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Regulation of arrestin-3 phosphorylation by casein kinase II
You-Me Kim1, Larry S Barak, Marc G Caron
1Department of Microbiology and Immunology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
Abstract:
Arrestins play an important role in regulating the function of G protein-coupled receptors including receptor desensitization, internalization, down-regulation, and signaling via nonreceptor tyrosine kinases and mitogen-activated protein kinases. Previous studies have revealed that arrestins themselves are also subject to regulation. In the present study, we focused on identifying potential mechanisms involved in regulating the function of arrestin-3. Using metabolic labeling, phosphoamino acid analysis, and mutagenesis studies, we found that arrestin-3 is constitutively phosphorylated at Thr-382 and becomes dephosphorylated upon beta(2)-adrenergic receptor activation in COS-1 cells. Casein kinase II (CKII) appears to be the major kinase mediating arrestin-3 phosphorylation, since 1) Thr-382 is contained within a canonical consensus sequence for CKII phosphorylation and 2) wild type arrestin-3 but not a T382A mutant is phosphorylated by CKII in vitro. Functional analysis reveals that mutants mimicking the phosphorylated (T382E) and dephosphorylated (T382A or T382V) states of arrestin-3 promote beta(2)-adrenergic receptor internalization and bind clathrin, beta-adaptin, and Src to comparable levels as wild type arrestin-3. This suggests that the phosphorylation of arrestin-3 does not directly regulate interaction with endocytic (clathrin, beta-adaptin) or signaling (Src) components and is in contrast to arrestin-2, where phosphorylation appears to regulate interaction with clathrin and Src. However, additional analysis reveals that arrestin-3 phosphorylation may regulate formation of a large arrestin-3-containing protein complex. Differences between the regulatory roles of arrestin-2 and -3 phosphorylation may contribute to the different cellular functions of these proteins in G protein-coupled receptor signaling and regulation.
Insights
Arrestin-3 is constitutively phosphorylated by Casein kinase II (CKII) at Thr-382, and this dephosphorylation upon receptor activation does not affect binding to key proteins. However, arrestin-3 phosphorylation may influence larger protein complex formation.
Area of Science:
- Cellular Biology
- Molecular Biology
- Biochemistry
Background:
- Arrestins regulate G protein-coupled receptor (GPCR) function, including desensitization and internalization.
- Arrestins themselves are subject to regulatory modifications, such as phosphorylation.
- Understanding arrestin-3 regulation is crucial for elucidating GPCR signaling pathways.
Purpose of the Study:
- To identify mechanisms regulating arrestin-3 function.
- To investigate the role of arrestin-3 phosphorylation in GPCR regulation.
- To compare the regulatory mechanisms of arrestin-3 with arrestin-2.
Main Methods:
- Metabolic labeling and phosphoamino acid analysis to identify phosphorylation sites.
- Site-directed mutagenesis to create phosphorylation-mimicking and non-phosphorylatable mutants.
- In vitro kinase assays using purified Casein kinase II (CKII).
- Functional assays measuring receptor internalization and protein-protein interactions (clathrin, beta-adaptin, Src).
Main Results:
- Arrestin-3 is constitutively phosphorylated at Thr-382, and dephosphorylated upon beta(2)-adrenergic receptor activation.
- CKII is identified as the primary kinase responsible for arrestin-3 phosphorylation at Thr-382.
- Mutants mimicking phosphorylated or dephosphorylated states did not alter interactions with clathrin, beta-adaptin, or Src.
- Arrestin-3 phosphorylation may regulate the formation of large, multi-protein complexes.
Conclusions:
- Arrestin-3 phosphorylation at Thr-382 by CKII is regulated by GPCR activation.
- Unlike arrestin-2, arrestin-3 phosphorylation does not appear to directly modulate interactions with endocytic or signaling partners.
- Arrestin-3 phosphorylation might play a distinct role in regulating the assembly of larger signaling complexes, contributing to differential GPCR regulation by arrestin isoforms.