scholarly journals Regulation of APC Activity by Phosphorylation and Regulatory Factors

1999 ◽  
Vol 146 (4) ◽  
pp. 791-800 ◽  
Author(s):  
Shuji Kotani ◽  
Hirofumi Tanaka ◽  
Hideyo Yasuda ◽  
Kazuo Todokoro
Keyword(s):  
Protein Kinase ◽  
Sister Chromatid ◽  
Regulatory Mechanism ◽  
Cyclin B ◽  
G1 Phase ◽  
Anaphase Promoting Complex ◽  
Dependent Protein Kinase ◽  
Regulatory Factors ◽  
Dependent Protein ◽  
The Right

Ubiquitin-dependent proteolysis of Cut2/Pds1 and Cyclin B is required for sister chromatid separation and exit from mitosis, respectively. Anaphase-promoting complex/cyclosome (APC) specifically ubiquitinates Cut2/Pds1 at metaphase–anaphase transition, and ubiquitinates Cyclin B in late mitosis and G1 phase. However, the exact regulatory mechanism of substrate-specific activation of mammalian APC with the right timing remains to be elucidated. We found that not only the binding of the activators Cdc20 and Cdh1 and the inhibitor Mad2 to APC, but also the phosphorylation of Cdc20 and Cdh1 by Cdc2-Cyclin B and that of APC by Polo-like kinase and cAMP-dependent protein kinase, regulate APC activity. The cooperation of the phosphorylation/dephosphorylation and the regulatory factors in regulation of APC activity may thus control the precise progression of mitosis.

scholarly journals Blockade of CaMKII depresses conduction preferentially in the right ventricular outflow tract and promotes ischemic ventricular fibrillation in the rabbit heart

2017 ◽  
Vol 312 (4) ◽  
pp. H752-H767 ◽  
Author(s):  
Mark Warren ◽  
Katie J. Sciuto ◽  
Tyson G. Taylor ◽  
Vivek Garg ◽  
Natalia S. Torres ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Ventricular Fibrillation ◽  
Right Ventricular ◽  
Right Ventricular Outflow Tract ◽  
Ventricular Outflow Tract ◽  
Outflow Tract ◽  
Dependent Protein Kinase ◽  
Calcium Calmodulin Dependent ◽  
Dependent Protein ◽  
The Right

Calcium/calmodulin-dependent protein kinase II (CaMKII) regulates the principle ion channels mediating cardiac excitability and conduction, but how this regulation translates to the normal and ischemic heart remains unknown. Diverging results on CaMKII regulation of Na+ channels further prevent predicting how CaMKII activity regulates excitability and conduction in the intact heart. To address this deficiency, we tested the effects of the CaMKII blocker KN93 (1 and 2.75 μM) and its inactive analog KN92 (2.75 μM) on conduction and excitability in the left (LV) and right (RV) ventricles of rabbit hearts during normal perfusion and global ischemia. We used optical mapping to determine local conduction delays and the optical action potential (OAP) upstroke velocity (d V/d tmax). At baseline, local conduction delays were similar between RV and LV, whereas the OAP d V/d tmax was lower in RV than in LV. At 2.75 μM, KN93 heterogeneously slowed conduction and reduced d V/d tmax, with the largest effect in the RV outflow tract (RVOT). This effect was further exacerbated by ischemia, leading to recurrent conduction block in the RVOT and early ventricular fibrillation (at 6.7 ± 0.9 vs. 18.2 ± 0.8 min of ischemia in control, P < 0.0001). Neither KN92 nor 1 μM KN93 depressed OAP d V/d tmax or conduction. Rabbit cardiomyocytes isolated from RVOT exhibited a significantly lower d V/d tmax than those isolated from the LV. KN93 (2.75 μM) significantly reduced d V/d tmax in cells from both locations. This led to frequency-dependent intermittent activation failure occurring predominantly in RVOT cells. Thus CaMKII blockade exacerbates intrinsically lower excitability in the RVOT, which is proarrhythmic during ischemia. NEW & NOTEWORTHY We show that calcium/calmodulin-dependent protein kinase II (CaMKII) blockade exacerbates intrinsically lower excitability in the right ventricular outflow tract, which causes highly nonuniform chamber-specific slowing of conduction and facilitates ventricular fibrillation during ischemia. Constitutive CaMKII activity is necessary for uniform and safe ventricular conduction, and CaMKII block is potentially proarrhythmic.

scholarly journals Analysis of BHK cell growth kinetics after microinjection of catalytic subunit of cyclic AMP-dependent protein kinase.

1984 ◽  
Vol 4 (6) ◽  
pp. 1079-1085 ◽  
Author(s):  
J K McClung ◽  
R F Kletzien
Keyword(s):  
Protein Kinase ◽  
Cell Growth ◽  
Cyclic Amp ◽  
Growth Kinetics ◽  
S Phase ◽  
G1 Phase ◽  
Dependent Protein Kinase ◽  
Bhk Cells ◽  
Dependent Protein ◽  
Cell Growth Kinetics

The effect of catalytic subunit (C) of cyclic AMP-dependent protein kinase on cell growth kinetics of BHK cells was assessed by microinjection with chicken erythrocyte ghosts as vehicles for introduction of the protein into the cytosol of large populations of cells. The advantage in using chicken erythrocytes for microinjection is that the inactive erythrocyte nuclei serve as a probe for identifying and analyzing microinjection events. By utilizing this procedure, BHK cells were microinjected with an amount of C that was 5- to 10-fold greater than their endogenous levels. Growth kinetics were analyzed by [3H]thymidine incorporation and autoradiography. Cells were stained after autoradiography to more clearly reveal the chicken nuclei, and at each time point, cells were categorized into four groups: (i) not microinjected, not in S phase, (ii) not microinjected, in S phase, (iii) microinjected, not in S phase, (iv) microinjected, in S phase. Those cells not microinjected served as internal controls. Two experimental protocols were used to test the notion that C is involved in blocking cell progression through G1 phase of the cell cycle. First, cells were arrested in G0 phase by serum deprivation, microinjected with C or control proteins, and stimulated to proceed to S phase by the addition of serum or purified growth factors. Second, cells were collected in mitosis, microinjected with C or control proteins, and stimulated to proceed to S phase by the addition of serum. The results of these studies indicate that a 5- to 10-fold increase in the intracellular concentration of C is not a sufficient signal to arrest cell growth in G1 phase. Thus, growth-inhibitory effects of cyclic AMP on BHK cells are unlikely to be the result of activation of cyclic AMP-dependent protein kinase.

scholarly journals Conduction in the right and left ventricle is differentially regulated by protein kinases and phosphatases: implications for arrhythmogenesis

2019 ◽  
Vol 316 (6) ◽  
pp. H1507-H1527 ◽  
Author(s):  
Alexey V. Zaitsev ◽  
Natalia S. Torres ◽  
Keiko M. Cawley ◽  
Amira D. Sabry ◽  
Junco S. Warren ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Dependent Manner ◽  
Dependent Protein Kinase ◽  
Specific Expression ◽  
Stress Kinases ◽  
Calcium Calmodulin Dependent ◽  
Protein Kinase Ii ◽  
Dependent Protein ◽  
The Right ◽  
Ventricular Conduction

The “stress” kinases cAMP-dependent protein kinase (PKA) and calcium/calmodulin-dependent protein kinase II (CaMKII), phosphorylate the Na+ channel Nav1.5 subunit to regulate its function. However, how the channel regulation translates to ventricular conduction is poorly understood. We hypothesized that the stress kinases positively and differentially regulate conduction in the right (RV) and the left (LV) ventricles. We applied the CaMKII blocker KN93 (2.75 μM), PKA blocker H89 (10 μM), and broad-acting phosphatase blocker calyculin (30 nM) in rabbit hearts paced at a cycle length (CL) of 150-8,000 ms. We used optical mapping to determine the distribution of local conduction delays (inverse of conduction velocity). Control hearts exhibited constant and uniform conduction at all tested CLs. Calyculin (15-min perfusion) accelerated conduction, with greater effect in the RV (by 15.3%) than in the LV (by 4.1%; P < 0.05). In contrast, both KN93 and H89 slowed down conduction in a chamber-, time-, and CL-dependent manner, with the strongest effect in the RV outflow tract (RVOT). Combined KN93 and H89 synergistically promoted conduction slowing in the RV (KN93: 24.7%; H89: 29.9%; and KN93 + H89: 114.2%; P = 0.0016) but not the LV. The progressive depression of RV conduction led to conduction block and reentrant arrhythmias. Protein expression levels of both the CaMKII-δ isoform and the PKA catalytic subunit were higher in the RVOT than in the apical LV ( P < 0.05). Thus normal RV conduction requires a proper balance between kinase and phosphatase activity. Dysregulation of this balance due to pharmacological interventions or disease is potentially proarrhythmic. NEW & NOTEWORTHY We show that uniform ventricular conduction requires a precise physiological balance of the activities of calcium/calmodulin-dependent protein kinase II (CaMKII), PKA, and phosphatases, which involves region-specific expression of CaMKII and PKA. Inhibiting CaMKII and/or PKA activity elicits nonuniform conduction depression, with the right ventricle becoming vulnerable to the development of conduction disturbances and ventricular fibrillation/ventricular tachycardia.

Analysis of BHK cell growth kinetics after microinjection of catalytic subunit of cyclic AMP-dependent protein kinase

1984 ◽  
Vol 4 (6) ◽  
pp. 1079-1085
Author(s):  
J K McClung ◽  
R F Kletzien
Keyword(s):  
Protein Kinase ◽  
Cell Growth ◽  
Cyclic Amp ◽  
Growth Kinetics ◽  
S Phase ◽  
G1 Phase ◽  
Dependent Protein Kinase ◽  
Bhk Cells ◽  
Dependent Protein ◽  
Cell Growth Kinetics

The effect of catalytic subunit (C) of cyclic AMP-dependent protein kinase on cell growth kinetics of BHK cells was assessed by microinjection with chicken erythrocyte ghosts as vehicles for introduction of the protein into the cytosol of large populations of cells. The advantage in using chicken erythrocytes for microinjection is that the inactive erythrocyte nuclei serve as a probe for identifying and analyzing microinjection events. By utilizing this procedure, BHK cells were microinjected with an amount of C that was 5- to 10-fold greater than their endogenous levels. Growth kinetics were analyzed by [3H]thymidine incorporation and autoradiography. Cells were stained after autoradiography to more clearly reveal the chicken nuclei, and at each time point, cells were categorized into four groups: (i) not microinjected, not in S phase, (ii) not microinjected, in S phase, (iii) microinjected, not in S phase, (iv) microinjected, in S phase. Those cells not microinjected served as internal controls. Two experimental protocols were used to test the notion that C is involved in blocking cell progression through G1 phase of the cell cycle. First, cells were arrested in G0 phase by serum deprivation, microinjected with C or control proteins, and stimulated to proceed to S phase by the addition of serum or purified growth factors. Second, cells were collected in mitosis, microinjected with C or control proteins, and stimulated to proceed to S phase by the addition of serum. The results of these studies indicate that a 5- to 10-fold increase in the intracellular concentration of C is not a sufficient signal to arrest cell growth in G1 phase. Thus, growth-inhibitory effects of cyclic AMP on BHK cells are unlikely to be the result of activation of cyclic AMP-dependent protein kinase.

scholarly journals Cdc2 and Mos Regulate Emi2 Stability to Promote the Meiosis I–Meiosis II Transition

2008 ◽  
Vol 19 (8) ◽  
pp. 3536-3543 ◽  
Author(s):  
Wanli Tang ◽  
Judy Qiju Wu ◽  
Yanxiang Guo ◽  
David V. Hansen ◽  
Jennifer A. Perry ◽  
...  
Keyword(s):  
Kinase Activity ◽  
Cyclin B ◽  
Anaphase Promoting Complex ◽  
Cdc2 Kinase ◽  
Dependent Protein Kinase ◽  
Dependent Protein ◽  
Cytostatic Factor ◽  
Cdc2 Activity ◽  
Phase Entry ◽  
Meiosis I

The transition of oocytes from meiosis I (MI) to meiosis II (MII) requires partial cyclin B degradation to allow MI exit without S phase entry. Rapid reaccumulation of cyclin B allows direct progression into MII, producing a cytostatic factor (CSF)-arrested egg. It has been reported that dampened translation of the anaphase-promoting complex (APC) inhibitor Emi2 at MI allows partial APC activation and MI exit. We have detected active Emi2 translation at MI and show that Emi2 levels in MI are mainly controlled by regulated degradation. Emi2 degradation in MI depends not on Ca2+/calmodulin-dependent protein kinase II (CaMKII), but on Cdc2-mediated phosphorylation of multiple sites within Emi2. As in MII, this phosphorylation is antagonized by Mos-mediated recruitment of PP2A to Emi2. Higher Cdc2 kinase activity in MI than MII allows sufficient Emi2 phosphorylation to destabilize Emi2 in MI. At MI anaphase, APC-mediated degradation of cyclin B decreases Cdc2 activity, enabling Cdc2-mediated Emi2 phosphorylation to be successfully antagonized by Mos-mediated PP2A recruitment. These data suggest a model of APC autoinhibition mediated by stabilization of Emi2; Emi2 proteins accumulate at MI exit and inhibit APC activity sufficiently to prevent complete degradation of cyclin B, allowing MI exit while preventing interphase before MII entry.

scholarly journals Regulatory Mechanism of Ca2+/Calmodulin-dependent Protein Kinase Kinase

2000 ◽  
Vol 275 (26) ◽  
pp. 20090-20095 ◽  
Author(s):  
Hiroshi Tokumitsu ◽  
Masa-aki Muramatsu ◽  
Mitsuhiko Ikura ◽  
Ryoji Kobayashi
Keyword(s):  
Protein Kinase ◽  
Regulatory Mechanism ◽  
Dependent Protein Kinase ◽  
Dependent Protein ◽  
Protein Kinase Kinase

scholarly journals Nocodazole-Induced Expression and Phosphorylation of Anillin and Other Mitotic Proteins Are Decreased in DNA-Dependent Protein Kinase Catalytic Subunit-Deficient Cells and Rescued by Inhibition of the Anaphase-Promoting Complex/Cyclosome with proTAME but Not Apcin

2020 ◽  
Vol 40 (13) ◽  
Author(s):  
Pauline Douglas ◽  
Ruiqiong Ye ◽  
Suraj Radhamani ◽  
Alexander Cobban ◽  
Nicole P. Jenkins ◽  
...  
Keyword(s):  
Protein Kinase ◽  
A549 Cells ◽  
Cyclin B1 ◽  
Strand Break ◽  
Catalytic Subunit ◽  
Aurora A ◽  
Anaphase Promoting Complex ◽  
Dependent Protein Kinase ◽  
Induced Expression ◽  
Dependent Protein

ABSTRACT The DNA-dependent protein kinase catalytic subunit (DNA-PKcs) has well-established roles in DNA double-strand break repair, and recently, nonrepair functions have also been reported. To better understand its cellular functions, we deleted DNA-PKcs from HeLa and A549 cells using CRISPR/Cas9. The resulting cells were radiation sensitive, had reduced expression of ataxia-telangiectasia mutated (ATM), and exhibited multiple mitotic defects. Mechanistically, nocodazole-induced upregulation of cyclin B1, anillin, and securin was decreased in DNA-PKcs-deficient cells, as were phosphorylation of Aurora A on threonine 288, phosphorylation of Polo-like kinase 1 (PLK1) on threonine 210, and phosphorylation of targeting protein for Xenopus Klp2 (TPX2) on serine 121. Moreover, reduced nocodazole-induced expression of anillin, securin, and cyclin B1 and phosphorylation of PLK1, Aurora A, and TPX2 were rescued by inhibition of the anaphase-promoting complex/cyclosome (APC/C) by proTAME, which prevents binding of the APC/C-activating proteins Cdc20 and Cdh1 to the APC/C. Altogether, our studies suggest that loss of DNA-PKcs prevents inactivation of the APC/C in nocodazole-treated cells.

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