Differential influence of tyrosine residues of the common receptor beta subunit on multiple signals induced by human GM-CSF.
BACKGROUND
GM-CSF induces the proliferation and differentiation of a broad range of hematopoietic cells. Stimulation with this cytokine results in the induction of rapid tyrosine phosphorylation of cellular proteins, including the common beta subunit of its receptor. However, the significance of beta subunit phosphorylation with regard to regulation of signaling molecules and the functions of GM-CSF is less well understood.


OBJECTIVE
To investigate the role of cytoplasmic tyrosine residues, we performed mutational analyses of the beta subunit.


METHODS
A series of beta subunit mutants with 1, some, or all of 8 cytoplasmic tyrosines converted to phenylalanines were constructed. These beta subunit mutants were expressed in murine BA/F3 cells together with the wild-type human alpha subunit, and their potentials to transmit signals were examined.


RESULTS
A mutant beta subunit lacking all cytoplasmic tyrosines (Fall) activated JAK2, but not Shc, SHP-2, the Raf/ERK cascade, signal transducer and activator of transcription 5 (STAT5), or the c-fos promoter, nor did it effectively induce proliferation. Adding back each tyrosine to Fall revealed that (1) Tyr577 was necessary and sufficient for Shc phosphorylation; (2) Tyr577, Tyr612, and Tyr695 were involved in activation of SHP-2, the Raf/ERK cascade, and c-fos transcription; and (3) all tyrosines, but particularly Tyr612, Tyr695, Tyr750, and Tyr806, facilitated STAT5 activation. Impaired cell growth was also partly restored by any of the tyrosines. Interestingly, the Fall mutant was still capable of suppressing apoptosis and maintaining cell viability.


CONCLUSIONS
These results provide evidence that beta subunit tyrosines possess distinct, yet overlapping, roles in the activation of multiple signaling pathways induced by GM-CSF. In addition, GM-CSF may activate both beta subunit tyrosine-dependent and -independent pathways, with the latter being sufficient to transmit survival signals.
D000818:Animals; D002470:Cell Survival / Q000187:drug effects; D004268:DNA-Binding Proteins / Q000235:genetics; D005786:Gene Expression Regulation; D016762:Genes, fos / Q000235:genetics; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000502:physiology*; D006801:Humans; D051379:Mice; D008894:Milk Proteins; D011401:Promoter Regions, Genetic / Q000235:genetics; D016028:Receptors, Leukocyte-Adhesion / Q000187:drug effects*; D050799:STAT5 Transcription Factor; D015398:Signal Transduction; D015534:Trans-Activators / Q000235:genetics; D014443:Tyrosine / Q000502:physiology*