scholarly journals Phosphorylation and modulation of hyperpolarization-activated HCN4 channels by protein kinase A in the mouse sinoatrial node

2010 ◽  
Vol 136 (3) ◽  
pp. 247-258 ◽  
Author(s):  
Zhandi Liao ◽  
Dean Lockhead ◽  
Eric D. Larson ◽  
Catherine Proenza
Keyword(s):  
Heart Rate ◽  
Protein Kinase ◽  
Cyclic Nucleotide ◽  
Sinoatrial Node ◽  
Voltage Dependence ◽  
C Terminus ◽  
Sympathetic Regulation ◽  
Direct Binding ◽  
End Effectors

The sympathetic nervous system increases heart rate by activating β adrenergic receptors and increasing cAMP levels in myocytes in the sinoatrial node. The molecular basis for this response is not well understood; however, the cardiac funny current (If) is thought to be among the end effectors for cAMP signaling in sinoatrial myocytes. If is produced by hyperpolarization-activated cyclic nucleotide–sensitive (HCN4) channels, which can be potentiated by direct binding of cAMP to a conserved cyclic nucleotide binding domain in the C terminus of the channels. β adrenergic regulation of If in the sinoatrial node is thought to occur via this direct binding mechanism, independent of phosphorylation. Here, we have investigated whether the cAMP-activated protein kinase (PKA) can also regulate sinoatrial HCN4 channels. We found that inhibition of PKA significantly reduced the ability of β adrenergic agonists to shift the voltage dependence of If in isolated sinoatrial myocytes from mice. PKA also shifted the voltage dependence of activation to more positive potentials for heterologously expressed HCN4 channels. In vitro phosphorylation assays and mass spectrometry revealed that PKA can directly phosphorylate at least 13 sites on HCN4, including at least three residues in the N terminus and at least 10 in the C terminus. Functional analysis of truncated and alanine-substituted HCN4 channels identified a PKA regulatory site in the distal C terminus of HCN4, which is required for PKA modulation of If. Collectively, these data show that native and expressed HCN4 channels can be regulated by PKA, and raise the possibility that this mechanism could contribute to sympathetic regulation of heart rate.

scholarly journals Shenxian-Shengmai Oral Liquid Improves Sinoatrial Node Dysfunction through the PKC/NOX-2 Signaling Pathway

2021 ◽  
Vol 2021 ◽  
pp. 1-10
Author(s):  
Heng Zhang ◽  
Miao Hao ◽  
Lingkang Li ◽  
Keyan Chen ◽  
Jing Qi ◽  
...  
Keyword(s):  
Heart Rate ◽  
Signaling Pathway ◽  
Sinoatrial Node ◽  
Cardiac Conduction ◽  
Potential Mechanism ◽  
Cardiac Syncope ◽  
Common Causes ◽  
Oral Liquid ◽  
Masson Staining

Sick sinus syndrome (SSS) is one of the common causes of cardiac syncope and sudden death; the occurrence of SSS is associated with the accumulation of ROS in the sinoatrial node (SAN). Shenxian-shengmai (SXSM) is a traditional Chinese medicine available as oral liquid that causes a significant increase in heart rate. The objective of this study is to observe the improvement of SXSM on SAN function in SSS mice and explore its potential mechanism. In the current study, SSS was simulated in mice by inducing SAN dysfunction using a micro-osmotic pump to inject angiotensin II (Ang II). The mouse model with SSS was used to determine the effect of SXSM on SAN function and to explore its potential mechanism. Furthermore, the HL-1 cell line, derived from mouse atrial myocytes, was used to simulate SAN pacemaker cells. Our results indicated that SXSM significantly increased the heart rate of SSS mice by reducing the AngII-induced accumulation of ROS in the SAN and by inhibiting the expression of HDAC4, thereby reducing the loss of HCN4, a critical component of the cardiac conduction system. MASSON staining revealed a reduction of SAN damage in SSS mice that were treated with SXSM compared with controls. In vitro experiments showed that AngII treatment caused an upregulation of the PKC/NOX-2 signaling pathway in HL-1 cells which could be prevented by pretreatment with SXSM. The protective effect of SXSM was attenuated upon treatment with the PCK agonist PMA. In conclusion, SXSM reduced the AngII-induced accumulation of ROS in the SAN through the PKC/NOX2 signaling pathway, improving the functioning of the SAN and preventing the decrease of heart rate in SSS mice.

scholarly journals Functional Characterization of the Interaction of Ste50p with Ste11p MAPKKK inSaccharomyces cerevisiae

1999 ◽  
Vol 10 (7) ◽  
pp. 2425-2440 ◽  
Author(s):  
Cunle Wu ◽  
Ekkehard Leberer ◽  
David Y. Thomas ◽  
Malcolm Whiteway
Keyword(s):  
Protein Kinase ◽  
Functional Characterization ◽  
Hog Pathway ◽  
C Terminus ◽  
Extracellular Signal ◽  
Osmotic Stress Response ◽  
N Terminus

The Saccharomyces cerevisiae Ste11p protein kinase is a homologue of mammalian MAPK/extracellular signal-regulated protein kinase kinase kinases (MAPKKKs or MEKKs) as well as theSchizosaccharomyces pombe Byr2p kinase. Ste11p functions in several signaling pathways, including those for mating pheromone response and osmotic stress response. The Ste11p kinase has an N-terminal domain that interacts with other signaling molecules to regulate Ste11p function and direct its activity in these pathways. One of the Ste11p regulators is Ste50p, and Ste11p and Ste50p associate through their respective N-terminal domains. This interaction relieves a negative activity of the Ste11p N terminus, and removal of this negative function is required for Ste11p function in the high-osmolarity glycerol (HOG) pathway. The Ste50p/Ste11p interaction is also important (but not essential) for Ste11p function in the mating pathway; in this pathway binding of the Ste11p N terminus with both Ste50p and Ste5p is required, with the Ste5p association playing the major role in Ste11p function. In vitro, Ste50p disrupts an association between the catalytic C terminus and the regulatory N terminus of Ste11p. In addition, Ste50p appears to modulate Ste11p autophosphorylation and is itself a substrate of the Ste11p kinase. Therefore, both in vivo and in vitro data support a role for Ste50p in the regulation of Ste11p activity.

scholarly journals Phosphorylation of the c-Fos transrepression domain by mitogen-activated protein kinase and 90-kDa ribosomal S6 kinase

1993 ◽  
Vol 90 (23) ◽  
pp. 10952-10956 ◽  
Author(s):  
R H Chen ◽  
C Abate ◽  
J Blenis
Keyword(s):  
Protein Kinase ◽  
Map Kinase ◽  
Mitogen Activated Protein Kinase ◽  
S6 Kinase ◽  
Downstream Signaling ◽  
C Terminus ◽  
Mitogen Activated Protein ◽  
Ribosomal S6 Kinase

Phosphorylation of the C terminus of c-Fos has been implicated in serum response element-mediated repression of c-fos transcription after its induction by serum growth factors. The growth-regulated enzymes responsible for this phosphorylation in early G1 phase of the cell cycle and the sites of phosphorylation have not been identified. We now provide evidence that two growth-regulated, nucleus- and cytoplasm-localized protein kinases, 90-kDa ribosomal S6 kinase (RSK) and mitogen-activated protein kinase (MAP kinase), contribute to the serum-induced phosphorylation of c-Fos. The major phosphopeptides derived from biosynthetically labeled c-Fos correspond to phosphopeptides generated after phosphorylation of c-Fos in vitro with both RSK and MAP kinase. The phosphorylation sites identified for RSK (Ser-362) and MAP kinase (Ser-374) are in the transrepression domain. Cooperative phosphorylation at these sites by both enzymes was observed in vitro and reflected in vivo by the predominance of the peptide phosphorylated on both sites, as opposed to singly phosphorylated peptides. This study suggests a role for nuclear RSK and MAP kinase in modulating newly synthesized c-Fos phosphorylation and downstream signaling.

Angiotensin regulates the selectivity of the Na+-K+ pump for intracellular Na+

1999 ◽  
Vol 277 (3) ◽  
pp. C461-C468 ◽  
Author(s):  
Kerrie A. Buhagiar ◽  
Peter S. Hansen ◽  
David F. Gray ◽  
Anastasia S. Mihailidou ◽  
Helge H. Rasmussen
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Voltage Dependence ◽  
Surface Protein ◽  
Pump Current ◽  
Ang Ii ◽  
Kinase C ◽  
Cytoplasmic Surface ◽  
In Vitro Exposure

Treatment of rabbits with angiotensin-converting enzyme (ACE) inhibitors increases the apparent affinity of the Na+-K+pump for Na+. To explore the mechanism, we voltage clamped myocytes from control rabbits and rabbits treated with captopril with patch pipettes containing 10 mM Na+. When pipette solutions were K+ free, pump current ( I p) for myocytes from captopril-treated rabbits was nearly identical to that for myocytes from controls. However, treatment caused a significant increase in I pmeasured with pipettes containing K+. A similar difference was observed when myocytes from rabbits treated with the ANG II receptor antagonist losartan and myocytes from controls were compared. Treatment-induced differences in I p were eliminated by in vitro exposure to ANG II or phorbol 12-myristate 13-acetate or inclusion of the protein kinase C fragment composed of amino acids 530–558 in pipette solutions. Treatment with captopril had no effect on the voltage dependence of I p. We conclude that ANG II regulates the pump’s selectivity for intracellular Na+ at sites near the cytoplasmic surface. Protein kinase C is implicated in the messenger cascade.

Structure and transforming potential of the human cot oncogene encoding a putative protein kinase

1991 ◽  
Vol 11 (8) ◽  
pp. 4088-4096
Author(s):  
J Miyoshi ◽  
T Higashi ◽  
H Mukai ◽  
T Ohuchi ◽  
T Kakunaga
Keyword(s):  
Protein Kinase ◽  
Transcriptional Control ◽  
Thyroid Tumor ◽  
Kinase Assay ◽  
Human Thyroid ◽  
Protein Kinase Activity ◽  
Amino Acid Residues ◽  
Morphological Transformation ◽  
C Terminus

A new transforming gene has been molecularly cloned from hamster SHOK cells transformed with DNA extracted from a human thyroid carcinoma cell line and named the cot (cancer Osaka thyroid) oncogene. cDNA sequencing disclosed that this oncogene codes for a protein with 415 amino acid residues, and computer matching showed 42 to 48% similarity matches with serine protein kinases. Its gene product was identified as a 52-kDa protein by transcription and translation in vitro. Expression of cot cDNA under transcriptional control by a retroviral long terminal repeat induced morphological transformation of NIH 3T3 cells as well as SHOK cells. Protein kinase activity associated with constructed p60gag-cot was detected by immune complex kinase assay with anti-gag antiserum. The cot oncogene was overexpressed in transformed SHOK cells and found to have a rearranged 3' end in the last coding exon, which probably resulted in a deletion and an altered C' terminus in the transforming protein. This DNA rearrangement appeared to have occurred during transfection of the tumor DNA into hamster SHOK cells and not in the original thyroid tumor.

scholarly journals Dosage-dependent modulation of glucose repression by MSN3 (STD1) in Saccharomyces cerevisiae.

1994 ◽  
Vol 14 (3) ◽  
pp. 1972-1978 ◽  
Author(s):  
E J Hubbard ◽  
R Jiang ◽  
M Carlson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Kinase ◽  
Glucose Repression ◽  
Binding Studies ◽  
Glucose Limitation ◽  
Snf1 Kinase ◽  
Multicopy Suppressor ◽  
C Terminus ◽  
In The Wild

The SNF1 protein kinase of Saccharomyces cerevisiae is required to relieve glucose repression of transcription. To identify components of the SNF1 pathway, we isolated multicopy suppressors of defects caused by loss of SNF4, an activator of the SNF1 kinase. Increased dosage of the MSN3 gene restored invertase expression in snf4 mutants and also relieved glucose repression in the wild type. Deletion of MSN3 caused no substantial phenotype, and we identified a homolog, MTH1, encoding a protein 61% identical to MSN3. Both are also homologous to chicken fimbrin, human plastin, and yeast SAC6 over a 43-residue region. Deletion of MSN3 and MTH1 together impaired derepression of invertase in response to glucose limitation. Finally, MSN3 physically interacts with the SNF1 protein kinase, as assayed by a two-hybrid system and by in vitro binding studies. MSN3 is the same gene as STD1, a multicopy suppressor of defects caused by overexpression of the C terminus of TATA-binding protein (R. W. Ganster, W. Shen, and M. C. Schmidt, Mol. Cell. Biol. 13:3650-3659, 1993). Taken together, these data suggest that MSN3 modulates the regulatory response to glucose and may couple the SNF1 pathway to transcription.

scholarly journals NEK2A Interacts with MAD1 and Possibly Functions as a Novel Integrator of the Spindle Checkpoint Signaling

2004 ◽  
Vol 279 (19) ◽  
pp. 20049-20057 ◽  
Author(s):  
Yang Lou ◽  
Jianhui Yao ◽  
Arzhang Zereshki ◽  
Zhen Dou ◽  
Kashif Ahmed ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Chromosome Segregation ◽  
Leucine Zipper ◽  
Spindle Checkpoint ◽  
Mitotic Checkpoint ◽  
Checkpoint Signaling ◽  
C Terminus ◽  
Chromosome Bridge

Chromosome segregation in mitosis is orchestrated by protein kinase signaling cascades. A biochemical cascade named spindle checkpoint ensures the spatial and temporal order of chromosome segregation during mitosis. Here we report that spindle checkpoint protein MAD1 interacts with NEK2A, a human orthologue of theAspergillus nidulansNIMA kinase. MAD1 interacts with NEK2Ain vitroandin vivovia a leucine zipper-containing domain located at the C terminus of MAD1. Like MAD1, NEK2A is localized to HeLa cell kinetochore of mitotic cells. Elimination of NEK2A by small interfering RNA does not arrest cells in mitosis but causes aberrant premature chromosome segregation. NEK2A is required for MAD2 but not MAD1, BUB1, and HEC1 to associate with kinetochores. These NEK2A-eliminated or -suppressed cells display a chromosome bridge phenotype with sister chromatid inter-connected. Moreover, loss of NEK2A impairs mitotic checkpoint signaling in response to spindle damage by nocodazole, which affected mitotic escape and led to generation of cells with multiple nuclei. Our data demonstrate that NEK2A is a kinetochore-associated protein kinase essential for faithful chromosome segregation. We hypothesize that NEK2A links MAD2 molecular dynamics to spindle checkpoint signaling.

scholarly journals Structure and transforming potential of the human cot oncogene encoding a putative protein kinase.

1991 ◽  
Vol 11 (8) ◽  
pp. 4088-4096 ◽  
Author(s):  
J Miyoshi ◽  
T Higashi ◽  
H Mukai ◽  
T Ohuchi ◽  
T Kakunaga
Keyword(s):  
Protein Kinase ◽  
Transcriptional Control ◽  
Thyroid Tumor ◽  
Kinase Assay ◽  
Human Thyroid ◽  
Protein Kinase Activity ◽  
Amino Acid Residues ◽  
Morphological Transformation ◽  
C Terminus

A new transforming gene has been molecularly cloned from hamster SHOK cells transformed with DNA extracted from a human thyroid carcinoma cell line and named the cot (cancer Osaka thyroid) oncogene. cDNA sequencing disclosed that this oncogene codes for a protein with 415 amino acid residues, and computer matching showed 42 to 48% similarity matches with serine protein kinases. Its gene product was identified as a 52-kDa protein by transcription and translation in vitro. Expression of cot cDNA under transcriptional control by a retroviral long terminal repeat induced morphological transformation of NIH 3T3 cells as well as SHOK cells. Protein kinase activity associated with constructed p60gag-cot was detected by immune complex kinase assay with anti-gag antiserum. The cot oncogene was overexpressed in transformed SHOK cells and found to have a rearranged 3' end in the last coding exon, which probably resulted in a deletion and an altered C' terminus in the transforming protein. This DNA rearrangement appeared to have occurred during transfection of the tumor DNA into hamster SHOK cells and not in the original thyroid tumor.

scholarly journals Regulation of heart rate and the pacemaker current by phosphoinositide 3-kinase signaling

2019 ◽  
Vol 151 (8) ◽  
pp. 1051-1058 ◽  
Author(s):  
Richard Z. Lin ◽  
Zhongju Lu ◽  
Evgeny P. Anyukhovsky ◽  
Ya-Ping Jiang ◽  
Hong Zhan Wang ◽  
...  
Keyword(s):  
Heart Rate ◽  
Sinoatrial Node ◽  
Cardiac Pacing ◽  
Pacemaker Current ◽  
Physiological Conditions ◽  
Diastolic Depolarization ◽  
Important Regulator ◽  
Phosphoinositide 3 Kinase

Heart rate in physiological conditions is set by the sinoatrial node (SN), the primary cardiac pacing tissue. Phosphoinositide 3-kinase (PI3K) signaling is a major regulatory pathway in all normal cells, and its dysregulation is prominent in diabetes, cancer, and heart failure. Here, we show that inhibition of PI3K slows the pacing rate of the SN in situ and in vitro and reduces the early slope of diastolic depolarization. Furthermore, inhibition of PI3K causes a negative shift in the voltage dependence of activation of the pacemaker current, IF, while addition of its second messenger, phosphatidylinositol 3,4,5-trisphosphate, induces a positive shift. These shifts in the activation of IF are independent of, and larger than, those induced by the autonomic nervous system. These results suggest that PI3K is an important regulator of heart rate, and perturbations in this signaling pathway may contribute to the development of arrhythmias.

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