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Beta-chemokine receptor CCR5 signals through SHP1, SHP2, and Syk

R K Ganju1, S A Brubaker, R D Chernock

  • 1Division of Experimental Medicine, Hematology/Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02115, USA. rganju@caregroup.harvard.edu

The Journal of Biological Chemistry
|April 5, 2000
PubMed

Insights

The beta-chemokine receptor CCR5, a target for HIV, activates signaling pathways involving RAFTK and Syk kinases. This study identifies a signaling complex including RAFTK, Syk, SHP1, and Grb2, crucial for MIP1beta-induced cell migration.

Area of Science:

  • Immunology
  • Cell Biology
  • Molecular Signaling

Background:

  • The beta-chemokine receptor CCR5 plays a role in cell migration, proliferation, and immune responses.
  • CCR5 also functions as a co-receptor for the human immunodeficiency virus (HIV).
  • Previous research indicated CCR5 activates related adhesion focal tyrosine kinase (RAFTK)/Pyk2/CAK-beta.

Purpose of the Study:

  • To further characterize signaling molecules activated by CCR5 upon binding to its ligand, macrophage inflammatory protein-1beta (MIP1beta).
  • To elucidate the roles of phosphatases SHP1 and SHP2, and the kinase Syk in CCR5-mediated signaling.
  • To determine the upstream or downstream relationship between RAFTK and Syk in this pathway.

Main Methods:

  • Stimulation of CCR5-expressing cells (L1.2 transfectants and T-cells) with MIP1beta.
  • Analysis of tyrosine phosphorylation of SHP1 and SHP2.
  • Co-immunoprecipitation assays to study protein-protein interactions (SHP1-RAFTK, SHP1/SHP2-Grb2-Syk, Syk-RAFTK).
  • Use of dominant-negative mutants of RAFTK (RAFTK(m906), RAFTK(m402)) and phosphatase inhibitor (orthovanadate).
  • Assessment of MIP1beta-induced chemotaxis.

Main Results:

  • MIP1beta stimulation enhanced tyrosine phosphorylation of SHP1 and SHP2 in CCR5-expressing cells.
  • SHP1 associated with RAFTK, but RAFTK did not mediate SHP1/SHP2 phosphorylation.
  • SHP1 and SHP2 associated with Grb2 and Syk; Syk was activated by MIP1beta and associated with RAFTK.
  • Dominant-negative RAFTK mutants attenuated Syk activation, while wild-type RAFTK enhanced it, indicating RAFTK acts upstream of Syk.
  • Orthovanadate pretreatment abolished MIP1beta-induced chemotaxis.

Conclusions:

  • MIP1beta stimulation of CCR5 leads to the activation of phosphatases SHP1 and SHP2.
  • The signaling pathway involves the formation of a complex including RAFTK, Syk, SHP1, and Grb2.
  • RAFTK acts upstream of CCR5-mediated Syk activation, playing a key role in MIP1beta-induced cell migration.

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