Multiple Signals Mediate Proliferation, Differentiation, and Survival from the Granulocyte Colony-stimulating Factor Receptor in Myeloid 32D Cells

Abstract

Granulocyte colony-stimulating factor (G-CSF) regulates neutrophil production through activation of its cognate receptor, the G-CSF-R. Previous studies with deletion mutants have shown that the membrane-proximal cytoplasmic domain of the receptor is sufficient for mitogenic signaling, whereas the membrane-distal domain is required for differentiation signaling. However, the function of the four cytoplasmic tyrosines of the G-CSF-R in the control of proliferation, differentiation, and survival has remained unclear. Here we investigated the role of these tyrosines by expressing a tyrosine "null" mutant and single tyrosine "add back" mutants in maturation-competent myeloid 32D cells. Clones expressing the null mutant showed only minimal proliferation and differentiation, with survival also reduced at low G-CSF concentrations. Analysis of clones expressing the add-back mutants revealed that multiple tyrosines contribute to proliferation, differentiation, and survival signals from the G-CSF-R. Analysis of signaling pathways downstream of these tyrosines suggested a positive role for STAT3 activation in both differentiation and survival signaling, whereas SHP-2, Grb2 and Shc appear important for proliferation signaling. In addition, we show that a tyrosine-independent "differentiation domain" in the membrane-distal region of the G-CSF-R appears necessary but not sufficient for mediating neutrophilic differentiation in these cells.

STAT3-mediated differentiation and survival of myeloid cells in response to granulocyte colony-stimulating factor: role for the cyclin-dependent kinase inhibitor p27Kip1

“…Mutant mO, which hardly activated STAT3, also failed to induce p27 expression (Figure 7). Importantly, mO supported neither di erentiation nor proliferation (Ward et al, 1999b), showing that the inability to induce proliferation does not automatically result in p27 expression. In clones expressing the`add-back' mutants, G-CSFinduced expression of p27 protein was greatest with mA (Y704) and mC (Y744).…”

“…Several independent clones were expanded for further analysis. Establishment of 32D.8.6 transfectants expressing di erent tyrosine-to-phenylalanine (Y-to-F) substitution mutants of the G-CSF-R has been described previously (Ward et al, 1999b). Clones expressing a quadruple Y-to-F or`null' mutant, with no cytoplasmic tyrosines (mO), or a series of triple Y-to-F or`add-back' mutants, which each retain a single cytoplasmic tyrosine (mA, mB, mC, mD) were used.…”

“…Expression in 32D cells revealed that signals from the G-CSF-R for di erentiation and survival are mediated most strongly by Y704 (mA) and Y744 (mC) (Ward et al, 1999b). Strikingly, the di erentiation and survivalinducing capacity of these mutants correlated strongly with their ability to activate STAT3 upon G-CSF stimulation, as measured by both tyrosine and serine phosphorylation, consistent with the observation that overexpression of DN-STAT3 totally blocked neutrophilic di erentiation and impaired survival of 32D cells (Figures 3 ± 5).…”

“…We and others have recently reported that Y704 and Y744 are involved in recruitment and activation of STAT3 (Chakraborty et al, 1999;de Koning et al, 1996a;Ward et al, 1999a). These tyrosines are also important for di erentiation and survival signals from the G-CSF-R (Ward et al, 1999b). …”

Multiple pathways contribute to the hyperproliferative responses from truncated granulocyte colony-stimulating factor receptors

“…In addition, the cytoplasmic domain of the human G-CSF-R contains four conserved tyrosine residues (Y704, Y729, Y744 and Y764) that serve as potential docking sites for Src homology 2 (SH2) domains of signaling proteins. 2,3 Pathways known to be stimulated by the G-CSF-R include the Janus kinase (JAK)/signal transducers and activators of transcription (STAT) pathway, [4][5][6][7][8] the Ras/Raf/MEK/mitogen-activated protein kinase (MAPK) pathway [9][10][11][12][13] and the phosphatidylinositol (PI) 3-kinase/Akt pathway. [14][15][16] We have previously identified a subset of severe congenital neutropenia (SCN) patients that harbor acquired mutations in the gene encoding the G-CSF-R, which serve to truncate the carboxyl-terminus of the receptor.…”