Liberated PKA Catalytic Subunits Associate with the Membrane via Myristoylation to Preferentially Phosphorylate Membrane Substrates

Tillo, Shane; Xiong, Wei; Takahashi, Mitsuaki; Miao, Sheng; Andrade, Adriana L.; Fortin, Dale A.; Yang, Guang; Qin, Maozhen; Smoody, Barbara F.; Stork, Philip J.S.; Zhong, Haining

doi:10.1016/j.celrep.2017.03.070

Cited by 73 publications

(90 citation statements)

References 76 publications

(101 reference statements)

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“…Depending on the stimuli, either Gs or Gi/o pathways are activated, and their effects are related to cytoskeletal contraction or relaxation, respectively [124,127]. Together with Cdc42 and G proteins, these signals are able to Other molecules that must also suffer N-terminal processing, such as Src [129], PKA [130], ARF [131], and calcineurin [132], are also involved in the same processes. Depending on the stimuli, either Gs or Gi/o pathways are activated, and their effects are related to cytoskeletal contraction or relaxation, respectively [124,127].…”

Section: Effects Of Fumagillin Activity On Host Cellsmentioning

confidence: 99%

Fumagillin, a Mycotoxin of Aspergillus fumigatus: Biosynthesis, Biological Activities, Detection, and Applications

Guruceaga

Perez-Cuesta

Abad‐Diaz‐de‐Cerio

et al. 2019

Toxins

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Fumagillin is a mycotoxin produced, above all, by the saprophytic filamentous fungus Aspergillus fumigatus. This mold is an opportunistic pathogen that can cause invasive aspergillosis, a disease that has high mortality rates linked to it. Its ability to adapt to environmental stresses through the production of secondary metabolites, including several mycotoxins (gliotoxin, fumagillin, pseurotin A, etc.) also seem to play an important role in causing these infections. Since the discovery of the A. fumigatus fumagillin in 1949, many studies have focused on this toxin and in this review we gather all the information currently available. First of all, the structural characteristics of this mycotoxin and the different methods developed for its determination are given in detail. Then, the biosynthetic gene cluster and the metabolic pathway involved in its production and regulation are explained. The activity of fumagillin on its target, the methionine aminopeptidase type 2 (MetAP2) enzyme, and the effects of blocking this enzyme in the host are also described. Finally, the applications that this toxin and its derivatives have in different fields, such as the treatment of cancer and its microsporicidal activity in the treatment of honeybee hive infections with Nosema spp., are reviewed. Therefore, this work offers a complete review of all the information currently related to the fumagillin mycotoxin secreted by A. fumigatus, important because of its role in the fungal infection process but also because it has many other applications, notably in beekeeping, the treatment of infectious diseases, and in oncology. Key Contribution:Fumagillin is a mycotoxin produced during infections caused by A. fumigatus and in the adaptation processes of this species to multiple environmental stresses. In this review; we include detailed information on the fungal mechanisms employed in the production of this mycotoxin and those present in its regulation, how it acts on its target, and how it can be detected, all of which may be useful for diagnosis or the development of new treatments. Its usefulness in different fields is also described; because of its anti-angiogenic activity it is used to attack different types of tumors; at the same time its antibiotic activity is useful against several parasites.

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Section: Effects Of Fumagillin Activity On Host Cellsmentioning

confidence: 99%

Fumagillin, a Mycotoxin of Aspergillus fumigatus: Biosynthesis, Biological Activities, Detection, and Applications

Guruceaga

Perez-Cuesta

Abad‐Diaz‐de‐Cerio

et al. 2019

Toxins

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“…Subsequent to our investigations of linker effects on expression and activity, we sought to analyze the impact of linker sequence upon the subcellular localization of PKAc‐VLR‐GFP fusions. PKAc possesses an N‐terminal myristoylation sequence, which is post‐translationally modified to render the protein membrane bound . We observed this anticipated localization pattern in transfected HeLa cells expressing PKAc‐GFP fusions containing no linker, the flexible GGS linker series, and the flexible GGSD linker series (Fig.…”

Section: Resultsmentioning

confidence: 57%

protaTETHER – a method for the incorporation of variable linkers in protein fusions reveals impacts of linker flexibility in a PKAc‐GFP fusion protein

Norris

Hughes

2018

FEBS Open Bio

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Protein fusions are of fundamental importance in the study of cellular biology and the elucidation of cell signaling pathways, and the importance of linkers for the proper function of protein fusions is well documented in the literature. However, there are few convenient methods available to experimentalists for the systematic implementation of linkers in protein fusions. In this work, we describe a universal approach to the creation and insertion of focused linker libraries into protein fusions. This process, deemed protaTETHER, utilizes reiterative oligomer design, PCR‐mediated linker library generation, and restriction enzyme‐free cloning methods in a straightforward, three‐step cloning process. We utilize a fusion between the catalytic subunit of cAMP‐dependent protein kinase A (PKAc) and green fluorescent protein (GFP) for the development of the protaTETHER method, implementing small linker libraries that vary by length, sequence, and predicted secondary structural elements. We analyze the impact of linker length and sequence on the expression, activity, and subcellular localization of the PKAc‐GFP fusions, and use these results to select a PKAc‐GFP fusion construct with robust expression and enzymatic activity. Based upon the results of both biochemical experiments and molecular modeling, we determine that linker flexibility is more important than linker length for optimal kinase activity and expression.

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“…Another important property of reporters is kinase specificity. In our study, we have used a well-characterized substrate sequence of PKA that has been used in previous FRET reporters 7–11 . A phosphorylation prediction algorithm, NetPhos3.1 21 , strongly suggests PKA as the target kinase for this reporter.…”

Section: Discussionmentioning

confidence: 99%

“…Upon phosphorylation, the binding affinity between the phosphopeptide binding domain and the substrate sequence dramatically increase, leading to a conformational change, which is then measured through changes in FRET. This design has been the basis for a number of generations of PKA FRET reporters in the literature 7–11 .…”

Section: Introductionmentioning

confidence: 99%

Rational Design of a Protein Kinase A Nuclear-cytosol Translocation Reporter

Kim

Inoue

2019

Preprint

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Protein Kinase A (PKA) exists as a tetrameric holoenzyme which activates with increase of cAMP and plays an important role in many physiological processes including cardiac physiology, neuronal development, and adipocyte function. Although this kinase has been the subject of numerous biosensor designs, a single-fluorophore reporter that performs comparably to Förster resonance energy transfer (FRET) has not yet been reported. Here, we have used basic observations of electrostatic interactions in PKA substrate recognition mechanism and nucleus localization sequence motif to design a phosphorylation switch that shuttles between the cytosol and the nucleus, a strategy that should be generalizable to all basophilic kinases. The resulting reporter yielded comparable kinetics and dynamic range to the PKA FRET reporter, AKAR3EV. We also performed basic characterization and demonstrated its potential use in monitoring multiple signaling molecules inside cells using basic fluorescence microscopy. Due to the single-fluorophore nature of this reporter, we envision that this could find broad applications in studies involving single cell analysis of PKA activity.

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Liberated PKA Catalytic Subunits Associate with the Membrane via Myristoylation to Preferentially Phosphorylate Membrane Substrates

Cited by 73 publications

References 76 publications

Fumagillin, a Mycotoxin of Aspergillus fumigatus: Biosynthesis, Biological Activities, Detection, and Applications

Fumagillin, a Mycotoxin of Aspergillus fumigatus: Biosynthesis, Biological Activities, Detection, and Applications

protaTETHER – a method for the incorporation of variable linkers in protein fusions reveals impacts of linker flexibility in a PKAc‐GFP fusion protein

Rational Design of a Protein Kinase A Nuclear-cytosol Translocation Reporter

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