sepharose and microcystin

Mammalian sperm contain the serine/threonine phosphatases PP1γ2 and PP2A. The role of sperm PP1γ2 is relatively well studied. Here we confirm the presence of PP2A in sperm and show that it undergoes marked changes in methylation (leucine 309), tyrosine phosphorylation (tyrosine 307) and catalytic activity during epididymal sperm maturation. Spermatozoa isolated from proximal caput, distal caput and caudal regions of the epididymis contain equal immuno-reactive amounts of PP2A. Using demethyl sensitive antibodies we show that PP2A is methylated at its carboxy terminus in sperm from the distal caput and caudal regions but not in sperm from the proximal caput region of the epididymis. The methylation status of PP2A was confirmed by isolation of PP2A with microcystin agarose followed by alkali treatment, which causes hydrolysis of protein carboxy methyl esters. Tyrosine phosphorylation of sperm PP2A varied inversely with methylation. That is, PP2A was tyrosine phosphorylated when it was demethylated but not when methylated. PP2A demethylation and its reciprocal tyrosine phosphorylation were also affected by treatment of sperm with L-homocysteine and adenosine, which are known to elevate intracellular S-adenosylhomocysteine, a feedback inhibitor of methyltransferases. Catalytic activity of PP2A declined during epididymal sperm maturation. Inhibition of PP2A by okadaic acid or by incubation of caudal epididymal spermatozoa with L-homocysteine and adenosine resulted in increase of sperm motility parameters including percent motility, velocity, and lateral head amplitude. Demethylation or pharmacological inhibition of PP2A also leads to an increase in phosphorylation of glycogen synthase kinase-3 (GSK3). Our results show for the first time that changes in PP2A activity due to methylation and tyrosine phosphorylation occur in sperm and that these changes may play an important role in the regulation of sperm function.

Topics: Animals; Catalysis; Cattle; DNA Methylation; Epididymis; Glycogen Synthase Kinase 3; Homocysteine; Leucine; Male; Methylation; Microcystins; Okadaic Acid; Phosphoprotein Phosphatases; Phosphorylation; Protein Phosphatase 2; Protein Structure, Tertiary; Sepharose; Sperm Maturation; Sperm Motility; Spermatozoa; Tyrosine

The development of effective array biosensors relies heavily on careful control of the density of surface-immobilized ligands on the transducing platform. In this paper we describe the synthesis of new dextran-lipase conjugates for use in immobilizing low molecular weight haptens onto glass planar waveguides for immunosensor development. The conjugates were synthesized by immobilizing bacterial thermoalkalophilic lipases (Geobacillus thermocatenulatus lipase 2, BTL2) on agarose macroporous beads, followed by covalent coupling to dextran networks of variable molecular weight (1500-40000). The chimeras were immobilized via nonspecific hydrophobic interactions onto glass planar waveguides modified with 1,1,1,3,3,3-hexamethyldisilazane to obtain highly ordered and homogeneous molecular architectures as confirmed by atomic force microscopy. Microcystin LR (MCLR) was covalently bound to the dextran-BTL2 conjugates. The usefulness of this approach in immunosensor development was demonstrated by determining amounts of MCLR down to a few picograms per liter with an automated array biosensor and evanescent wave excitation for fluorescence measurements of attached DyLight649-labeled secondary antibody. Modifying BTL2 with dextrans of an increased molecular weight (>6000) provided surfaces with an increased loading capacity that was ascribed to the production of three-dimensional surfaces by the effect of analyte binding deep in the volume, leading to expanded dynamic ranges (0.09-136.56 ng L(-1)), lower limits of detection (0.007 ± 0.001 ng L(-1)), and lower IC50 values (4.4 ± 0.7 ng L(-1)). These results confirm the effectiveness of our approach for the development of high-performance biosensing platforms.

Topics: Dextrans; Geobacillus; Glass; Immobilized Proteins; Ligands; Lipase; Microarray Analysis; Microcystins; Molecular Weight; Porosity; Sepharose; Surface Properties

Protein phosphatase one (PP1) is a ubiquitously expressed, highly conserved protein phosphatase that dephosphorylates target protein serine and threonine residues. PP1 is localized to its site of action by interacting with targeting or regulatory proteins, a majority of which contains a primary docking site referred to as the RVXF/W motif.. We demonstrate that a peptide based on the RVXF/W motif can effectively displace PP1 bound proteins from PP1 retained on the phosphatase affinity matrix microcystin-Sepharose. Subsequent co-immunoprecipitation experiments confirmed that each identified binding protein was either a direct PP1 interactor or was in a complex that contains PP1. Our results have linked PP1 to numerous new nuclear functions and proteins, including Ki-67, Rif-1, topoisomerase IIalpha, several nuclear helicases, NUP153 and the TRRAP complex.. This modification of the microcystin-Sepharose technique offers an effective means of purifying novel PP1 regulatory subunits and associated proteins and provides a simple method to uncover a link between PP1 and additional cellular processes.

Topics: Amino Acid Motifs; Animals; Binding Sites; Chromatography, Affinity; Glycogen; HeLa Cells; Humans; Microcystins; Muscle, Skeletal; Protein Phosphatase 1; Protein Subunits; Rabbits; Sepharose

Microcystin-based affinity matrices have been utilized to demonstrate the association of signaling proteins with protein phosphatases and for the purification of low-abundance microcystin-sensitive protein phosphatases. Here, we describe the procedure for the synthesis and use of microcystin-Sepharose and microcystin-biotin-Sepharose.

Topics: Biotin; Chromatography, Affinity; Microcystins; Peptides, Cyclic; Phosphoprotein Phosphatases; Reproducibility of Results; Sepharose

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.

Topics: Animals; Binding Sites; Cell Nucleus; Chromatography, Agarose; Male; Mass Spectrometry; Microcystins; Nuclear Proteins; Peptides, Cyclic; Phosphoprotein Phosphatases; Phosphorylation; Protein Binding; Protein Phosphatase 1; Proteomics; Rats; Rats, Wistar; RNA Precursors; Sepharose

Mitogen-activated protein kinase (MAPK) signaling cascades are multifunctional signaling networks that influence cell growth, differentiation, apoptosis, and cellular responses to stress. Since the activation/propagation of MAPK signaling requires the sequential phosphorylation of many downstream proteins, the phosphatases that dephosphorylate MAPKs represent critical elements in the control of MAPK-signaling networks. Here we show that hypoxia induces a transient increase in the activity of apoptosis signal-regulating kinase 1 (ASK-1), a MAPKKK that responds to oxidative stress by triggering cascades leading to the phosphorylation/activation of c-Jun N-terminal kinases (JNK) and p38-MAPK. Hypoxia-induced ASK-1/MKK-4/JNK signaling is suppressed by serine/threonine protein phosphatase type 5 (PP5), which acts to turn off ASK-1/MKK-4/JNK signaling via two mechanisms. First, in a rapid response hypoxia facilitates the association of endogenous PP5 with ASK-1. PP5 binds to the C-terminal domain of ASK-1, and studies with siRNA targeting PP5 indicate that PP5 acts to suppress the phosphorylation of MKK4 (Thr-261), JNK (Thr-183/Tyr-185), and c-Jun (Ser-63) without affecting the activating phosphorylation of p38 MAPK (Thr-180/Tyr-182), p44/p42-MAPK/ERK1/2 (Thr-202/Tyr-204), or c-Jun protein levels. If hypoxia is prolonged, the expression of PP5 is increased due to the activation of a transcriptional activator, which was identified as hypoxia-inducible factor-1. Together, these studies indicate that PP5 plays an important role in the survival of cells in a low oxygen environment by suppressing a hypoxia-induced ASK-1/MKK4/JNK signaling cascade that promotes an apoptotic response.

Topics: Apoptosis; Base Sequence; Blotting, Western; Cell Line; Cell Line, Tumor; Enzyme Activation; Genes, Reporter; Humans; Hypoxia; JNK Mitogen-Activated Protein Kinases; Luciferases; MAP Kinase Kinase 4; MAP Kinase Kinase Kinase 5; Microcystins; Mitogen-Activated Protein Kinase Kinases; Models, Biological; Molecular Sequence Data; Nuclear Proteins; Oxygen; Peptides, Cyclic; Phosphoprotein Phosphatases; Phosphorylation; Promoter Regions, Genetic; Protein Binding; Protein Structure, Tertiary; Reverse Transcriptase Polymerase Chain Reaction; RNA, Double-Stranded; RNA, Small Interfering; Sepharose; Sequence Homology, Nucleic Acid; Signal Transduction; Threonine; Time Factors; Transcriptional Activation

Serine/threonine phosphatase PP1gamma2 is a testis-specific protein phosphatase isoform in spermatozoa. This enzyme appears to play a key role in motility initiation and stimulation. Catalytic activity of PP1gamma2 is higher in immotile compared with motile spermatozoa. Inhibition of PP1gamma2 activity causes both motility initiation and motility stimulation. Protein phosphatases, in general, are regulated by their binding proteins. The objective of this article is to understand the mechanisms by which PP1gamma2 is regulated, first by identifying its regulatory proteins. We had previously shown that a portion of bovine sperm PP1gamma2 is present in the cytosolic fraction of sperm sonicates. We purified PP1gamma2 from soluble bovine sperm extracts by immunoaffinity chromatography. Gel electrophoresis of the purified enzyme showed that it was complexed to a protein 43 M(r) x 10(-3) in size. Microsequencing revealed that this protein is a mammalian homologue of sds22, which is a yeast PP1 binding protein. Phosphatase activity measurements showed that PP1gamma2 complexed to sds22 is catalytically inactive. The complex cannot be activated by limited proteolysis. The complex is unable to bind to microcystin sepharose. This suggests that sds22 may block the microcystin binding site in PP1gamma2. A proportion of PP1gamma2 in sperm extracts, which is presumably not complexed to sds22, is catalytically active. Fluorescence immunocytochemistry was used to determine the intrasperm localization of PP1gamma2 and sds22. Both proteins are present in the tail. They are also present in distinct locations in the head. Our data suggest that PP1gamma2 binding to sds22 inhibits its catalytic activity. Mechanisms regulating sds22 binding to PP1gamma2 are likely to be important in understanding the biochemical basis underlying development and regulation of sperm function.

Topics: Amino Acid Sequence; Animals; Catalysis; Cattle; Cell Cycle Proteins; Chromatography, Affinity; Epididymis; Fluorescent Antibody Technique; Fungal Proteins; Homeostasis; Male; Microcystins; Molecular Sequence Data; Nuclear Proteins; Peptides, Cyclic; Phosphoprotein Phosphatases; Schizosaccharomyces pombe Proteins; Sepharose; Spermatozoa

During mitotic arrest induced by paclitaxel, most of the mitochondrial Bcl-2 is phosphorylated. This mitotic arrest is transient; exit from mitosis, due to mitotic slippage, occurs and Bcl-2 is rapidly dephosphorylated. In the present study, we characterized PP1 as the cytosolic phosphatase involved in Bcl-2 dephosphorylation. When mitochondria and cytosol prepared from mitotic arrested cells were incubated in vitro, the proportion of phosphorylated forms of Bcl-2 in mitochondria remained unchanged. In contrast, cytosol prepared from cells during mitotic slippage led to a dose-dependent loss of phosphorylated forms of Bcl-2. Depletion of these cytosol extracts by microcystin-Sepharose maintained Bcl-2 phosphorylated forms, indicating that this cytosol possessed phosphatase activity. Furthermore, the dephosphorylation of Bcl-2 by cytosol prepared from cells exiting mitotic block was inhibited by okadaic acid, at a dose known to inhibit PP1, and by inhibitor 2, a specific inhibitor of PP1 and by immunodepletion of PP1. Finally, we showed that PP1 is associated with mitochondrial Bcl-2 in vivo. Taken together, these results demonstrate that PP1 is directly involved in Bcl-2 dephosphorylation during mitotic slippage.

Topics: Antineoplastic Agents; Breast Neoplasms; Cytosol; Enzyme Inhibitors; Female; Humans; Microcystins; Mitochondria; Mitosis; Paclitaxel; Peptides, Cyclic; Phosphoprotein Phosphatases; Phosphorylation; Precipitin Tests; Protein Binding; Proto-Oncogene Proteins c-bcl-2; Sepharose; Tumor Cells, Cultured

At the onset of mitosis, the nuclear lamins are hyperphosphorylated leading to nuclear lamina disassembly, a process required for nuclear envelope breakdown and entry into mitosis. Multiple lamin kinases have been identified, including protein kinase C, that mediate mitotic lamin phosphorylation and mitotic nuclear lamina disassembly. Conversely, lamin dephosphorylation is required for nuclear lamina reassembly at the completion of mitosis. However, the protein phosphatase(s) responsible for the removal of mitotic phosphates from the lamins is unknown. In this study, we use human lamin B phosphorylated at mitosis-specific sites as a substrate to identify and characterize a lamin phosphatase activity from mitotic human cells. Several lines of evidence demonstrate that the mitotic lamin phosphatase corresponds to type 1 protein phosphatase (PP1). First, mitotic lamin phosphatase activity is inhibited by high nanomolar concentrations of okadaic acid and the specific PP1 peptide inhibitor, inhibitor-2. Second, mitotic lamin phosphatase activity cofractionates with PP1 after ion exchange chromatography. Third, microcystin-agarose depletes mitotic extracts of both PP1 and lamin phosphatase activity. Our results demonstrate that PP1 is the major mitotic lamin phosphatase responsible for removal of mitotic phosphates from lamin B, a process required for nuclear lamina reassembly.

Topics: Chromatography, Ion Exchange; Enzyme Inhibitors; HL-60 Cells; Humans; Lamin Type B; Lamins; Microcystins; Mitosis; Nuclear Proteins; Okadaic Acid; Peptides, Cyclic; Phosphoprotein Phosphatases; Protein Phosphatase 1; Sepharose

A microcystin (MC)-Sepharose column was prepared by addition of 2-aminoethanethiol to the alpha, beta-unsaturated carbonyl of the N-methyldehydroalanine residue of MC-LR, followed by reaction of the introduced amino group with N-hydroxysuccinimide-activated CH-Sepharose. The MC-Sepharose bound protein phosphatase-1 (PP1) with high capacity and purified human PP1 gamma in one step from E. coli extracts. It was also used to purify forms of PP1 bound to myofibrils from skeletal muscle. Two of these comprised PP1 complexed to N-terminal fragments of the M-subunit which enhance its myosin phosphatase activity, while the third comprised PP1 and an N-terminal fragment of the glycogen-binding (G)-subunit.

Topics: Animals; Chromatography, Affinity; Escherichia coli; Microcystins; Muscle, Skeletal; Peptides, Cyclic; Phosphoprotein Phosphatases; Protein Phosphatase 1; Rabbits; Recombinant Proteins; Sepharose