Site-specific Phosphorylation of CXCR4 Is Dynamically Regulated by Multiple Kinases and Results in Differential Modulation of CXCR4 Signaling

• Published in: The Journal of biological chemistry Vol. 285; no. 10; pp. 7805 - 7817
• Main Authors: Busillo, John M., Armando, Sylvain, Sengupta, Rajarshi, Meucci, Olimpia, Bouvier, Michel, Benovic, Jeffrey L.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 05.03.2010; American Society for Biochemistry and Molecular Biology
• Subjects: Amino Acid Sequence; Animals; Antibodies, Phospho-Specific - metabolism; Arrestin; Arrestins - genetics; Arrestins - metabolism; Astrocytes - cytology; Astrocytes - metabolism; Calcium; Cell Line; Chemokines; ERK; Extracellular Signal-Regulated MAP Kinases - genetics; Extracellular Signal-Regulated MAP Kinases - metabolism; G Protein-coupled Receptors (GPCR); G-Protein-Coupled Receptor Kinases - genetics; G-Protein-Coupled Receptor Kinases - metabolism; Humans; Isoenzymes - genetics; Isoenzymes - metabolism; Molecular Sequence Data; Phosphorylation; Protein Kinase C - genetics; Protein Kinase C - metabolism; Receptor Regulation; Receptor Serine Threonine Kinase; Receptors, CXCR4 - genetics; Receptors, CXCR4 - metabolism; RNA, Small Interfering - genetics; RNA, Small Interfering - metabolism; Signal Transduction; Signal Transduction - physiology; Tandem Mass Spectrometry; Receptor Regulation; G Protein-coupled Receptors (GPCR); Receptor Serine Threonine Kinase; Calcium; Signal Transduction; Arrestin; Chemokines; ERK
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M109.091173

The chemokine receptor CXCR4 is a widely expressed G protein-coupled receptor that has been implicated in a number of diseases including human immunodeficiency virus, cancer, and WHIM syndrome, with the latter two involving dysregulation of CXCR4 signaling. To better understand the role of phosphorylation in regulating CXCR4 signaling, tandem mass spectrometry and phospho-specific antibodies were used to identify sites of agonist-promoted phosphorylation. These studies demonstrated that Ser-321, Ser-324, Ser-325, Ser-330, Ser-339, and two sites between Ser-346 and Ser-352 were phosphorylated in HEK293 cells. We show that Ser-324/5 was rapidly phosphorylated by protein kinase C and G protein-coupled receptor kinase 6 (GRK6) upon CXCL12 treatment, whereas Ser-339 was specifically and rapidly phosphorylated by GRK6. Ser-330 was also phosphorylated by GRK6, albeit with slower kinetics. Similar results were observed in human astroglia cells, where endogenous CXCR4 was rapidly phosphorylated on Ser-324/5 by protein kinase C after CXCL12 treatment, whereas Ser-330 was slowly phosphorylated. Analysis of CXCR4 signaling in HEK293 cells revealed that calcium mobilization was primarily negatively regulated by GRK2, GRK6, and arrestin3, whereas GRK3, GRK6, and arrestin2 played a primary role in positively regulating ERK1/2 activation. In contrast, GRK2 appeared to play a negative role in ERK1/2 activation. Finally, we show that arrestin association with CXCR4 is primarily driven by the phosphorylation of far C-terminal residues on the receptor. These studies reveal that site-specific phosphorylation of CXCR4 is dynamically regulated by multiple kinases resulting in both positive and negative modulation of CXCR4 signaling.