Hierarchical organization of multi-site phosphorylation at the CXCR4 C terminus

The chemokine receptor CXCR4 regulates cell migration during ontogenesis and disease states including cancer and inflammation. Upon stimulation by the endogenous ligand CXCL12, CXCR4 becomes phosphorylated at multiple sites in its C-terminal domain. Mutations in the CXCR4 gene affecting C-terminal phosphorylation sites are a hallmark of WHIM syndrome, a genetic disorder characterized by a gain-of-CXCR4-function. To better understand how multi-site phosphorylation of CXCR4 is organized and how perturbed phosphorylation might affect CXCR4 function, we developed novel phosphosite-specific CXCR4 antibodies and studied the differential regulation and interaction of three C-terminal phosphorylation sites in human embryonic kidney cells (HEK293). CXCL12 promoted a robust phosphorylation at S346/347 which preceded phosphorylation at S324/325 and S338/339. After CXCL12 washout, the phosphosites S338/339 and S324/325 were rapidly dephosphorylated whereas phosphorylation at S346/347 was long-lasting. CXCL12-induced phosphorylation at S346/347 was staurosporine-insensitive and mediated by GRK2/3. WHIM syndrome-associated CXCR4 truncation mutants lacking the S346/347 phosphosite and the recently identified E343K WHIM mutant displayed strongly impaired phosphorylation at S324/325 and S338/339 as well as reduced CXCL12-induced receptor internalization. Relevance of the S346-S348 site was confirmed by a S346-348A mutant showing strongly impaired CXCL12-promoted phosphorylation at S324/325 and S338/339, defective internalization, gain of calcium mobilization, and reduced desensitization. Thus, the triple serine motif S346-S348 contains a major initial CXCR4 phosphorylation site and is required for efficient subsequent multi-site phosphorylation and receptor regulation. Hierarchical organization of CXCR4 phosphorylation explains why small deletions at the extreme CXCR4 C terminus typically associated with WHIM syndrome severely alter CXCR4 function.