Regulation of arrestin-3 phosphorylation by casein kinase II.
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.
Kim
You-Me
YM
Department of Microbiology and Immunology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
Barak
Larry S
LS
Caron
Marc G
MG
Benovic
Jeffrey L
JL
D033966:Adaptor Protein Complex beta Subunits; D000595:Amino Acid Sequence; D000818:Animals; D019390:Arrestins / Q000737:chemistry* / Q000378:metabolism*; D019556:COS Cells; D047390:Casein Kinase II; D002460:Cell Line; D002966:Clathrin / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008565:Membrane Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D014176:Protein Biosynthesis; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D015398:Signal Transduction; D013347:Subcellular Fractions / Q000378:metabolism; D013912:Threonine / Q000737:chemistry; D013997:Time Factors; D014162:Transfection
D033966:Adaptor Protein Complex beta Subunits; D019390:Arrestins; D002966:Clathrin; D008565:Membrane Proteins; C082333:arrestin3; D013912:Threonine; D005982:Glutathione Transferase; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases; D019204:GTP-Binding Proteins