scholarly journals Characterization of A-kinase-anchoring disruptors using a solution-based assay

2006 ◽  
Vol 400 (3) ◽  
pp. 493-499 ◽  
Author(s):  
Anne J. Stokka ◽  
Frank Gesellchen ◽  
Cathrine R. Carlson ◽  
John D. Scott ◽  
Friedrich W. Herberg ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Interactions ◽  
Protein Protein Interactions ◽  
Regulatory Subunits ◽  
Anchoring Proteins ◽  
Ic50 Values ◽  
Order Of Magnitude ◽  
Dependent Protein

Subcellular localization of PKA (cAMP-dependent protein kinase or protein kinase A) is determined by protein–protein interactions between its R (regulatory) subunits and AKAPs (A-kinase-anchoring proteins). In the present paper, we report the development of the Amplified Luminescent Proximity Homogeneous Assay (AlphaScreen™) as a means to characterize AKAP-based peptide competitors of PKA anchoring. In this assay, the prototypic anchoring disruptor Ht31 efficiently competed in RIIα isoform binding with RII-specific and dual-specificity AKAPs (IC50 values of 1.4±0.2 nM and 6±1 nM respectively). In contrast, RIα isoform binding to a dual-specific AKAP was less efficiently competed (IC50 of 156±10 nM). Characterization of two RI-selective anchoring disruptors, RIAD (RI-anchoring disruptor) and PV-38 revealed that RIAD (IC50 of 13±1 nM) was 20-fold more potent than PV-38 (IC50 of 304±17 nM) and did not compete in the RIIα–AKAP interaction. We also observed that the kinetics of RII displacement from pre-formed PKA–AKAP complexes and competition of RII–AKAP complex formation by Ht31 differed by an order of magnitude when the component parts were mixed in vitro. No such difference in potency was seen for RIα–AKAP complexes. Thus the AlphaScreen assay may prove to be a valuable tool for detailed characterization of a variety of PKA–AKAP complexes.

Subcellular Localization of the Type II cAMP-Dependent Protein Kinase

Physiology ◽  
1992 ◽  
Vol 7 (4) ◽  
pp. 143-148 ◽  
Author(s):  
JD Scott ◽  
DW Carr
Keyword(s):  
Protein Kinase ◽  
Protein Interactions ◽  
Regulatory Subunit ◽  
Type Ii ◽  
Protein Protein Interactions ◽  
Dependent Protein Kinase ◽  
Biochemical Effects ◽  
Camp Dependent Protein Kinase ◽  
Anchoring Proteins ◽  
Dependent Protein

Diverse biochemical effects of different neurotransmitters or hormones that stimulate cAMP production may occur through activation of compartmentalized pools of cAMP-dependent protein kinase (PKA). Evidence suggests that compartmentalization of type II PKA is maintained through protein-protein interactions between the regulatory subunit and specific anchoring proteins.

Regulation of the exocytotic machinery by cAMP-dependent protein kinase: implications for presynaptic plasticity

2003 ◽  
Vol 31 (4) ◽  
pp. 824-827 ◽  
Author(s):  
G.J.O. Evans ◽  
A. Morgan
Keyword(s):  
Protein Kinase ◽  
Protein Interactions ◽  
Cell Types ◽  
Protein Protein Interactions ◽  
Dependent Protein Kinase ◽  
Camp Dependent Protein Kinase ◽  
Cysteine String Protein ◽  
Presynaptic Plasticity ◽  
Dependent Protein

For over a decade, the enhancement of regulated exocytosis by cAMP-dependent protein kinase (PKA) has remained unexplained at the molecular level. The fact that this phenomenon has been observed in such a wide variety of secretory cell types, from pancreatic β-cells to neurons, suggests that it is an important and fundamental mechanism. Extensive analysis of the phosphorylation of exocytotic proteins has yielded few substrates of PKA in vitro, and fewer still have had physiological effects attributed to their phosphorylation. Here we review two proteins that do fulfil these criteria: the synaptic vesicle proteins cysteine string protein (CSP) and Snapin. Phosphorylation of these proteins by PKA produces changes in their respective protein–protein interactions, and has been attributed to modulation of the vesicle priming (Snapin) and vesicle fusion (CSP) stages of exocytosis. We also discuss how the function of CSP and Snapin phosphorylation might fit into an interesting aspect of the PKA-dependent enhancement of exocytosis: presynaptic plasticity in the brain.

scholarly journals Cloning and characterization of BCY1, a locus encoding a regulatory subunit of the cyclic AMP-dependent protein kinase in Saccharomyces cerevisiae.

1987 ◽  
Vol 7 (4) ◽  
pp. 1371-1377 ◽  
Author(s):  
T Toda ◽  
S Cameron ◽  
P Sass ◽  
M Zoller ◽  
J D Scott ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Kinase ◽  
Cyclic Amp ◽  
Carbon Sources ◽  
Regulatory Subunit ◽  
Dependent Protein Kinase ◽  
Yeast Saccharomyces Cerevisiae ◽  
Regulatory Subunits ◽  
Dependent Protein

We have cloned a gene (BCY1) from the yeast Saccharomyces cerevisiae that encodes a regulatory subunit of the cyclic AMP-dependent protein kinase. The encoded protein has a structural organization similar to that of the RI and RII regulatory subunits of the mammalian cyclic AMP-dependent protein kinase. Strains of S. cerevisiae with disrupted BCY1 genes do not display a cyclic AMP-dependent protein kinase in vitro, fail to grow on many carbon sources, and are exquisitely sensitive to heat shock and starvation.

scholarly journals Label-free methods for optical in vitro characterization of protein–protein interactions

2021 ◽  
Author(s):  
Fabian Soltermann ◽  
Weston B. Struwe ◽  
Philipp Kukura
Keyword(s):  
Protein Interactions ◽  
Label Free ◽  
Protein Protein Interactions ◽  
Cellular Processes ◽  
P Protein ◽  
In Vitro Characterization ◽  
And Function

Protein–protein interactions are involved in the regulation and function of the majority of cellular processes.

scholarly journals Phosphorylation of hepatitis B virus Cp at Ser87 facilitates core assembly

2006 ◽  
Vol 398 (2) ◽  
pp. 311-317 ◽  
Author(s):  
Hee Yong Kang ◽  
Seungkeun Lee ◽  
Sung Gyoo Park ◽  
Jaehoon Yu ◽  
Youngsoo Kim ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Protein Kinase ◽  
Hepatitis B ◽  
Protein Interactions ◽  
Present Report ◽  
Protein Protein Interactions ◽  
B Virus ◽  
Dimeric Form ◽  
Genome Encapsidation

Protein–protein interactions can be regulated by protein modifications such as phosphorylation. Some of the phosphorylation sites (Ser155, Ser162 and Ser170) of HBV (hepatitis B virus) Cp have been discovered and these sites are implicated in the regulation of viral genome encapsidation, capsid localization and nucleocapsid maturation. In the present report, the dimeric form of HBV Cp was phosphorylated by PKA (protein kinase A), but not by protein kinase C in vitro, and the phosphorylation of dimeric Cp facilitated HBV core assembly. Matrix-assisted laser-desorption ionization–time-of-flight analysis revealed that the HBV Cp was phosphorylated at Ser87 by PKA. This was further confirmed using a mutant HBV Cp with S87G mutation. The S87G mutation inhibited the phosphorylation and, as a result, the in vitro HBV core assembly was not facilitated by PKA. In addition, when either pCMV/FLAG–Core(WT) or pCMV/FLAG–Core(S87G) was transfected into HepG2 cells, few mutant Cps (S87G) assembled into capsids compared with the wild-type (WT) Cps, although the same level of total Cps was expressed in both cases. In conclusion, PKA facilitates HBV core assembly through phosphorylation of the HBV Cp at Ser87.

scholarly journals Structural and functional characterization of Rpn12 identifies residues required for Rpn10 proteasome incorporation

2012 ◽  
Vol 448 (1) ◽  
pp. 55-65 ◽  
Author(s):  
Jonas Boehringer ◽  
Christiane Riedinger ◽  
Konstantinos Paraskevopoulos ◽  
Eachan O. D. Johnson ◽  
Edward D. Lowe ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Functional Characterization ◽  
Ubiquitin Proteasome System ◽  
26S Proteasome ◽  
Structural Motif ◽  
Protein Protein Interactions ◽  
Ubiquitin Proteasome

The ubiquitin–proteasome system targets selected proteins for degradation by the 26S proteasome. Rpn12 is an essential component of the 19S regulatory particle and plays a role in recruiting the extrinsic ubiquitin receptor Rpn10. In the present paper we report the crystal structure of Rpn12, a proteasomal PCI-domain-containing protein. The structure helps to define a core structural motif for the PCI domain and identifies potential sites through which Rpn12 might form protein–protein interactions. We demonstrate that mutating residues at one of these sites impairs Rpn12 binding to Rpn10 in vitro and reduces Rpn10 incorporation into proteasomes in vivo.

scholarly journals Zebrafish xenografts to isolate unique human breast cancer metastatic cell populations

2021 ◽  
Author(s):  
Jerry Xiao ◽  
Joseph R. McGill ◽  
Apsra Nasir ◽  
Alexander Lekan ◽  
Bailey Johnson ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Cancer Metastasis ◽  
Circulatory System ◽  
Cell Populations ◽  
Protein Protein Interactions ◽  
Zebrafish Model ◽  
Metastatic Cell ◽  
Metastatic Cells

Cancer metastasis is a critical culprit frequently blamed for treatment failure, drug resistance, poor prognosis, and high mortality rate among all human cancers. Laboratory efforts to isolate metastatic cell populations have typically been confined to mouse models, which are time-consuming and expensive. Here, we present a model system based on xenografting zebrafish embryos to select for cells that are predisposed to progress through the early stages of metastasis. This model requires only 3-5 days to achieve distinct intravasation to the zebrafish circulatory system. The metastatic cells are easily tracked in real-time as they migrate, as well as isolated and propagated in vitro. Once expanded, molecular characterization of the serially derived invasive cell populations from the tails of the zebrafish accurately predicts genes, signaling pathways, protein-protein interactions, and differential splicing products that are important for an invasive phenotype. This zebrafish model therefore offers a high-throughput and robust method for identifying gene targets critical for cancer metastasis.

scholarly journals GSKIP-Mediated Anchoring Increases Phosphorylation of Tau by PKA but Not by GSK3beta via cAMP/PKA/GSKIP/GSK3/Tau Axis Signaling in Cerebrospinal Fluid and iPS Cells in Alzheimer Disease

2019 ◽  
Vol 8 (10) ◽  
pp. 1751 ◽  
Author(s):  
Ko ◽  
Chiou ◽  
Wong ◽  
Wang ◽  
Lai ◽  
...  
Keyword(s):  
Cerebrospinal Fluid ◽  
Protein Kinase ◽  
Glycogen Synthase ◽  
Tau Phosphorylation ◽  
In Vitro Assays ◽  
Anchoring Proteins ◽  
Dependent Protein ◽  
Induced Pluripotent ◽  
Isogenic Mutants

Based on the protein kinase A (PKA)/GSK3β interaction protein (GSKIP)/glycogen synthase kinase 3β (GSK3β) axis, we hypothesized that these might play a role in Tau phosphorylation. Here, we report that the phosphorylation of Tau Ser409 in SHSY5Y cells was increased by overexpression of GSKIP WT more than by PKA- and GSK3β-binding defective mutants (V41/L45 and L130, respectively). We conducted in vitro assays of various kinase combinations to show that a combination of GSK3β with PKA but not Ca2+/calmodulin-dependent protein kinase II (CaMK II) might provide a conformational shelter to harbor Tau Ser409. Cerebrospinal fluid (CSF) was evaluated to extend the clinical significance of Tau phosphorylation status in Alzheimer’s disease (AD), neurological disorders (NAD), and mild cognitive impairment (MCI). We found higher levels of different PKA–Tau phosphorylation sites (Ser214, Ser262, and Ser409) in AD than in NAD, MCI, and normal groups. Moreover, we used the CRISPR/Cas9 system to produce amyloid precursor protein (APPWT/D678H) isogenic mutants. These results demonstrated an enhanced level of phosphorylation by PKA but not by the control. This study is the first to demonstrate a transient increase in phosphor-Tau caused by PKA, but not GSK3β, in the CSF and induced pluripotent stem cells (iPSCs) of AD, implying that both GSKIP and GSK3β function as anchoring proteins to strengthen the cAMP/PKA/Tau axis signaling during AD pathogenesis.

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