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Updated: Aug 18, 2026
Identification of Kinase-substrate Pairs Using High Throughput Screening
Published on: August 29, 2015
Phosphorylation screening identifies translational initiation factor 4GII as an intracellular target of
Hui Qin1, Brian Raught, Nahum Sonenberg
1Department of Pharmacology and Toxicology, State University of New York at Buffalo, Buffalo, New York 14214, USA.
Abstract:
CaMKI is a Ca2+/calmodulin-dependent protein kinase that is widely expressed in eukaryotic cells and tissues but for which few, if any, physiological substrates are known. We screened a human lung cDNA expression library for potential CaMKI substrates by solid phase in situ phosphorylation ("phosphorylation screening"). Multiple overlapping partial length cDNAs encoding three proteins were detected. Two of these proteins are known: 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase and eukaryotic translation initiation factor (eIF) 4GII. To determine whether CaMKI substrates identified by phosphorylation screening represent authentic physiological targets, we examined the potential for [Ca2+]i- and CaMKI-dependent phosphorylation of eIF4GII in vitro and in vivo. Endogenous eIF4GII immunoprecipitated from HEK293T cells was phosphorylated by CaMKI, in vitro as was a recombinant fragment of eIF4GII encompassing the central and C-terminal regions. The latter phosphorylation occurred with favorable kinetics (Km = 1 microm; kcat = 1.8 s-1) at a single site, Ser1156, located in a segment of eIF4GII aligning with the phosphoregion of eIF4GI. Phosphopeptide mapping and back phosphorylation experiments revealed [Ca2+]i-dependent, CaMKI site-specific, eIF4GII phosphorylation in vivo. This phosphorylation was blocked by kinase-negative CaMKI consistent with a requirement for endogenous CaMKI for in vivo eIF4GII phosphorylation. We conclude that phosphorylation screening is an effective method for searching for intracellular targets of CaMKI and may have identified a new role of Ca2+ signaling to the translation apparatus.
Insights
Researchers identified eukaryotic translation initiation factor (eIF) 4GII as a novel substrate for Ca2+/calmodulin-dependent protein kinase I (CaMKI). This discovery reveals a new link between calcium signaling and the translation machinery.
Area of Science:
- Molecular Biology
- Cell Signaling
- Biochemistry
Background:
- Ca2+/calmodulin-dependent protein kinase I (CaMKI) is crucial in cellular signaling but has few known physiological substrates.
- Identifying CaMKI substrates is essential for understanding its diverse cellular roles.
Purpose of the Study:
- To identify novel physiological substrates of CaMKI using a phosphorylation screening approach.
- To validate eukaryotic translation initiation factor (eIF) 4GII as a direct substrate of CaMKI in vitro and in vivo.
Main Methods:
- Utilized a human lung cDNA expression library for in situ phosphorylation screening to identify potential CaMKI substrates.
- Performed in vitro kinase assays with purified CaMKI and eIF4GII fragments.
- Conducted in vivo phosphorylation studies using HEK293T cells, including immunoprecipitation and phosphopeptide mapping.
Main Results:
- Identified eIF4GII as a novel CaMKI substrate through phosphorylation screening.
- Demonstrated direct phosphorylation of eIF4GII by CaMKI in vitro at a specific site (Ser1156) with efficient kinetics.
- Confirmed Ca2+-dependent and CaMKI-mediated phosphorylation of eIF4GII in intact cells, which was blocked by a kinase-negative CaMKI mutant.
Conclusions:
- Phosphorylation screening is an effective strategy for discovering intracellular CaMKI targets.
- eIF4GII is a physiological substrate of CaMKI, suggesting a role for Ca2+ signaling in regulating protein translation.
- This finding opens new avenues for research into calcium's impact on the translational apparatus.
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