scholarly journals Global Forebrain Ischemia Results in Decreased Immunoreactivity of Calcium/Calmodulin-Dependent Protein Kinase II

1992 ◽  
Vol 12 (5) ◽  
pp. 784-793 ◽  
Author(s):  
Severn B. Churn ◽  
Amy Yaghmai ◽  
John Povlishock ◽  
Azhar Rafiq ◽  
Robert J. DeLorenzo
Keyword(s):  
Monoclonal Antibody ◽  
Protein Kinase ◽  
Neuronal Cell ◽  
Cam Kinase Ii ◽  
Forebrain Ischemia ◽  
Cam Kinase ◽  
Dependent Protein Kinase ◽  
Calcium Calmodulin Dependent ◽  
Protein Kinase Ii ◽  
Dependent Protein

Previous studies utilizing crude brain homogenates have shown that forebrain ischemia results in inhibition of calcium/calmodulin-dependent protein kinase II (CaM kinase II) activity without large-scale proteolysis of the enzyme. In this report, a monoclonal antibody (1C3-3D6) directed against the β- (60-kDa) subunit of CaM kinase II that does not recognize ischemically altered enzyme was utilized to further investigate the ischemia-induced inhibition of CaM kinase II. Immunohistochemical investigations showed that the ischemia-induced decreased immunoreactivity of CaM kinase II occurred immediately following ischemic insult in ischemia-sensitive cells such as pyramidal cells of the hippocampus. No decrease in CaM kinase II immunoreactivity was observed in ischemia-resistant cells such as granule cells of the dentate gyrus. The decreased immunoreactivity was observed for CaM kinase II balanced for protein staining and calmodulin binding in vitro. In addition, autophosphorylation of CaM kinase II in the presence of low (7 μ M) or high (500 μ M) ATP did not alter immunoreactivity of the enzyme with 1C3-3D6. The data demonstrate the production of a monoclonal antibody that recognizes the β-subunit of CaM kinase II in a highly specific manner, but does not recognize ischemic enzyme. Together with previous studies, the data support the hypothesis that rapid, ischemia-induced inhibition of CaM kinase II activity may be involved in the cascade of events that lead to selective neuronal cell loss in stroke.

scholarly journals Muscarinic activation of Ca2+/calmodulin-dependent protein kinase II in pancreatic islets. Temporal dissociation of kinase activation and insulin secretion

1996 ◽  
Vol 317 (1) ◽  
pp. 167-172 ◽  
Author(s):  
Eric L. BABB ◽  
Jim TARPLEY ◽  
Michael LANDT ◽  
Richard A. EASOM
Keyword(s):  
Insulin Secretion ◽  
Protein Kinase ◽  
Pancreatic Islets ◽  
Cam Kinase Ii ◽  
Muscarinic Agonist ◽  
Cam Kinase ◽  
Dependent Protein Kinase ◽  
Kinase Activation ◽  
Protein Kinase Ii ◽  
Dependent Protein

We have demonstrated previously that glucose activates the multifunctional Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) in isolated rat pancreatic islets in a manner consistent with a role of this enzyme in the regulation of insulin secretion [Wenham, Landt and Easom (1994) J. Biol. Chem. 269, 4947–4952]. In the current study, the muscarinic agonist, carbachol, has been shown to induce the conversion of CaM kinase II into a Ca2+-independent, autonomous form indicative of its activation. Maximal activation (2-fold) was achieved by 15 s, followed by a rapid return to basal levels by 1 min. This response was primarily the result of the mobilization of Ca2+ from intracellular stores since it was not affected by a concentration (20 μM) of verapamil that completely prevented the activation of CaM kinase II by glucose. Surprisingly, carbachol added prior to, or simultaneously with, glucose attenuated nutrient activation of CaM kinase II. This effect was mimicked by cholecystokinin-8 (CCK-8) and thapsigargin, suggesting its mediation by phospholipase C and the mobilization of intracellular Ca2+. In contrast, carbachol, CCK-8 and thapsigargin markedly potentiated glucose (12 mM)-induced insulin secretion. These results suggest that CaM kinase II activation can be temporally dissociated from insulin secretion but do not exclude the potential dependence of insulin exocytosis on CaM kinase II-mediated protein phosphorylation.

scholarly journals Co-distribution of calmodulin-dependent protein kinase II and inositol trisphosphate receptors in an apical domain of gastrointestinal mucosal cells.

1996 ◽  
Vol 44 (11) ◽  
pp. 1243-1250 ◽  
Author(s):  
L M Matovcik ◽  
A R Maranto ◽  
C J Soroka ◽  
F S Gorelick ◽  
J Smith ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Pancreatic Acinar Cells ◽  
Apical Region ◽  
Cam Kinase Ii ◽  
Cam Kinase ◽  
Dependent Protein Kinase ◽  
Protein Kinase Ii ◽  
Dependent Protein ◽  
Type 3 ◽  
Insp3 Receptor

The Type 3 inositol 1,4,5-trisphosphate (InsP3) receptor is expressed at high levels in gastrointestinal tissues. This receptor has 16 potential phosphorylation sites for calcium/calmodulin-dependent protein kinase II (CaM kinase II). To determine if the Type 3 InsP3 receptor is likely to be a physiologic substrate for CaM kinase II, localizations of the Type 3 InsP3 receptor and CaM kinase II were compared in tissues of the gastrointestinal tract. Cellular and subcellular localizations were determined by immunofluorescence microscopy in rat intestine, pancreas, and stomach, and in isolated rabbit gastric glands. Both proteins were found in the apical region of intestinal enterocytes, pancreatic acinar cells, and gastric parietal, chief, and surface mucous cells. CaM kinase II was found throughout the entire intracellular canalicular F-actin domain of parietal cells, whereas the type 3 InsP3 receptor was restricted to the neck region. Thus, in several gastrointestinal tissues the Type 3 InsP3 receptor is specifically localized to a portion of the apical cytoskeletal domain in which resides the calcium-responsive effector CaM kinase II.

Immunohistochemical detection of calmodulin and calmodulin-dependent protein kinase II in the mouse testis

Zygote ◽  
2000 ◽  
Vol 8 (4) ◽  
pp. 303-314 ◽  
Author(s):  
M. Moriya ◽  
C. Katagiri ◽  
M. Ikebe ◽  
K. Yagi
Keyword(s):  
Protein Kinase ◽  
Mouse Testis ◽  
Seminiferous Tubules ◽  
Cam Kinase Ii ◽  
Cam Kinase ◽  
High Concentration ◽  
Dependent Protein Kinase ◽  
Calcium Calmodulin Dependent ◽  
Cell Tissue ◽  
Dependent Protein

We reported previously that in mouse testis calmodulin-dependent protein phosphatase (calcineurin) is localised in the nuclei of round and elongating spermatids (Cell Tissue Res. 1995; 281: 273-81). In this study, we studied the immunohistochemical localisation of calcium/calmodulin-dependent protein kinase (CaM kinase II) using antibodies against CaM kinase IIγ from chicken gizzard and specific antibodies raised against the amino acid sequence Ileu480–Ala493 of this enzyme, and compared it with the distribution of calmodulin. Indirect immunofluorescence was most concentrated in early spermatocytes and localised in the outermost layer of seminiferous tubules where the calmodulin level was relatively low. Measurements of immuno-gold particle densities on electron micrographs revealed that CaM kinase II is transiently increased in the nucleus of zygotene spermatocytes. These observations suggest the involvement of CaM kinase II in the meiotic chromosomal pairing process. An extremely high concentration of calmodulin in spermatogenic cells undergoing meiosis may not be directly related to activation of calmodulin-dependent kinases and phosphatases.

scholarly journals Anti-immunoglobulin M activates nuclear calcium/calmodulin-dependent protein kinase II in human B lymphocytes.

1995 ◽  
Vol 182 (6) ◽  
pp. 1943-1949 ◽  
Author(s):  
M A Valentine ◽  
A J Czernik ◽  
N Rachie ◽  
H Hidaka ◽  
C L Fisher ◽  
...  
Keyword(s):  
Protein Kinase ◽  
B Lymphocytes ◽  
Intracellular Free Calcium ◽  
Free Calcium ◽  
Cam Kinase Ii ◽  
Dependent Manner ◽  
Cam Kinase ◽  
Dependent Protein Kinase ◽  
Calcium Calmodulin Dependent ◽  
Dependent Protein

We and others have previously shown that the nuclear protein, Ets-1, is phosphorylated in a calcium-dependent manner after ligation of immunoglobulin (Ig) M on B lymphocytes. As this phosphorylation was independent of protein kinase C activity, we tested whether a calcium/calmodulin-dependent protein kinase (CaM kinase) might phosphorylate the Ets-1 protein after elevation of intracellular free calcium concentrations. The dephosphorylated form of Ets-1 has been shown to bind to chromatin, suggesting that the operative kinase should be detectable in the nucleus. We prepared nuclear extracts from two human B cell lines in which increased intracellular free calcium levels correlated with increased phosphorylation of the Ets-1 protein. Activity of the CaM kinases was determined using a synthetic peptide substrate both in the absence and presence of an inhibitor specific for the CaM kinase family, KN-62. Stimulation of cells with anti-IgM led to increased activity of a nuclear kinase that could phosphorylate the peptide, and this activity was reduced by 10 microM KN-62. Kinase activity was reduced in lysates preadsorbed using an antibody specific for CaM kinase II. Two-dimensional phosphopeptide maps of the Ets-1 protein from cells incubated with ionomycin or anti-IgM contained two unique phosphopeptides that were absent in untreated cells. Incubation of isolated Ets-1 protein with purified CaM kinase II produced phosphorylation of peptides that migrated identically to those found in cells incubated with either anti-IgM or ionomycin. These data suggest a model of signal transduction by the antigen receptor on B lymphocytes in which increased intracellular free calcium can rapidly activate nuclear CaM kinase II, potentially resulting in phosphorylation and regulation of DNA-binding proteins.

scholarly journals Correlation of the Activation of Ca2+/Calmodulin-Dependent Protein Kinase II with the Initiation of Insulin Secretion from Perifused Pancreatic Islets*

Endocrinology ◽  
1997 ◽  
Vol 138 (6) ◽  
pp. 2359-2364 ◽  
Author(s):  
Richard A. Easom ◽  
Natalie R. Filler ◽  
Emma M. Ings ◽  
Jim Tarpley ◽  
Michael Landt
Keyword(s):  
Insulin Secretion ◽  
Protein Kinase ◽  
Second Phase ◽  
Cam Kinase Ii ◽  
Cam Kinase ◽  
Dependent Protein Kinase ◽  
Kinase Activation ◽  
Protein Kinase Ii ◽  
Dependent Protein ◽  
Perifused Islets

Abstract An experimental procedure has been designed to permit the simultaneous assessment of the activation status of the multifunctional Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) with insulin secretion in perifused islets. By this procedure, the activation of CaM kinase II by glucose correlated closely with the initial and sustained phases of insulin secretion within a 30-min test period. By contrast, islets (160–200/tube) in static incubations neither supported second-phase insulin secretion nor CaM kinase II activation beyond 10–15 min. This was not the result of the accumulation of insulin, because the introduction of insulin (40–160 ng/ml) into the perifusion medium failed to mimic the suppression of glucose-induced insulin secretion or CaM kinase II activation. A similar addition of SRIF (0.01–1 μm) or epinephrine (1μ m) profoundly suppressed insulin secretion although failing to significantly influence CaM kinase II activation. Finally, on withdrawal of glucose from perifused islets, insulin secretion rapidly returned to basal rates, but CaM kinase II deactivation was significantly delayed. The correlation of kinase activation with the initiation of insulin secretion suggests that CaM kinase II may be important in the regulation of glucose-induced insulin secretion. The observed dissociation of these parameters in the presence of inhibitory hormones or after the withdrawal of a glucose stimulus, however, suggests that the kinase is not directly involved in the final steps of insulin exocytosis.

scholarly journals Ischemia-Induced Neuronal Damage: A Role for Calcium/Calmodulin-Dependent Protein Kinase II

1996 ◽  
Vol 16 (1) ◽  
pp. 1-6 ◽  
Author(s):  
M. Neal Waxham ◽  
James C. Grotta ◽  
Alcino J. Silva ◽  
Roger Strong ◽  
Jaroslaw Aronowski
Keyword(s):  
Protein Kinase ◽  
Neuronal Damage ◽  
Alpha Subunit ◽  
Cam Kinase ◽  
Wild Type ◽  
Dependent Protein Kinase ◽  
Knock Out ◽  
Calcium Calmodulin Dependent ◽  
Protein Kinase Ii ◽  
Dependent Protein

Calcium/calmodulin-dependent protein kinase II (CaM-kinase) is a central enzyme in regulating neuronal processes. Imbalances in the activity and distribution of this enzyme have been reported following in vivo ischemia, and sustained decreases in activity correlate with subsequent neuronal death. In this report, mice that had been rendered deficient in the alpha subunit of CaM-kinase using gene knock-out technology were utilized to determine whether this enzyme is causally related to ischemic damage. Using a focal model of cerebral ischemia, we showed that homozygous knock-out mice lacking the alpha subunit exhibited an infarct volume almost twice that of wild-type litter mates. Heterozygous mice exhibited slightly less damage following ischemia than did homozygous mice, but infarct volumes remained significantly larger than those of wild-type litter mates. We conclude that reduced amounts of the alpha subunit of CaM-kinase predisposes neurons to increased damage following ischemia and that any perturbation that decreases the amount or activity of the enzyme will produce enhanced susceptibility to neuronal damage.

scholarly journals Dephosphorylation and deactivation of Ca2+/calmodulin-dependent protein kinase II in βTC3-cells is mediated by Mg2+- and okadaic-acid-sensitive protein phosphatases

1998 ◽  
Vol 329 (2) ◽  
pp. 283-288 ◽  
Author(s):  
A. Richard EASOM ◽  
L. Jim TARPLEY ◽  
R. Natalie FILLER ◽  
Harshika BHATT
Keyword(s):  
Protein Kinase ◽  
Okadaic Acid ◽  
Protein Phosphatases ◽  
Cam Kinase Ii ◽  
Calyculin A ◽  
Cam Kinase ◽  
Dependent Protein Kinase ◽  
Protein Kinase Ii ◽  
Dependent Protein ◽  
Combined Action

The α-toxin-permeabilized βTC3 cell has been utilized as an experimental model for the identification of protein phosphatases responsible for the dephosphorylation and deactivation of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) in situ. In this model, the elevation of Ca2+ from 0.05 to 10 μM induced the near-total conversion of CaM kinase II into a Ca2+/calmodulin-independent (autonomous) form characteristic of autophosphorylated, activated enzyme. On the removal of Ca2+, the activation state of CaM Kinase II rapidly returned to prestimulated levels. This reversal was slowed, but not prevented, by the inhibitors of protein phosphatase-1 (PP-1) and PP-2A, okadaic acid and calyculin A, and by the selective chelation of Mg2+ by the addition of EDTA. Near-complete prevention of enzyme deactivation, however, was observed in the combined presence of both okadaic acid and EDTA. Under these conditions, CaM kinase II phosphatase was more sensitive to calyculin A relative to okadaic acid, characteristic of the involvement of PP-1. CaM kinase II deactivation was not affected by FK-506, eliminating the involvement of PP-2B in this process. These data suggest that CaM kinase II dephosphorylation and deactivation in the pancreatic β-cell is mediated by the combined action of an okadaic-acid-sensitive phosphatase and a Mg2+-dependent phosphatase, such as PP-2C.

scholarly journals Regulation of endothelial cell barrier function by calcium/calmodulin-dependent protein kinase II

2001 ◽  
Vol 280 (5) ◽  
pp. L983-L990 ◽  
Author(s):  
Talaibek Borbiev ◽  
Alexander D. Verin ◽  
Shu Shi ◽  
Feng Liu ◽  
Joe G. N. Garcia
Keyword(s):  
Endothelial Cell ◽  
Protein Kinase ◽  
Phosphorylation Site ◽  
Cam Kinase Ii ◽  
Dependent Manner ◽  
Cam Kinase ◽  
Barrier Dysfunction ◽  
Dependent Protein Kinase ◽  
Protein Kinase Ii ◽  
Dependent Protein

Thrombin-induced endothelial cell barrier dysfunction is tightly linked to Ca2+-dependent cytoskeletal protein reorganization. In this study, we found that thrombin increased Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) activities in a Ca2+- and time-dependent manner in bovine pulmonary endothelium with maximal activity at 5 min. Pretreatment with KN-93, a specific CaM kinase II inhibitor, attenuated both thrombin-induced increases in monolayer permeability to albumin and decreases in transendothelial electrical resistance (TER). We next explored potential thrombin-induced CaM kinase II cytoskeletal targets and found that thrombin causes translocation and significant phosphorylation of nonmuscle filamin (ABP-280), which was attenuated by KN-93, whereas thrombin-induced myosin light chain phosphorylation was unaffected. Furthermore, a cell-permeable N-myristoylated synthetic filamin peptide (containing the COOH-terminal CaM kinase II phosphorylation site) attenuated both thrombin-induced filamin phosphorylation and decreases in TER. Together, these studies indicate that CaM kinase II activation and filamin phosphorylation may participate in thrombin-induced cytoskeletal reorganization and endothelial barrier dysfunction.

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