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PMID: 20048153 Published · ppublish English Journal Article Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't

Site-specific phosphorylation of CXCR4 is dynamically regulated by multiple kinases and results in differential modulation of CXCR4 signaling.

The Journal of biological chemistry ·Vol. 285 ·No. 10 ·2010-03-05 ·Pages 7805-17

Busillo JM, Armando S, Sengupta R, Meucci O, Bouvier M, Benovic JL

Abstract

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.

MeSH Terms
Amino Acid Sequence Animals Antibodies, Phospho-Specific/metabolism Arrestins/genetics,metabolism Astrocytes/cytology,metabolism Cell Line Extracellular Signal-Regulated MAP Kinases/genetics,metabolism G-Protein-Coupled Receptor Kinases/genetics,metabolism Humans Isoenzymes/genetics,metabolism Molecular Sequence Data Phosphorylation Protein Kinase C/genetics,metabolism RNA, Small Interfering/genetics,metabolism Receptors, CXCR4/genetics,metabolism Signal Transduction/physiology Tandem Mass Spectrometry
Chemicals
Antibodies, Phospho-Specific Arrestins Isoenzymes RNA, Small Interfering Receptors, CXCR4 Protein Kinase C G-Protein-Coupled Receptor Kinases G-protein-coupled receptor kinase 6 Extracellular Signal-Regulated MAP Kinases
Authors & Affiliations
6 authors, click to expand affiliations / ORCID
Busillo John M
Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
Armando Sylvain
Sengupta Rajarshi
Meucci Olimpia
Bouvier Michel
Benovic Jeffrey L
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Article Info
Journal
The Journal of biological chemistry
Abbr.
J Biol Chem
ISSN
1083-351X
DOI
10.1074/jbc.M109.091173
Published
2010-03-05
Epub
2010-00-04
Pages
7805-17
Language
English
Region
United States
NLM ID
2985121R
PMCID
PMC2844224
Subset
IM
Grants
NIDA NIH HHS · DA15014 · United States
NIDA NIH HHS · R01 DA015014 · United States
NIGMS NIH HHS · R01 GM044944 · United States
NIDA NIH HHS · R01 DA019808 · United States
NIDA NIH HHS · DA19808 · United States
NIGMS NIH HHS · R01 GM047417 · United States
NIGMS NIH HHS · R37 GM047417 · United States
NCI NIH HHS · R01 CA129626 · United States
NIGMS NIH HHS · GM47417 · United States
NCI NIH HHS · CA129626 · United States
NIGMS NIH HHS · GM44944 · United States
External Links
PubMed source DOI: 10.1074/jbc.M109.091173 PMC Full Text View journal details
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