Showing papers by "Pál Gergely published in 2000"

Inhibition of serine/threonine-specific protein phosphatases causes premature activation of cdc2MsF kinase at G2/M transition and early mitotic microtubule organisation in alfalfa

Abstract: Reversible phosphorylation of serine/threonine residues of cell cycle-regulatory proteins is one of the key molecular mechanisms controlling eukaryotic cell division. In plants, the protein kinase partners (i.e. p34cdc2/CDC28-related kinases) have been extensively studied, while the role of counter-acting protein phosphatases is less well understood. We used endothall (ET) as a cell-permeable inhibitor of serine/threonine-specific protein phosphatases to alter cytological and biochemical characteristics of cell division in cultured alfalfa cells. A high concentration of ET (10 and 50 microM) inhibited both protein phosphatases 1 and 2 (PP1 and PP2A), while a low concentration (1 microM) of ET-treatment primarily reduced the PP2A activity. High concentrations of the inhibitor increased the frequency of hypercondensed early and late prophase chromosomes that could not enter metaphase. In contrast, a low concentration of ET did not interfere with chromosomal events but caused significant alterations in the organisation of microtubules. Exposure of cells to 1 microM ET resulted in disturbance of preprophase band formation, increase in the number of nuclei with prophase microtubule assembly, premature polarisation of the spindle, and abnormal phragmoplast maturation. Under the same conditions, the ET-treated cells exhibited an early increase in cdc2MsF kinase activity. These results suggest that PP2A contributes to the control of mitotic kinase activities and microtubule organisation. Normal chromosome condensation and mitotic progression are dependent on both PP1 and PP2A activities. The presented data support the functional role of protein phosphatases in the co-ordination of chromosomal and microtubule events in dividing plant cells.

Study of the subunit interactions in myosin phosphatase by surface plasmon resonance.

Abstract: The interactions of the catalytic subunit of type 1 protein phosphatase (PP1c) and the N-terminal half (residues 1–511) of myosin phosphatase target subunit 1 (MYPT1) were studied. Biotinylated MYPT1 derivatives were immobilized on streptavidin-biosensor chips, and binding parameters with PP1c were determined by surface plasmon resonance (SPR). The affinity of binding of PP1c was: MYPT11–296 > MYPT11–38 > MYPT123–38. No binding was detected with MYPT11–34, suggesting a critical role for residues 35–38, i.e. the PP1c binding motif. Binding of residues 1–22 was inferred from: a higher affinity binding to PP1c for MYPT11–38 compared to MYPT123–38, as deduced from SPR kinetic data and ligand competition assays; and an activation of the myosin light chain phosphatase activity of PP1c by MYPT11–38, but not by MYPT123–38. Residues 40–296 (ankyrin repeats) in MYPT11–296 inhibited the phosphorylase phosphatase activity of PP1c (IC50 = 0.2 nm), whereas MYPT11–38, MYPT123–38 or MYPT11–34 were without effect. MYPT140–511, which alone did not bind to PP1c, showed facilitated binding to the complexes of PP1c–MYPT11–38 and PP1c–MYPT123–38. The inhibitory effect of MYPT140–511 on the phosphorylase phosphatase activity of PP1c also was increased in the presence of MYPT11–38. The binding of MYPT1304–511 to complexes of PP1c and MYPT11–38, or MYPT11–296, was detected by SPR. These results suggest that within the N-terminal half of MYPT1 there are at least four binding sites for PP1c. The essential interaction is with the PP1c-binding motif and the other interactions are facilitated in an ordered and cooperative manner.

Gram-scale synthesis of a glucopyranosylidene-spiro-thiohydantoin and its effect on hepatic glycogen metabolism studied in vitro and in vivo

Abstract: A high yielding, simple synthesis is described starting from d -glucose to produce gram quantities of a glucopyranosylidene-spiro-thiohydantoin. This compound efficiently inhibited the activity of rat liver glycogen phosphorylase a ; moreover, it also activated phosphorylase phosphatase which, in turn, decreased the amount of glycogen phosphorylase a . Both effects result in the inhibition of glycogen mobilization and the formation of glucose from glycogen.

Immunohistochemical localisation of two phosphatidylinositol 4-kinase isoforms, PI4K230 and PI4K92, in the central nervous system of rats

Abstract: The distribution and cellular localisation of the phosphatidylinositol 4-kinase isoforms, PI4K230 and PI4K92, that are believed to play important roles in the intracellular signalling mechanisms were studied in the rat brain (cortex, cerebellum, hippocampus and spinal cord) using immunocytochemistry with light and electron microscopy. PI4K230 was detected with a specific antibody purified by affinity chromatography from the egg yolk of chicken immunised with a 33-kDa fragment of bovine PI4K230, comprising amino acids 873-1175 of the native protein. PI4K92 was immunostained with a commercially available antibody raised in rabbit against amino acid residues 410-537 of human PI4K92. At the light microscopic level, the immunostaining of PI4K230 and PI4K92 showed a very similar distribution throughout the neurons and appeared as dense punctate labelling in the cytoplasm of perikarya and stem dendrites of various neurons. In addition to neurons, a strongly stained cell population was observed in the molecular layer of the cerebellar cortex that resembled Bergmann glia cells. Electron microscopy of neurons in the ventral horn of the spinal cord showed dense granular immunoprecipitates for both PI4K230 and PI4K92, mostly associated with the outer membrane of mitochondria and membranes of the rough endoplasmic reticulum. In addition, immunostaining of PI4K92 was also frequently found on the outer surface of cisterns and vesicles of Golgi complexes, whereas PI4K230 immunoreactivity was colocalised with some multivesicular bodies. Neither nuclear localisation nor a regular attachment to the cell membrane of these enzymes were observed. Our findings indicate that PI4K230 and PI4K92 are not involved directly in the ligand-stimulated turnover of phosphoinositides at the plasma membrane of neurons. However, they may provide regulatory phosphoinositides for intracellular vesicular traffic being associated with various organelles.

Protein phosphatase 2A holoenzyme and its subunits from Medicago sativa.

Abstract: We detected an about 200 kDa holoenzyme of protein phosphatase 2A (PP2A) in the crude extract of Medicago sativa microcallus cells by gel permeation chromatography. By polymerase chain reaction (PCR) we isolated two M. sativa cDNA fragments corresponding to the catalytic (C) subunit, and one each coding for the A and the B regulatory subunits of PP2A. The C subunit sequences were different from that published previously, indicating the existence of at least three different isoforms in M. sativa. Using the PCR fragments as probes, we obtained two distinct full-length clones for both the A and B subunits from an alfalfa cDNA library. Our results demonstrate that the components of the PP2A holoenzyme, namely the catalytic and regulatory subunits, are present in alfalfa in several isoforms and that their sequences are highly similar to their plant, yeast and animal counterparts. The distinct regulatory subunit genes are constitutively expressed during the cell cycle. Interestingly, two A-B subunit pairs had parallel mRNA steady-state levels in different plant tissues suggesting that not all of the possible isoform combinations are present in all tissues. The expression of the MsPP2A Bβ subunit form was induced by abscisic acid indicating a specific function for this protein in the stress response.