Tyrosine 729 of the G‐CSF receptor controls the duration of receptor signaling: involvement of SOCS3 and SOCS1

Mutations in the granulocyte‐colony stimulating factor receptor (G‐CSF‐R) gene resulting in carboxy terminal truncation have been associated with acute myeloid leukemia (AML). The truncated G‐CSF‐R from AML patients mediate enhanced and prolonged activation of signal transducer and activator of transcription 5 (Stat5). It has been shown that Src homology‐2 (SH2)‐containng tyrosine phosphatase‐1 attenuates the intensity of G‐CSF‐induced Stat5 activation through interacting with the carboxy terminus of the G‐CSF‐R. Using a series of tyrosine‐to‐phenylalanine substitution mutants, we show here that tyrosine (Tyr) 729, located in the carboxy terminus of the G‐CSF‐R, controls the duration of G‐CSF‐stimulated activation of Stat5, Akt, and extracellular signal‐regulated kinase 1/2. It is interesting that activation of these signaling molecules by G‐CSF was prolonged by pretreating cells with actinomycin D or cyclohexamide, suggesting that de novo protein synthesis is required for appropriate termination of G‐CSF‐R signaling. The transcripts for suppressor of cytokine signaling 3 (SOCS3) and SOCS1 were up‐regulated rapidly upon G‐CSF stimulation. Expression of SOCS3 or SOCS1, but not SOCS2 and cytokine‐inducible SH2 domain‐containing protein, completely suppressed G‐CSF‐induced Stat5 activation but had only a weak effect on Stat5 activation mediated by the receptor mutant lacking Tyr 729. SOCS1 and SOCS3 also inhibited G‐CSF‐dependent cell proliferation, but the inhibitory effect of the two SOCS proteins on cell proliferation was diminished when Tyr 729 of the G‐CSF‐R was mutated. These data indicate that Tyr 729 of the G‐CSF‐R is required for SOCS1‐ and SOCS3‐mediated negative regulation of G‐CSF‐R signaling and that the duration and intensity of G‐CSF‐induced Stat5 activation are regulated by two distinct mechanisms.