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Characterizing PKA-Mediated Phosphorylation of Plexin Using Purified Proteins
Taehong Yang1,2, Jonathan R Terman3
1Departments of Neuroscience and Pharmacology and Neuroscience Graduate Program, The University of Texas Southwestern Medical Center, Dallas, TX, 75390, USA.
Methods in Molecular Biology (Clifton, N.J.)
|October 28, 2016
Summary
This study details how protein phosphorylation modifies Plexin A function. We developed a method using radioactive ATP to detect and quantify this crucial modification in vitro.
Area of Science:
- Molecular Biology
- Cell Signaling
- Neuroscience
Background:
- Protein phosphorylation is a key posttranslational modification regulating protein function.
- Plexin A mediates repulsive guidance, crucial for neuronal development.
- Cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) phosphorylates Plexin A.
Purpose of the Study:
- To describe a method for detecting and quantifying Plexin A phosphorylation.
- To investigate the functional consequences of Plexin A phosphorylation.
Main Methods:
- In vitro kinase assay.
- Use of radioactive [γ-P32] adenosine 5'-triphosphate (ATP) for detection.
- Analysis of GTPase-activating protein (GAP) domain activity.
Main Results:
- Plexin A phosphorylation by PKA occurs in the GAP domain's active site.
- Phosphorylation inhibits GAP activity towards Ras/Rap small GTP-binding proteins.
- Recruitment of 14-3-3ε to the phosphorylated site.
Conclusions:
- Phosphorylation of Plexin A by PKA is a regulatory mechanism for repulsive guidance.
- This modification affects downstream signaling by inhibiting GAP activity.
- An in vitro assay allows for the study of this critical regulatory event.
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