Phosphorylation/dephosphorylation of Komatsuna (Brassica campestris ) leaf nitrate reductase in vivo and in vitro in response to environmental light conditions: Effects of protein kinase and protein phosphatase inhibitors

1995 ◽  
Vol 93 (1) ◽  
pp. 139-145 ◽  
Author(s):  
Michiko Kojima ◽  
Sheng-Jun Wu ◽  
Hajime Fukui ◽  
Toshio Sugimoto ◽  
Takashi Nanmori ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Phosphatase ◽  
Brassica Campestris ◽  
Light Conditions ◽  
Phosphatase Inhibitors ◽  
Environmental Light ◽  
Protein Phosphatase Inhibitors ◽  
Leaf Nitrate

In vitro and in vivo effects of protein phosphatase inhibitors, microcystins and nodularin, on mouse skin and fibroblasts

1990 ◽  
Vol 171 (2) ◽  
pp. 867-874 ◽  
Author(s):  
Rie Matsushima ◽  
Shigeru Yoshizawa ◽  
Mariyo F. Watanabe ◽  
Ken-ichi Harada ◽  
Mitsuru Furusawa ◽  
...  
Keyword(s):  
Protein Phosphatase ◽  
Mouse Skin ◽  
Phosphatase Inhibitors ◽  
Protein Phosphatase Inhibitors ◽  
In Vivo Effects

scholarly journals Dissecting the Role of 5′-AMP for Allosteric Stimulation, Activation, and Deactivation of AMP-activated Protein Kinase

2006 ◽  
Vol 281 (43) ◽  
pp. 32207-32216 ◽  
Author(s):  
Marianne Suter ◽  
Uwe Riek ◽  
Roland Tuerk ◽  
Uwe Schlattner ◽  
Theo Wallimann ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Phosphatase ◽  
Energy Homeostasis ◽  
Adenine Nucleotides ◽  
Enzyme Preparations ◽  
Activity Measurements ◽  
Ampk Activity ◽  
Amp Activated Protein Kinase

AMP-activated protein kinase (AMPK) is a heterotrimeric protein kinase that is crucial for cellular energy homeostasis of eukaryotic cells and organisms. Here we report on the activation of AMPK α1β1γ1 and α2β2γ1 by their upstream kinases (Ca2+/calmodulin-dependent protein kinase kinase-β and LKB1-MO25α-STRADα), the deactivation by protein phosphatase 2Cα, and on the extent of stimulation of AMPK by its allosteric activator AMP, using purified recombinant enzyme preparations. An accurate high pressure liquid chromatography-based method for AMPK activity measurements was established, which allowed for direct quantitation of the unphosphorylated and phosphorylated artificial peptide substrate, as well as the adenine nucleotides. Our results show a 1000-fold activation of AMPK by the combined effects of upstream kinase and saturating concentrations of AMP. The two AMPK isoforms exhibit similar specific activities (6 μmol/min/mg) and do not differ significantly by their responsiveness to AMP. Due to the inherent instability of ATP and ADP, it proved impossible to assay AMPK activity in the absolute absence of AMP. However, the half-maximal stimulatory effect of AMP is reached below 2 μm. AMP does not appear to augment phosphorylation by upstream kinases in the purified in vitro system, but deactivation by dephosphorylation of AMPK α-subunits at Thr-172 by protein phosphatase 2Cα is attenuated by AMP. Furthermore, it is shown that neither purified NAD+ nor NADH alters the activity of AMPK in a concentration range of 0–300 μm, respectively. Finally, evidence is provided that ZMP, a compound formed in 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside-treated cells to activate AMPK in vivo, allosterically activates purified AMPK in vitro, but compared with AMP, maximal activity is not reached. These data shed new light on physiologically important aspects of AMPK regulation.

scholarly journals Specific enzymatic dephosphorylation of the retinoblastoma protein.

1993 ◽  
Vol 13 (1) ◽  
pp. 367-372 ◽  
Author(s):  
J W Ludlow ◽  
C L Glendening ◽  
D M Livingston ◽  
J A DeCarprio
Keyword(s):  
Phosphatase Activity ◽  
Large Population ◽  
Mitotic Cell ◽  
Phosphatase Inhibitors ◽  
Cell Extracts ◽  
Protein Phosphatase Inhibitors ◽  
Cycle Dependent

The retinoblastoma gene product (RB) undergoes cell cycle-dependent phosphorylation and dephosphorylation. Pulse-chase experiments revealed that the change in RB gel electrophoretic migration which occurs near mitosis is due to enzymatic dephosphorylation (J. W. Ludlow, J. Shon, J. M. Pipas, D. M. Livingston, and J. A. DeCaprio, Cell 60:387-396, 1990). To determine the precise timing of RB dephosphorylation and whether a specific phosphatase is active in this process, we have utilized a nocodazole block and release protocol which allows a large population of cells to progress synchronously through mitosis. In such experiments, RB dephosphorylation began during anaphase and continued until complete dephosphorylation was apparent in the ensuing G1 period. In addition, late mitotic cell extracts were capable of dephosphorylating RB in vitro. This RB-specific mitotic phosphatase activity was more active in anaphase extracts than in pro- or metaphase extracts, which is consistent with the results obtained in vivo. Okadaic acid and protein phosphatase inhibitors 1 and 2 inhibited this specific RB phosphatase activity. These results suggest a role for serine and threonine phosphoprotein phosphatase type 1 in the late mitotic dephosphorylation of RB.

Identification of rat epidermal profilaggrin phosphatase as a member of the protein phosphatase 2A family

1993 ◽  
Vol 106 (1) ◽  
pp. 219-226 ◽  
Author(s):  
E. Kam ◽  
K.A. Resing ◽  
S.K. Lim ◽  
B.A. Dale
Keyword(s):  
Enzymatic Activity ◽  
Intermediate Filaments ◽  
Protein Phosphatase ◽  
Protein Phosphatase 2A ◽  
Gel Filtration ◽  
Phosphatase Inhibitors ◽  
Rate Analysis ◽  
Phosphatase 2A

The aggregation of cellular intermediate filaments is an important step in the terminal differentiation of keratinocytes. It has been shown that epidermal filaggrin can cause intermediate filaments to aggregate in vitro and may also have the same function in vivo. Filaggrin is derived via dephosphorylation and proteolysis from a highly phosphorylated precursor, profilaggrin, which is found in the granular layer of the epidermis. Using casein kinase II phosphorylated filaggrin as substrate, a profilaggrin phosphatase has been partially purified from rat epidermal homogenate by three chromatographic steps (DE52, hydroxylapatite and S200 gel filtration). Profilaggrin phosphatase activity eluted from the last column has a Km of 0.12 mM and a Vmax of 8 nmol/mg/min with respect to phosphofilaggrin. Results obtained by initial rate analysis showed that the enzymatic activity is not affected by phospho-tyrosyl phosphatase inhibitors and the active fractions preferentially dephosphorylate the alpha subunit of phosphorylase kinase which has been phosphorylated by cAMP-dependent kinase. These results suggest that epidermal profilaggrin phosphatase is not a phospho-tyrosyl phosphatase or a type 1 phospho-seryl/phospho-threonyl phosphatase. Dephosphorylation is not affected by EDTA, calcium or magnesium, but is very sensitive to okadaic acid inhibition (IC50 = 80 pM), suggesting that the enzymatic activity is related to that of the protein phosphatase 2A (PP2A).(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals A tightly associated serine/threonine protein kinase regulates phosphoinositide 3-kinase activity.

1993 ◽  
Vol 13 (3) ◽  
pp. 1657-1665 ◽  
Author(s):  
C L Carpenter ◽  
K R Auger ◽  
B C Duckworth ◽  
W M Hou ◽  
B Schaffhausen ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Phosphatase ◽  
Kinase Activity ◽  
Protein Phosphatase 2A ◽  
T Antigen ◽  
Serine Kinase ◽  
Phosphatase 2A ◽  
Phosphoinositide 3 Kinase

We identified a serine/threonine protein kinase that is associated with and phosphorylates phosphoinositide 3-kinase (PtdIns 3-kinase). The serine kinase phosphorylates both the 85- and 110-kDa subunits of PtdIns 3-kinase and purifies with it from rat liver and immunoprecipitates with antibodies raised to the 85-kDa subunit. Tryptic phosphopeptide maps indicate that p85 from polyomavirus middle T-transformed cells is phosphorylated in vivo at three sites phosphorylated in vitro by the associated serine kinase. The 85-kDa subunit of PtdIns 3-kinase is phosphorylated in vitro on serine at a stoichiometry of approximately 1 mol of phosphate per mol of p85. This phosphorylation results in a three- to sevenfold decrease in PtdIns 3-kinase activity. Dephosphorylation with protein phosphatase 2A reverses the inhibition. This suggests that the association of protein phosphatase 2A with middle T antigen may function to activate PtdIns 3-kinase.

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