Radiolabelling of bovine myristoylated alanine-rich protein kinase C substrate (MARCKS) in an ADP-ribosylation reaction

1994 ◽  
Vol 72 (9-10) ◽  
pp. 391-396 ◽  
Author(s):  
D. Chao ◽  
D. L. Severson ◽  
H. Zwiers ◽  
M. D. Hollenberg
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Brain Homogenate ◽  
Bovine Brain ◽  
Two Dimensional Gel Electrophoresis ◽  
Adp Ribosylation ◽  
Substrate Protein ◽  
Kinase C ◽  
Neuronal Proteins ◽  
Functional Consequences

In an ADP-ribosylation reaction, we have observed the radiolabelling of a protein in a crude bovine brain homogenate, which upon two-dimensional gel electrophoresis migrated with an acidic pI (< 4.5) and an apparent molecular mass (80–90 kDa) consistent with the properties of the myristoylated, alanine-rich, protein kinase C substrate protein termed MARCKS. To establish the identity of this radiolabelled constituent in brain homogenates, we first purified bovine brain MARCKS using calmodulin-Sepharose affinity chromatography and we then supplemented the crude ADP-ribosylation reaction mixture with this purified MARCKS fraction. Concordant increases in radiolabelling and silver staining of the same protein component from the MARCKS-supplemented ADP-ribosylation reaction, as compared with the ADP-ribosylated crude homogenate, established the identity of this constituent as MARCKS. The radiolabelling of MARCKS was lower in comparison with the ADP-ribosylation of the related neuronal protein B-50/GAP-43 under identical reaction conditions. The potential functional consequences of the ADP-ribosylation of MARCKS are discussed and the possibility is raised that other members of the MARCKS family, such as the F52/MacMARCKS/MRP protein, may also be subject to ADP-ribosylation.Key words: MARCKS, ADP-ribosylation, neuronal proteins, B-50/GAP-43, kinase C, calmodulin.

scholarly journals Phosphoinositide-dependent protein kinase-1 (PDK1)-independent activation of the protein kinase C substrate, protein kinase D

FEBS Letters ◽  
2007 ◽  
Vol 581 (18) ◽  
pp. 3494-3498 ◽  
Author(s):  
C. David Wood ◽  
April P. Kelly ◽  
Sharon A. Matthews ◽  
Doreen A. Cantrell
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Protein Kinase D ◽  
Dependent Protein Kinase ◽  
Substrate Protein ◽  
Kinase C ◽  
Dependent Protein

scholarly journals Ca2+-binding proteins from bovine brain including a potent inhibitor of protein kinase C

1985 ◽  
Vol 232 (2) ◽  
pp. 559-567 ◽  
Author(s):  
J R McDonald ◽  
M P Walsh
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Large Scale ◽  
Binding Proteins ◽  
Potent Inhibitor ◽  
Binding Protein ◽  
Sodium Dodecyl ◽  
Bovine Brain ◽  
Heat Stable ◽  
Kinase C

We have previously described the use of Ca2+-dependent hydrophobic-interaction chromatography to isolate the Ca2+ + phospholipid-dependent protein kinase (protein kinase C) and a novel heat-stable 21 000-Mr Ca2+-binding protein from bovine brain [Walsh, Valentine, Ngai, Carruthers & Hollenberg (1984) Biochem. J. 224, 117-127]. The procedure described for purification of the 21 000-Mr calciprotein to electrophoretic homogeneity has been modified to permit the large-scale isolation of this Ca2+-binding protein, enabling further structural and functional characterization. The 21 000-Mr calciprotein was shown by equilibrium dialysis to bind approx. 1 mol of Ca2+/mol, with apparent Kd approx. 1 microM. The modified large-scale purification procedure revealed three additional, previously unidentified, Ca2+-binding proteins of Mr 17 000, 18 400 and 26 000. The 17 000-Mr and 18 400-Mr Ca2+-binding proteins are heat-stable, whereas the 26 000-Mr Ca2+-binding protein is heat-labile. Use of the transblot/45CaCl2 overlay technique [Maruyama, Mikawa & Ebashi (1984) J. Biochem. (Tokyo) 95, 511-519] suggests that the 18 400-Mr and 21 000-Mr Ca2+-binding proteins are high-affinity Ca2+-binding proteins, whereas the 17 000-Mr Ca2+-binding protein has a relatively low affinity for Ca2+. Consistent with this observation, the 18 400-Mr and 21 000-Mr Ca2+-binding proteins exhibit a Ca2+-dependent mobility shift on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, whereas the 17 000-Mr Ca2+-binding protein does not. The amino acid compositions of the 17 000-Mr, 18 400-Mr and 21 000-Mr Ca2+-binding proteins show some similarities to each other and to calmodulin and other members of the calmodulin superfamily; however, they are clearly distinct and novel calciproteins. In functional terms, none of the 17 000-Mr, 18 400-Mr or 21 000-Mr Ca2+-binding proteins activates either cyclic nucleotide phosphodiesterase or myosin light-chain kinase, both calmodulin-activated enzymes. However, the 17 000-Mr Ca2+-binding protein is a potent inhibitor of protein kinase C. It may therefore serve to regulate the activity of this important enzyme at elevated cytosolic Ca2+ concentrations.

scholarly journals Localization of non-conventional protein kinase C isoforms in bovine brain cell nuclei

1995 ◽  
Vol 305 (1) ◽  
pp. 269-275 ◽  
Author(s):  
U Rosenberger ◽  
M Shakibaei ◽  
K Buchner
Keyword(s):  
Cerebral Cortex ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Nuclear Envelope ◽  
Immunofluorescence Microscopy ◽  
Brain Cell ◽  
Bovine Brain ◽  
Cell Nuclei ◽  
Kinase C ◽  
Protein Kinase C Isoforms

Using Western blotting and immunofluorescence microscopy we detected the protein kinase C isoforms delta, epsilon and zeta in isolated cell nuclei from bovine cerebral cortex. Both protein kinase C (PKC) delta and PKC epsilon are present in higher concentrations in neuronal than in glial nuclei and are located inside the nucleus and at the nuclear envelope. There they give a punctate staining in immunofluorescence microscopy. PKC zeta is also present both in the nucleoplasm and at the nuclear envelope. PKC eta could not be detected in the cell nuclei and, even in the homogenate of cerebral cortex, this isoform is present only in very low concentrations. The antibody against PKC eta bound strongly to a nucleoplasmic protein with an apparent molecular mass of 99 kDa. The localization of non-conventional PKC isoforms at the cell nucleus strongly indicates that these isoforms are directly involved in the regulation of nuclear processes.

scholarly journals The mono-ADP-ribosyltransferase ARTD10 regulates the voltage-gated K+ channel Kv1.1 through protein kinase C delta

BMC Biology ◽  
2020 ◽  
Vol 18 (1) ◽  
Author(s):  
Yuemin Tian ◽  
Patricia Korn ◽  
Priyanka Tripathi ◽  
Daniel Komnig ◽  
Dominik Wiemuth ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Hippocampal Neurons ◽  
K Channel ◽  
Current Component ◽  
Post Translational Modification ◽  
Protein Kinase C Delta ◽  
Voltage Gated ◽  
Adp Ribosylation ◽  
Kinase C

Abstract Background ADP-ribosylation is a ubiquitous post-translational modification that involves both mono- and poly-ADP-ribosylation. ARTD10, also known as PARP10, mediates mono-ADP-ribosylation (MARylation) of substrate proteins. A previous screen identified protein kinase C delta (PKCδ) as a potential ARTD10 substrate, among several other kinases. The voltage-gated K+ channel Kv1.1 constitutes one of the dominant Kv channels in neurons of the central nervous system and the inactivation properties of Kv1.1 are modulated by PKC. In this study, we addressed the role of ARTD10-PKCδ as a regulator of Kv1.1. Results We found that ARTD10 inhibited PKCδ, which increased Kv1.1 current amplitude and the proportion of the inactivating current component in HeLa cells, indicating that ARTD10 regulates Kv1.1 in living cells. An inhibitor of ARTD10, OUL35, significantly decreased peak amplitude together with the proportion of the inactivating current component of Kv1.1-containing channels in primary hippocampal neurons, demonstrating that the ARTD10-PKCδ signaling cascade regulates native Kv1.1. Moreover, we show that the pharmacological blockade of ARTD10 increases excitability of hippocampal neurons. Conclusions Our results, for the first time, suggest that MARylation by ARTD10 controls neuronal excitability.

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