'α regulatory subunit in dephosphorylation of sphingosine kinase 1

Sphingosine kinase 1 (SK1) is an important regulator of cellular signalling that has gained recent attention as a potential target for anti-cancer therapies. SK1 activity, subcellular localization and oncogenic function are regulated by phosphorylation and dephosphorylation at Ser225. ERK1/2 have been identified as the protein kinases responsible for phosphorylation and activation of SK1. Conversely, dephosphorylation and deactivation of SK1 occurs by protein phosphatase 2A (PP2A). Active PP2A, however, is a heterotrimer, composed of tightly associated catalytic and structural subunits that can interact with an array of regulatory subunits, which are critical for determining holoenzyme substrate specificity and subcellular localization. Thus, PP2A represents a large family of holoenzyme complexes with different activities and diverse substrate specificities. To date the regulatory subunit essential for targeting PP2A to SK1 has remained undefined. Here, we demonstrate a critical role for the B'α (B56α/PR61α/PPP2R5A) regulatory subunit of PP2A in SK1 dephosphorylation. B'α was found to interact with the c-terminus of SK1, and reduce SK1 phosphorylation when overexpressed, while having no effect on upstream ERK1/2 activation. siRNA-mediated knockdown of B'α increased SK1 phosphorylation, activity and membrane localization of endogenous SK1. Furthermore, overexpression of B'α blocked agonist-induced translocation of SK1 to the plasma membrane and abrogated SK1-induced neoplastic transformation of NIH3T3 fibroblasts. Thus, the PP2A-B'α holoenzyme appears to function as an important endogenous regulator of SK1.