[41] Assay of cyclic AMP-dependent protein kinases

doi:10.1016/0076-6879(74)38044-5

Methods in Enzymology

Volume 38, 1974, Pages 287-290

https://doi.org/10.1016/0076-6879(74)38044-5 Get rights and content

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Most tissues contain protein kinases that are stimulated several-fold by cyclic 3', 5'-adenosine monophosphate (cAMP) and catalyze the transfer of phosphate from ATP to several proteins. Incorporation of phosphate into protein can be monitored by transfer of 32p to protein from [γ^-32P] ATP in the presence of magnesium. The phosphorylated protein is separated from the labeled precursor by adsorption of the precipitated protein on filter paper disks and washing the disks as described by several investigators. Several proteins may be used as substrates in the assay, including muscle phosphorylase kinase and glycogen synthetase, protamine, adipose tissue lipase, casein, specific histones, and histone mixtures. When either phosphorylase kinase, glycogen synthetase, or lipase are used, the phosphorylation causes enzymatic activity changes which may be developed into alternate methods for assay of protein kinases. In practice, however, such assays are difficult to quantirate. A histone mixture is a suitable substrate for several reasons: (1) it is available from commercial sources, (2) there is little if any protein kinase contamination, (3) an adequate amount of phosphate is incorporated, (4) it is a stable and easily precipitable protein mixture, and (5) the degree of stimulation of histone phosphorylation by cAMP is usually relatively high.

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Autoactivation of C-terminally truncated Ca <sup>2+</sup> /calmodulin-dependent protein kinase (CaMK) Iδ via CaMK kinase-independent autophosphorylation
2019, Archives of Biochemistry and Biophysics
Citation Excerpt :
The LC-MS/MS data were analyzed by MASCOT software (Matrix Science) using the amino acid sequence data of zCaMKIδ(1–299). Protein kinase assays were conducted as described [7,8,20]. SDS-PAGE and Western blotting were performed as described [19].
Ca²⁺/calmodulin-dependent protein kinase I isoforms (CaMKIα, β, γ, and δ) play important roles in Ca²⁺ signaling in eukaryotic cells by being activated by CaMK kinase (CaMKK) through phosphorylation at a Thr residue in the activation loop. However, we have recently found that, unlike rat CaMKIα (rCaMKIα), C-terminally truncated fragments of zebrafish and mouse CaMKIδ [zCaMKIδ(1–299) and mCaMKIδ(1–297)] produced by Escherichia coli exhibit almost full activity in the absence of CaMKK. To address the CaMKK-independent activation mechanism of CaMKIδ in E. coli cells, here we performed comparative analyses between recombinant zCaMKIδ(1–299) and rCaMKIα(1–294) in vitro. By using a kinase-dead mutant of zCaMKIδ(1–299) and λ phosphatase coexpression method, we elucidated that zCaMKIδ(1–299) was highly autophosphorylated and activated in E. coli during cell culture, but rCaMKIα(1–294) was not. The major autophosphorylation site leading to activation of the kinase was Ser²⁹⁶, determined using mass spectrometry analysis in conjunction with site-directed mutagenesis. Furthermore, mimicking phosphorylation at Ser²⁹⁶ in full-length zCaMKIδ resulted in additional activation of the kinase compared with CaMKI fully activated by CaMKK. Our results provide the first evidence that CaMKIδ is activated through CaMKK-independent phosphorylation at Ser²⁹⁶, which might be a clue to understand the physiological regulation of CaMKIδ isoform.
Facile preparation of highly active casein kinase 1 using Escherichia coli constitutively expressing lambda phosphatase
2018, Analytical Biochemistry
Citation Excerpt :
The amounts of enzyme and substrate for each reaction are described in the corresponding figure legends. The specific activity of CK1s and CK1s(ΔC) was determined essentially according to Corbin and Reimann [30]. In vitro kinase assays were carried out at 30 °C for 30 s in a standard reaction mixture (20 μl) using CK1s (10 ng) and α-casein (2 μg) as a substrate.
Casein kinase 1 (CK1) is a widely expressed Ser/Thr kinase in eukaryotic organisms that is involved in various cellular processes (e.g., circadian rhythm and apoptosis). Therefore, preparing highly active CK1 and investigating its properties in vitro have important implications for understanding the biological roles of the kinase. However, recombinant CK1 undergoes autoinactivation via autophosphorylation in Escherichia coli cells and thus is undesirably prepared as a phosphorylated and inactivated kinase. To circumvent this problem, we established a protein expression system using E. coli strain BL21(DE3)pλPP in which λ protein phosphatase (λPPase) is constitutively expressed. Using this system, recombinant CK1 isoforms (α, δ and ε) were readily prepared as unphosphorylated forms. Furthermore, we found that CK1s prepared using BL21(DE3)pλPP showed markedly higher activity than those prepared by the conventional BL21(DE3). Finally, we demonstrated that the kinase activity of CK1δ from BL21(DE3)pλPP was higher than that prepared by a conventional method consisting of troublesome steps such as in vitro λPPase treatment. Thus, this simple method using BL21(DE3)pλPP is valuable for preparing highly active CK1s. It may also be applicable to other kinases that are difficult to prepare because of phosphorylation in E. coli cells.
A protein kinase A-ezrin complex regulates connexin 43 gap junction communication in liver epithelial cells
2017, Cellular Signalling
Communication between adjacent cells can occur via gap junctions (GJ) composed of connexin (Cx) hexamers that allow passage of small molecules. One of the most widely and highly expressed Cxs in human tissues is Cx43, shown to be regulated through phosphorylation by several kinases including PKA. Ezrin is a membrane associated protein that can serve as an A-kinase anchoring protein (AKAP) and hold an anchored pool of PKA. Here, we used the liver epithelial cell line IAR20, which expresses Cx43 as the predominant GJ protein, to test the hypothesis that Ezrin may associate with Cx43 in cell types that form stable GJs and serve as an AKAP. Our biochemical and proteomics data indicate that Ezrin associates with Cx43 in epithelial cells. Analyses by confocal immunofluorescence microscopy and proximity ligation assays demonstrate that Ezrin and Cx43 co-localize, together with zonula occludens-1 (ZO-1) and PKA RIα and RIIα, at the cell membrane. Quantitative gap-FRAP experiments show increased GJ intercellular communication after cAMP stimulation. Moreover, loading of cells with the Ht31 peptide that displaces both PKA RIα and RIIα from the AKAP or a peptide that disrupts the Cx43-Ezrin interaction reverts the effect and reduces the level of communication, supporting the hypothesis that in IAR20 cells Ezrin associates with Cx43 (in complex with ZO-1) which places PKA in proximity to Cx43, enabling its phosphorylation and GJ opening.
Phosphorylated TandeMBP: A unique protein substrate for protein phosphatase assay
2016, Analytical Biochemistry
To analyze a variety of protein phosphatases, we developed phosphorylated TandeMBP (P-TandeMBP), in which two different mouse myelin basic protein isoforms were fused in tandem, as a protein phosphatase substrate. P-TandeMBP was prepared efficiently in four steps: (1) phosphorylation of TandeMBP by a protein kinase mixture (Ca²⁺/calmodulin-dependent protein kinase Iδ, casein kinase 1δ, and extracellular signal-regulated kinase 2); (2) precipitation of both P-TandeMBP and protein kinases to remove ATP, Pi, and ADP; (3) acid extraction of P-TandeMBP with HCl to remove protein kinases; and (4) neutralization of the solution that contains P-TandeMBP with Tris. In combination with the malachite green assay, P-TandeMBP can be used to detect protein phosphatase activity without using radioactive materials. Moreover, P-TandeMBP served as an efficient substrate for PPM family phosphatases (PPM1A, PPM1B, PPM1D, PPM1F, PPM1G, PPM1H, PPM1K, and PPM1M) and PPP family phosphatase PP5. Various phosphatase activities were also detected with high sensitivity in gel filtration fractions from mouse brain using P-TandeMBP. These results indicate that P-TandeMBP might be a powerful tool for the detection of protein phosphatase activities.
High-performance CaMKI: A highly active and stable form of CaMKIδ produced by high-level soluble expression in Escherichia coli
2016, Biochemical and Biophysical Research Communications
We describe here the expression and characterization of a constitutively active fragment of zebrafish Ca²⁺/calmodulin-dependent protein kinase (CaMK) Iδ designated zCaMKIδ(1–299) that lacks an autoinhibitory domain. We used a simple one-step purification method to isolate the recombinant enzyme at high yield (220 mg/l of the culture medium) from the soluble fraction of lysates prepared from Escherichia coli. Unlike the corresponding fragment of CaMKIα (CaMKΙα(1–294)), the kinase activity of zCaMKIδ(1–299), without activation procedures, was comparable to that of wild-type zCaMKIδ activated by CaMK kinase. zCaMKIδ(1–299) exhibited broad substrate specificity highly similar to that of wild-type zCaMKIδ, and complementary to that of the cAMP-dependent protein kinase catalytic subunit (PKAc). The protein kinase activity of zCaMKIδ(1–299) was higher compared with that of PKAc as well as CX-30K-CaMKII that comprises a constitutively active fragment of CaMKII fused to the N-terminal region of Xenopus CaMKI. Furthermore, kinase activity was highly stable against thermal inactivation and repeated freezing-thawing. Thus, zCaMKIδ(1–299) represents a readily available alternative that can be used as a “High-performance phosphorylating reagent” alone or in combination with PKAc in diverse experiments on protein phosphorylation and dephosphorylation.
Characterization of the wzc gene from Pantoea sp. strain PPE7 and its influence on extracellular polysaccharide production and virulence on Pleurotus eryngii
2015, Microbiological Research
To characterize of the pathogenicity gene from the soft rot pathogen Pantoea sp. PPE7 in Pleurotus eryngii, we constructed over 10,000 kanamycin-resistant transposon mutants of Pantoea sp. strain PPE7 by transposon mutagenesis. One mutant, Pantoea sp. NPPE9535, did not cause a soft rot disease on Pleurotus eryngii was confirmed by the pathogenicity test.
The transposon was inserted into the wzc gene and the disruption of the wzc gene resulted in the reduction of polysaccharide production and abolished the virulence of Pantoea sp. strain PPE7 in P. eryngii. Analysis of the hydropathic profile of this protein indicated that it is composed of two main domains: an N-terminal domain including two transmembrane α-helices and a C-terminal cytoplasmic domain consisting of a tyrosine-rich region. Comparative analysis indicated that the amino acid sequence of Wzc is similar to that of a number of proteins involved in the synthesis or export of polysaccharides in other bacterial species. Purified GST-Wzc was found to affect the phosphorylation of tyrosine residue in vivo. These results showed that the wzc gene might play an important role in the virulence of Pantoea sp. strain PPE7 in P. eryngii.

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[41] Assay of cyclic AMP-dependent protein kinases

Publisher Summary

Curr. Top. Cell. Regul.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.