scholarly journals Cardiac-specific phosphorylation site for multifunctional Ca2+/calmodulin-dependent protein kinase is conserved in the brain ryanodine receptor.

1992 ◽  
Vol 267 (7) ◽  
pp. 4963-4967
Author(s):  
D.R. Witcher ◽  
B.A. Strifler ◽  
L.R. Jones
Keyword(s):  
Protein Kinase ◽  
Ryanodine Receptor ◽  
Phosphorylation Site ◽  
Dependent Protein Kinase ◽  
Dependent Protein ◽  
The Brain

scholarly journals Phosphorylation of septin 3 on Ser-91 by cGMP-dependent protein kinase-I in nerve terminals

2004 ◽  
Vol 381 (3) ◽  
pp. 753-760 ◽  
Author(s):  
Jing XUE ◽  
Peter J. MILBURN ◽  
Bernadette T. HANNA ◽  
Mark E. GRAHAM ◽  
John A. P. ROSTAS ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Phosphorylation Site ◽  
Nerve Terminals ◽  
Dependent Protein Kinase ◽  
Cgmp Dependent Protein Kinase ◽  
Dependent Protein ◽  
Phosphoamino Acid Analysis ◽  
The Brain ◽  
Major Phosphorylation Site

The septins are a family of GTPase enzymes required for cytokinesis and play a role in exocytosis. Among the ten vertebrate septins, Sept5 (CDCrel-1) and Sept3 (G-septin) are primarily concentrated in the brain, wherein Sept3 is a substrate for PKG-I (cGMP-dependent protein kinase-I) in nerve terminals. There are two motifs for potential PKG-I phosphorylation in Sept3, Thr-55 and Ser-91, but phosphoamino acid analysis revealed that the primary site is a serine. Derivatization of phosphoserine to S-propylcysteine followed by N-terminal sequence analysis revealed Ser-91 as a major phosphorylation site. Tandem MS revealed a single phosphorylation site at Ser-91. Substitution of Ser-91 with Ala in a synthetic peptide abolished phosphorylation. Mutation of Ser-91 to Ala in recombinant Sept3 also abolished PKG phosphorylation, confirming that Ser-91 is the major site in vitro. Antibodies raised against a peptide containing phospho-Ser-91 detected phospho-Sept3 only in the cytosol of nerve terminals, whereas Sept3 was located in a peripheral membrane extract. Therefore Sept3 is phosphorylated on Ser-91 in nerve terminals and its phosphorylation may contribute to the regulation of its subcellular localization in neurons.

scholarly journals LKB1, a novel serine/threonine protein kinase and potential tumour suppressor, is phosphorylated by cAMP-dependent protein kinase (PKA) and prenylated in vivo

2000 ◽  
Vol 345 (3) ◽  
pp. 673-680 ◽  
Author(s):  
Sean P. COLLINS ◽  
Junewai L. REOMA ◽  
David M. GAMM ◽  
Michael D. UHLER
Keyword(s):  
Protein Kinase ◽  
Cellular Localization ◽  
Fluorescent Protein ◽  
Phosphorylation Site ◽  
Multiple Cancer ◽  
Dependent Protein Kinase ◽  
Rnase Protection ◽  
Camp Dependent Protein Kinase ◽  
Increased Risk ◽  
Dependent Protein

Peutz-Jeghers syndrome (PJS) is an autosomal dominant disease characterized by melanocytic macules, hamartomatous polyps and an increased risk for numerous cancers. The human LKB1 (hLKB1) gene encodes a serine/threonine protein kinase that is deficient in the majority of patients with PJS. The murine LKB1 (mLKB1) cDNA was isolated, sequenced and shown to produce a 2.4-kb transcript encoding a 436 amino acid protein with 90% identity with hLKB1. RNA blot and RNase-protection analysis revealed that mLKB1 mRNA is expressed in all tissues and cell lines examined. The widespread expression of LKB1 transcripts is consistent with the elevated risk of multiple cancer types in PJS patients. The predicted LKB1 protein sequence terminates with a conserved prenylation motif (Cys433-Lys-Gln-Gln436) directly downstream from a consensus cAMP-dependent protein kinase (PKA) phosphorylation site (Arg428-Arg-Leu-Ser431). The expression of enhanced green fluorescent protein (EGFP)-mLKB1 chimaeras demonstrated that LKB1 possesses a functional prenylation motif that is capable of targeting EGFP to cellular membranes. Mutation of Cys433 to an alanine residue, but not phosphorylation by PKA, blocked membrane localization. These findings suggest that PKA does phosphorylate LKB1, although this phosphorylation does not alter the cellular localization of LKB1.

scholarly journals Activation State-Dependent Substrate Gating in Ca2+/Calmodulin-Dependent Protein Kinase II

2017 ◽  
Vol 2017 ◽  
pp. 1-13 ◽  
Author(s):  
D. E. Johnson ◽  
A. Hudmon
Keyword(s):  
Protein Kinase ◽  
Catalytic Domain ◽  
Intrinsic Property ◽  
Dependent Protein Kinase ◽  
Calcium Calmodulin Dependent ◽  
State Dependent ◽  
Protein Kinase Ii ◽  
Dependent Protein ◽  
Substrate Phosphorylation ◽  
The Brain

Calcium/calmodulin-dependent protein kinase II (CaMKII) is highly concentrated in the brain where its activation by the Ca2+sensor CaM, multivalent structure, and complex autoregulatory features make it an ideal translator of Ca2+signals created by different patterns of neuronal activity. We provide direct evidence that graded levels of kinase activity and extent of T287(T286αisoform) autophosphorylation drive changes in catalytic output and substrate selectivity. The catalytic domains of CaMKII phosphorylate purified PSDs much more effectively when tethered together in the holoenzyme versus individual subunits. Using multisubstrate SPOT arrays, high-affinity substrates are preferentially phosphorylated with limited subunit activity per holoenzyme, whereas multiple subunits or maximal subunit activation is required for intermediate- and low-affinity, weak substrates, respectively. Using a monomeric form of CaMKII to control T287autophosphorylation, we demonstrate that increased Ca2+/CaM-dependent activity for all substrates tested, with the extent of weak, low-affinity substrate phosphorylation governed by the extent of T287autophosphorylation. Our data suggest T287autophosphorylation regulates substrate gating, an intrinsic property of the catalytic domain, which is amplified within the multivalent architecture of the CaMKII holoenzyme.

scholarly journals The substrate specificity of adenosine 3′:5′-cyclic monophosphate-dependent protein kinase of rabbit skeletal muscle

1977 ◽  
Vol 162 (2) ◽  
pp. 411-421 ◽  
Author(s):  
S J Yeaman ◽  
P Cohen ◽  
D C Watson ◽  
G H Dixon
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Protein Kinase ◽  
Phosphorylation Site ◽  
Amino Acid Sequences ◽  
Beta Subunit ◽  
Alpha Subunit ◽  
Phosphorylase Kinase ◽  
Dependent Protein Kinase ◽  
Dependent Protein

The known amino acid sequences at the two sites on phosphorylase kinase that are phosphorylated by cyclic AMP-dependent protein kinase were extended. The sequences of 42 amino acids around the phosphorylation site on the alpha-subunit and of 14 amino acids around the phosphorylation site on the beta-subunit were shown to be: alpha-subunit Phe-Arg-Arg-Leu-Ser(P)-Ile-Ser-Thr-Glu-Ser-Glx-Pro-Asx-Gly-Gly-His-Ser-Leu-Gly-Ala-Asp-Leu-Met-Ser-Pro-Ser-Phe-Leu-Ser-Pro-Gly-Thr-Ser-Val-Phe(Ser,Pro,Gly)His-Thr-Ser-Lys; beta-subunit, Ala-Arg-Thr-Lys-Arg-Ser-Gly-Ser(P)-VALIle-Tyr-Glu-Pro-Leu-Lys. The sites on histone H2B which are phosphorylated by cyclic AMP-dependent protein kinase in vitro were identified as serine-36 and serine-32. The amino acid sequence in this region is: Lys-Lys-Arg-Lys-Arg-Ser32(P)-Arg-Lys-Glu-Ser36(P)-Tyr-Ser-Val-Tyr-Val- [Iwai, K., Ishikawa, K. & Hayashi, H. (1970) Nature (London) 226, 1056-1058]. Serine-36 was phosphorylated at 50% of the rate at which the beta-subunit of phosphorylase kinase was phosphorylated, and it was phosphorylated 6-7-fold more rapidly than was serine-32. The amino acid sequences when compared with those at the phosphorylation sites of other physiological substrates suggest that the presence of two adjacent basic amino acids on the N-terminal side of the susceptible serine residue may be critical for specific substrate recognition in vivo.

scholarly journals Ca2+/calmodulin-dependent kinase II-dependent regulation of atrial myocyte late Na+ current, Ca2+ cycling, and excitability: a mathematical modeling study

2017 ◽  
Vol 313 (6) ◽  
pp. H1227-H1239 ◽  
Author(s):  
Birce Onal ◽  
Daniel Gratz ◽  
Thomas J. Hund
Keyword(s):  
Protein Kinase ◽  
Computational Model ◽  
Ryanodine Receptor ◽  
The United States ◽  
Atrial Myocytes ◽  
Open Probability ◽  
Dependent Protein Kinase ◽  
Protein Kinase Ii ◽  
Dependent Protein ◽  
Atrial Myocyte

Atrial fibrillation (AF) affects more than three million people per year in the United States and is associated with high morbidity and mortality. Both electrical and structural remodeling contribute to AF, but the molecular pathways underlying AF pathogenesis are not well understood. Recently, a role for Ca2+/calmodulin-dependent protein kinase II (CaMKII) in the regulation of persistent “late” Na+ current ( INa,L) has been identified. Although INa,L inhibition is emerging as a potential antiarrhythmic strategy in patients with AF, little is known about the mechanism linking INa,L to atrial arrhythmogenesis. A computational approach was used to test the hypothesis that increased CaMKII-activated INa,L in atrial myocytes disrupts Ca2+ homeostasis, promoting arrhythmogenic afterdepolarizations. Dynamic CaMKII activity and regulation of multiple downstream targets [ INa,L, L-type Ca2+ current, phospholamban, and the ryanodine receptor sarcoplasmic reticulum Ca2+-release channel (RyR2)] were incorporated into an existing well-validated computational model of the human atrial action potential. Model simulations showed that constitutive CaMKII-dependent phosphorylation of Nav1.5 and the subsequent increase in INa,L effectively disrupt intracellular atrial myocyte ion homeostasis and CaMKII signaling. Specifically, increased INa,L promotes intracellular Ca2+ overload via forward-mode Na+/Ca2+ exchange activity, which greatly increases RyR2 open probability beyond that observed for CaMKII-dependent phosphorylation of RyR2 alone. Increased INa,L promotes atrial myocyte repolarization defects (afterdepolarizations and alternans) in the setting of acute β-adrenergic stimulation. We anticipate that our modeling efforts will help identify new mechanisms for atrial NaV1.5 regulation with direct relevance for human AF. NEW & NOTEWORTHY Here, we present a novel computational model to study the effects of late Na+ current ( INa,L) in human atrial myocytes. Simulations predict that INa,L promotes intracellular accumulation of Ca2+, with subsequent dysregulation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) signaling and ryanodine receptor 2-mediated Ca2+ release. Although INa,L plays a small role in regulating atrial myocyte excitability at baseline, CaMKII-dependent enhancement of the current promoted arrhythmogenic dynamics. Listen to this article’s corresponding podcast at http://ajpheart.podbean.com/e/camkii-dependent-regulation-of-atrial-late-sodium-current-and-excitability/ .

scholarly journals Engineering a bioluminescent indicator for cyclic AMP-dependent protein kinase

1991 ◽  
Vol 279 (3) ◽  
pp. 727-732 ◽  
Author(s):  
G B Sala-Newby ◽  
A K Campbell
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Specific Activity ◽  
Phosphorylation Site ◽  
Live Cells ◽  
Phosphorylation Sites ◽  
Dependent Protein Kinase ◽  
Ph Optimum ◽  
Dependent Protein

cDNA coding for the luciferase in the firefly Photinus pyralis was amplified in vitro to generate cyclic AMP-dependent protein kinase phosphorylation sites. The DNA was transcribed and translated to generate light-emitting protein. A valine at position 217 was mutated to arginine to generate a site RRFS and the heptapeptide kemptide, the phosphorylation site of the porcine pyruvate kinase, was added at the N- or C-terminus of the luciferase. The proteins carrying phosphorylation sites were characterized for their specific activity, pI, effect of pH on the colour of the light emitted and effect of the catalytic subunit of protein kinase A in the presence of ATP. Only one of the recombinant proteins (RRFS) was significantly different from wild-type luciferase. The RRFS mutant had a lower specific activity, lower pH optimum, emitted greener light at low pH and when phosphorylated it decreased its activity by up to 80%. This latter effect was reversed by phosphatase. This recombinant protein is a good candidate to measure for the first time cyclic AMP-dependent phosphorylation in live cells.

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