scholarly journals Regulation of acrosome reaction of fowl spermatozoa: evidence for the involvement of protein kinase C and protein phosphatase-type 1 and/or -type 2A

Reproduction ◽  
2006 ◽  
Vol 131 (6) ◽  
pp. 1017-1024 ◽  
Author(s):  
K Ashizawa ◽  
G J Wishart ◽  
S Katayama ◽  
D Takano ◽  
A R A H Ranasinghe ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Okadaic Acid ◽  
Protein Phosphatase ◽  
Acrosome Reaction ◽  
Dependent Manner ◽  
Kinase C ◽  
Protein Phosphatase Type 1 ◽  
Protein Phosphatase Type

The signal transduction pathways involved in the regulation of the acrosome reaction and motility of fowl spermatozoa were investigated. The motility and acrosomal integrity of fowl spermatozoa in TES/NaCl buffer, with or without homogenised inner perivitelline layers (IPVL), prepared from laid fowl eggs, was almost negligible at 40 °C. In the presence of 2 mmol CaCl2/l at 40 °C, motility became vigorous and the acrosome reaction was stimulated when IPVL was added. In the absence of Ca2+, motility was stimulated by the addition of calyculin A and okadaic acid, both specific inhibitors of protein phosphatase-type 1 (PP1) and -type 2A (PP2A), but Okadaic acid, which is a weaker inhibitor of PP1, did not completely restore motility at 40 °C. However, the acrosome reaction was significantly and equally stimulated in a dose-dependent manner by both inhibitors in the range of 10–1000 nmol/l, when spermatozoa were incubated with IPVL but without Ca2+. These inhibitors did not stimulate the acrosome reaction in the absence of IPVL. The vigorous motility of spermatozoa, stimulated by the addition of Ca2+, was reduced gradually as the concentrations of SC-9, a selective activator of protein kinase C (PKC), were increased and a similar SC-9-induced inhibition was observed in the acrosome reaction in the presence of Ca2+ and IPVL. These results confirm that IPVL is necessary for the activation of the acrosome reaction in fowl spermatozoa and that Ca2+ plays an important role in the stimulation of motility and acrosomal exocytosis. Furthermore, it appears that the intracellular molecular mechanisms for the regulation of acrosome reaction of fowl spermatozoa are different from those for the restoration of motility, i.e., protein dephosporylation involving PP1 and/or PP2A in the former, and PP1 alone in the latter case. In addition, the activation of PKC may contribute to a decrease in the flagellar movement and acrosome reaction of fowl spermatozoa.

Phosphorylation of the Protein Phosphatase Type 1 Inhibitor Protein CPI-17 by Protein Kinase C

2007 ◽  
pp. 209-224 ◽  
Author(s):  
Michael P. Walsh ◽  
Marija Susnjar ◽  
Jingti Deng ◽  
Cindy Sutherland ◽  
Eniko Kiss ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Protein Phosphatase ◽  
Inhibitor Protein ◽  
Kinase C ◽  
Protein Phosphatase Type 1 ◽  
Protein Phosphatase Type

scholarly journals Functional Analysis of the Yeast Glc7-Binding Protein Reg1 Identifies a Protein Phosphatase Type 1-Binding Motif as Essential for Repression of ADH2 Expression

1999 ◽  
Vol 19 (9) ◽  
pp. 6029-6040 ◽  
Author(s):  
Kenneth M. Dombek ◽  
Valentina Voronkova ◽  
Alexa Raney ◽  
Elton T. Young
Keyword(s):  
Protein Kinase ◽  
Central Region ◽  
Protein Phosphatase ◽  
Kinase Activity ◽  
Protein Kinase Activity ◽  
Snf1 Kinase ◽  
Protein Phosphatase Type 1 ◽  
Mutant Cells ◽  
Protein Phosphatase Type

ABSTRACT In Saccharomyces cerevisiae, the protein phosphatase type 1 (PP1)-binding protein Reg1 is required to maintain complete repression of ADH2 expression during growth on glucose. Surprisingly, however, mutant forms of the yeast PP1 homologue Glc7, which are unable to repress expression of another glucose-regulated gene, SUC2, fully repressed ADH2. ConstitutiveADH2 expression in reg1 mutant cells did require Snf1 protein kinase activity like constitutive SUC2expression and was inhibited by unregulated cyclic AMP-dependent protein kinase activity like ADH2 expression in derepressed cells. To further elucidate the functional role of Reg1 in repressingADH2 expression, deletions scanning the entire length of the protein were analyzed. Only the central region of the protein containing the putative PP1-binding sequence RHIHF was found to be indispensable for repression. Introduction of the I466M F468A substitutions into this sequence rendered Reg1 almost nonfunctional. Deletion of the central region or the double substitution prevented Reg1 from significantly interacting with Glc7 in two-hybrid analyses. Previous experimental evidence had indicated that Reg1 might target Glc7 to nuclear substrates such as the Snf1 kinase complex. Subcellular localization of a fully functional Reg1-green fluorescent protein fusion, however, indicated that Reg1 is cytoplasmic and excluded from the nucleus independently of the carbon source. When the level of Adr1 was modestly elevated, ADH2 expression was no longer fully repressed in glc7 mutant cells, providing the first direct evidence that Glc7 can repress ADH2 expression. These results suggest that the Reg1-Glc7 phosphatase is a cytoplasmic component of the machinery responsible for returning Snf1 kinase activity to its basal level and reestablishing glucose repression. This implies that the activated form of the Snf1 kinase complex must cycle between the nucleus and the cytoplasm.

K-Cl cotransport in rabbit red cells: further evidence for regulation by protein phosphatase type 1

1993 ◽  
Vol 264 (1) ◽  
pp. C118-C124 ◽  
Author(s):  
L. C. Starke ◽  
M. L. Jennings
Keyword(s):  
Okadaic Acid ◽  
Protein Phosphatase ◽  
Inhibitory Concentration ◽  
Cell Swelling ◽  
Calyculin A ◽  
Phosphatase Inhibitor ◽  
Protein Phosphatase Type 1 ◽  
Pharmacological Studies ◽  
Protein Phosphatase Type

We have examined inhibition of swelling-induced K-Cl cotransport in rabbit red blood cells by calyculin A, a potent serine-threonine protein phosphatase inhibitor, to determine whether transport is regulated by phosphatase type 1 or type 2A. Calyculin A blocks K(Rb) influx [half-maximal inhibitory concentration (IC50) = 3-6 nM] 10 times more potently than a second phosphatase inhibitor, okadaic acid (IC50 = 40 nM), consistent with earlier pharmacological studies showing that calyculin A inhibits phosphatase type 1 10 times more effectively than does okadaic acid. Calyculin A always inhibits Rb influx when added either before or after cell swelling, indicating that the phosphatase must operate continually to first activate and then maintain high transport rates in swollen cells. Similarly, N-ethylmaleimide (NEM) fails to stimulate K-Cl cotransport only when added to cells pretreated with calyculin A. Therefore, like cell swelling, activation of K-Cl cotransport by NEM involves a phosphatase sensitive to calyculin A. We conclude that cell swelling and NEM activate K-Cl cotransport via a net dephosphorylation that appears to involve protein phosphatase type 1.

Hyperosmolarity inhibits the Na+/H+ exchanger isoforms NHE2 and NHE3: an effect opposite to that on NHE1

1996 ◽  
Vol 270 (3) ◽  
pp. G431-G441 ◽  
Author(s):  
S. K. Nath ◽  
C. Y. Hang ◽  
S. A. Levine ◽  
C. H. Yun ◽  
M. H. Montrose ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Okadaic Acid ◽  
Protein Phosphatase ◽  
Kinase Inhibitor ◽  
Reaction Velocity ◽  
Kinase C ◽  
Protein Kinase C Inhibitor ◽  
Nhe Isoforms ◽  
Menten Constant

The effect of hyperosmolarity on cloned Na+/H+ exchanger (NHE) isoforms NHE2 and NHE3 was studied in stably transfected PS120 fibroblasts. Na+/H+ exchanger activity was determined spectrofluorometrically in acidified cells that were exposed to isosmolar (300 mosmol/kg) or hyperosmolar (450 mosmol/kg) media, in which the only difference is the presence or absence of 150 mM mannitol. Hyperosmolar solution reversibly inhibited NHE2 and NHE3 with a delay of approximately 15 s. Hyperosmolarity significantly reduced their maximal reaction velocity compared with isosmolar medium but did not alter their Michaelis-Menten constant for intracellular H+. The Michaelis-Menten constant of the exchangers for extracellular Na+ in hyperosmolar medium was not different from that in isosmolar medium. Pretreatment of PS120/NHE3 cells with the protein kinase C inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, the tyrosine kinase inhibitor genistein, and the serine/threonine protein phosphatase inhibitor okadaic acid did not affect the hyperosmolar inhibition of NHE3. Hyperosmolar inhibition of Na+/H+ exchanger activity was also observed in PS120 cells transfected with truncated NHE3 cDNAs (E3/585, E3/543, E3509, and E3/475) and NHE2 cDNA (E2/499). We conclude that 1) hyperosmolarity inhibits NHE2 and NHE3, in contrast to the stimulatory effect on the housekeeping isoform NHE1, 2) this inhibition is reversible, and 3) the COOH termini of NHE2 and NHE3 are not necessary for hyperosmolar inhibition of NHE2 and NHE3.

Protein phosphorylation and dephosphorylation in type II pneumocytes

1991 ◽  
Vol 260 (6) ◽  
pp. L548-L554 ◽  
Author(s):  
D. Warburton ◽  
A. Tayag ◽  
S. Buckley ◽  
L. Cosico ◽  
R. Seth
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Protein Phosphorylation ◽  
Phorbol Ester ◽  
Okadaic Acid ◽  
Protein Phosphatase ◽  
Type Ii ◽  
Kinase C ◽  
Type Ii Pneumocytes ◽  
Specific Proteins

Protein phosphorylation and dephosphorylation are a major mechanism for regulating cellular activity. Substantial evidence exists for ascribing a key role of protein phosphorylation and dephosphorylation in the regulation of surfactant secretion from type II pneumocytes, yet understanding of the specific molecular mechanisms is generally lacking. Herein, we report two-dimensional electrophoretic mapping of proteins phosphorylated in type II pneumocytes in primary culture, the response to protein kinase C simulation with phorbol ester, and the response to protein phosphatase inhibition with okadaic acid. Exposure of cells for 15 min to phorbol ester at a concentration (10(-4) M) which maximally stimulated both translocation of protein kinase C from cytosol to membranes and surfactant secretion increased phosphorylation (50-80% compared with control) of three specific proteins (50 kDa, pI 5.8 and 5.7; 25 kDa, pI 5.7). Exposure of cells for 2.5 h to okadaic acid (10(-6) M), a concentration that inhibited 90% of protein phosphatase activity, resulted in greatly increased phosphorylation (200-1,500% compared with control) of five specific proteins (50 kDa, pI 5.7 and 5.6; 45 kDa, pI 5.5; 40 kDa, pI 5.5; 25 kDa, pI 5.5). Combined treatment with okadaic acid and phorbol ester resulted in further increases (145-3,080% compared with control) in phosphorylation of four specific proteins (50 kDa, pI 5.6; 45 kDa, pI 5.5; 40 kDa, pI 5.5; 25 kDa, pI 5.5). We conclude that these respective proteins comprise major substrates for protein kinase C-dependent phosphorylation and for protein phosphatases in type II pneumocytes in primary culture. Furthermore, we speculate that these proteins will prove to play key roles in the regulation of type II pneumocyte function.

scholarly journals Okadaic acid inhibits insulin-induced glucose transport in fetal brown adipocytes in an Akt-independent and protein kinase C ζ-dependent manner

FEBS Letters ◽  
2000 ◽  
Vol 472 (1) ◽  
pp. 153-158 ◽  
Author(s):  
Angela M. Valverde ◽  
Margarita Lorenzo ◽  
Paloma Navarro ◽  
Cecilia Mur ◽  
Manuel Benito
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Glucose Transport ◽  
Okadaic Acid ◽  
Dependent Manner ◽  
Brown Adipocytes ◽  
Kinase C

Endotoxin-Induced Tissue Factor in Human Monocytes is Dependent upon Protein Kinase C Activation

1993 ◽  
Vol 70 (05) ◽  
pp. 800-806 ◽  
Author(s):  
C Ternisien ◽  
M Ramani ◽  
V Ollivier ◽  
F Khechai ◽  
T Vu ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Tissue Factor ◽  
Factor Ix ◽  
Transcriptional Level ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Human Monocytes ◽  
Kinase C ◽  
Pkc Activation

SummaryTissue factor (TF) is a transmembrane receptor which, in association with factors VII and Vila, activates factor IX and X, thereby activating the coagulation protease cascades. In response to bacterial lipopolysaccharide (LPS) monocytes transcribe, synthesize and express TF on their surface. We investigated whether LPS-induced TF in human monocytes is mediated by protein kinase C (PKC) activation. The PKC agonists phorbol 12- myristate 13-acetate (PMA) and phorbol 12, 13 dibutyrate (PdBu) were both potent inducers of TF in human monocytes, whereas 4 alpha-12, 13 didecanoate (4 a-Pdd) had no such effect. Both LPS- and PMA-induced TF activity were inhibited, in a concentration dependent manner, by three different PKC inhibitors: H7, staurosporine and calphostin C. TF antigen determination confirmed that LPS-induced cell-surface TF protein levels decreased in parallel to TF functional activity under staurosporine treatment. Moreover, Northern blot analysis of total RNA from LPS- or PMA-stimulated monocytes showed a concentration-dependent decrease in TF mRNA levels in response to H7 and staurosporine. The decay rate of LPS-induced TF mRNA evaluated after the arrest of transcription by actinomycin D was not affected by the addition of staurosporine, suggesting that its inhibitory effect occurred at a transcriptional level. We conclude that LPS-induced production of TF and its mRNA by human monocytes are dependent on PKC activation.

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