Chemical tools selectively target components of the PKA system

Bertinetti, Daniela; Schweinsberg, Sonja; Hanke, Susanne E.; Schwede, Frank; Bertinetti, Oliver; Drewianka, Stephan; Genieser, Hans‐Gottfried

doi:10.1186/1472-6769-9-3

Cited by 40 publications

(40 citation statements)

References 59 publications

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“…Lanes were cut into 22 equally spaced pieces with an in-house made gelcutter. The sample was digested with trypsin (sequencing grade-modified trypsin; Promega) as described previously (26), and peptides were analyzed subsequently on a hybrid triple quadrupole/linear ion-trap mass spectrometer (4000 QTRAP; Applied Biosystems/MDS Sciex) coupled to a one-dimension (1D) nano-liquid chromatography (LC) system (Eksigent). Five microliters (10% sample) was injected onto a PepMap RPC18 trap column (300-m inside diameter [i.d.]…”

Section: Methodsmentioning

confidence: 99%

Dictyostelium Lipid Droplets Host Novel Proteins

Barisch

Paschke

et al. 2013

Eukaryot Cell

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Across all kingdoms of life, cells store energy in a specialized organelle, the lipid droplet. In general, it consists of a hydrophobic core of triglycerides and steryl esters surrounded by only one leaflet derived from the endoplasmic reticulum membrane to which a specific set of proteins is bound. We have chosen the unicellular organism Dictyostelium discoideum to establish kinetics of lipid droplet formation and degradation and to further identify the lipid constituents and proteins of lipid droplets. Here, we show that the lipid composition is similar to what is found in mammalian lipid droplets. In addition, phospholipids preferentially consist of mainly saturated fatty acids, whereas neutral lipids are enriched in unsaturated fatty acids. Among the novel protein components are LdpA, a protein specific to Dictyostelium, and Net4, which has strong homologies to mammalian DUF829/ Tmem53/NET4 that was previously only known as a constituent of the mammalian nuclear envelope. The proteins analyzed so far appear to move from the endoplasmic reticulum to the lipid droplets, supporting the concept that lipid droplets are formed on this membrane.

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Section: Methodsmentioning

confidence: 99%

Dictyostelium Lipid Droplets Host Novel Proteins

Barisch

Paschke

et al. 2013

Eukaryot Cell

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“…In brief, 8-AHA-cAMP (BioLog, Bremen, Germany) was covalently coupled to CM5 sensor chips (research grade) using NHS/EDC chemistry. RI or RII subunits, prepared according Bertinetti et al (28) using cAMP affinity chromatography, were injected in running buffer containing 1 mg/ml bovine serum albumin and reversibly captured on an 8-AHA-cAMP surface (surface concentration of 120 -200 resonance units for each subunit). GSKIP was injected at different concentrations (8 -256 nM) at a flow rate of 30 l/min.…”

Section: Methodsmentioning

confidence: 99%

Glycogen Synthase Kinase 3β Interaction Protein Functions as an A-kinase Anchoring Protein

Hundsrucker

Skroblin

Frank

et al. 2010

Journal of Biological Chemistry

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A-kinase anchoring proteins (AKAPs) include a family of scaffolding proteins that target protein kinase A (PKA) and other signaling proteins to cellular compartments and thereby confine the activities of the associated proteins to distinct regions within cells. AKAPs bind PKA directly. The interaction is mediated by the dimerization and docking domain of regulatory subunits of PKA and the PKA-binding domain of AKAPs. Analysis of the interactions between the dimerization and docking domain and various PKA-binding domains yielded a generalized motif allowing the identification of AKAPs. Our bioinformatics and peptide array screening approaches based on this signature motif identified GSKIP (glycogen synthase kinase 3␤ interaction protein) as an AKAP. GSKIP directly interacts with PKA and GSK3␤ (glycogen synthase kinase 3␤). It is widely expressed and facilitates phosphorylation and thus inactivation of GSK3␤ by PKA. GSKIP contains the evolutionarily conserved domain of unknown function 727. We show here that this domain of GSKIP and its vertebrate orthologues binds both PKA and GSK3␤ and thereby provides a mechanism for the integration of PKA and GSK3␤ signaling pathways. A-kinase anchoring proteins (AKAPs)3 are a family of scaffoldingproteinscharacterizedbytheabilitytobindcAMPdependent protein kinase (protein kinase A (PKA)). They tether PKA in the vicinity of its substrates, thereby facilitating their phosphorylation. In addition, AKAPs bind further signaling molecules, including other protein kinases (e.g. protein kinase C and protein kinase D), phosphodiesterases (e.g. PDE4D), and protein phosphatases (e.g. PP1 and PP2B/calcineurin). A few AKAPs possess catalytic activity. For example, AKAP-Lbc is a Rho guanine nucleotide exchange factor (1-3). Thus, AKAPs assemble multiprotein complexes and thereby coordinate cellular signaling. AKAPs are required for many cellular processes, including vasopressin-mediated water reabsorption in renal principal cells and ␤-adrenoreceptor-dependent increases of cardiac myocyte contractility (2, 4, 5).The PKA holoenzyme consists of a dimer of regulatory RI or RII subunits and two catalytic subunits, each bound to one R subunit. Upon binding of two molecules of cAMP to each R subunit, the catalytic subunits dissociate and phosphorylate their substrates (6, 7). The interaction of AKAPs with PKA is mediated by the PKA-anchoring domain of AKAPs and the dimerization and docking (DD) domain of R subunit dimers. Because most AKAPs preferentially anchor RII subunits, PKAanchoring domains are termed RII-binding domains (RIIBD). These domains are structurally conserved amphipathic helices, 14 -18 amino acid residues in length (8, 9). Based on recently described determinants of the RIIBD/DD domain interaction (8 -10), we developed a bioinformatics and peptide array screening approach to identify new AKAPs. For one of the discovered proteins, GSKIP (GSK3␤ interaction protein), we show that it functions as an AKAP.Mammalian cells express two isoforms of glycogen synthase kinase-3 (GSK3), GSK...

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“…8-(6-Aminohexylamino) cAMP (8-AHA-cAMP; BioLog) was dissolved in 100 mM HEPES (pH 8) by careful warming (32). CM5 (carboxymethylated dextran) sensor chip was activated with a mixture of 0.2 M 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 0.05 M N-hydroxysuccinimide.…”

Section: Mycobacterial Strains and Culture Conditions-m Smegmatis MCmentioning

confidence: 99%

A Universal Stress Protein (USP) in Mycobacteria Binds cAMP

Banerjee

Adolph

Gopalakrishnapai

et al. 2015

Journal of Biological Chemistry

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Background: Mycobacteria utilize cAMP for regulating transcription and protein acetylation. Results: We identify and structurally characterize a universal stress protein as an abundant and specific cAMP-binding protein. Conclusion:The USP domain is a novel cAMP-binding module. Significance: Certain members of the universal stress protein family serve to sequester cAMP, acting as sinks to regulate the availability of cAMP for downstream effectors.

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Chemical tools selectively target components of the PKA system

Cited by 40 publications

References 59 publications

Dictyostelium Lipid Droplets Host Novel Proteins

Dictyostelium Lipid Droplets Host Novel Proteins

Glycogen Synthase Kinase 3β Interaction Protein Functions as an A-kinase Anchoring Protein

A Universal Stress Protein (USP) in Mycobacteria Binds cAMP

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