Xavier F. Csar

Direct binding of Shc, Grb2, SHP-2 and p40 to the murine granulocyte colony-stimulating factor receptor.

Granulocyte colony-stimulating factor (G-CSF) mediates the proliferation, differentiation and activation of cells in the granulocytic lineage. However, knowledge about the specific signaling pathways utilized by the G-CSF receptor (G-CSF-R) upon ligand binding remains limited. In this report, we show rapid phosphorylation of Shc upon stimulation of NFS-60 cells with G-CSF, and inducible association of Shc and Grb2 with the G-CSF-R in these cells. Using a tyrosine-phosphorylated GST-G-CSF-R fusion we demonstrate that Shc, Grb2 and SHP-2 directly bind the receptor via their respective SH2 domains, suggesting multiple routes of MAPK activation from the G-CSF-R are possible. In addition, we have identified an unknown p40 molecule which is associated with the G-CSF-R transiently following G-CSF stimulation, and a constitutively-associated p37 molecule.

Copper/zinc superoxide dismutase is phosphorylated and modulated specifically by granulocyte-colony stimulating factor in myeloid cells.

Using two‐dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (2‐D SDS‐PAGE) of 32P‐labeled cytosolic and membrane extracts, we identified a 21.5 kDa phosphoprotein with an isoelectric point of 6.0 in NFS‐60 cells that was phosphorylated maximally at 15 min by treatment with granulocyte‐colony stimulating factor (G‐CSF) but not with interlevkin‐3 (IL‐3) or colony‐stimulating factor‐1 (macrophage‐colony stimulating factor (CSF‐1 (M‐CSF)). The phosphorylation of this protein, designated 21.5/6.0, was unaffected by a series of antiproliferative agents [32]. These findings suggested that the 21.5/6.0 phosphoprotein may be involved in specific G‐CSF‐mediated biological responses such as activation and/or differentiation. We sought to characterize this 21.5/6.0 by a novel combination of 2‐D SDS‐PAGE and hydroxyapatite (HTP)‐chromatography. Amino acid sequence determination of 21.5/6.0 revealed it to share a high level of homology with copper/zinc superoxide dismutase (Cu/Zn‐SOD), indicating that a Cu/Zn‐SOD is phosphorylated following treatment with G‐CSF. This is the first report of the phosphorylation and possible involvement of Cu/Zn‐SOD protein in granulocyte activation/differentiation events. In addition, Cu/Zn‐SOD levels and activity were diminished by G‐CSF but not IL‐3 treatment. This new protocol combining 2‐D SDS‐PAGE and HTP‐chromatography allows the characterization of low abundance phosphoproteins involved in the cellular responses to G‐CSF and presumably to other cytokines/growth factors.

Colony-stimulating factor-1 (CSF-1) receptor-mediated macrophage differentiation in myeloid cells: a role for tyrosine 559-dependent protein phosphatase 2A (PP2A) activity.

M1 myeloid cells transfected with the wild-type (WT) colony-stimulating factor-1 (CSF-1) receptor (CSF-1R; M1/WT cells) undergo CSF-1-dependent macrophage differentiation. By mutation studies, we have provided prior evidence that tyrosine 559 in the CSF-1R cytoplasmic domain governs the Src-dependent differentiation pathway. Further components of this pathway were then sought. We report that the extent of CSF-1-mediated tyrosine phosphorylation of protein phosphatase 2A (PP2A), and the associated loss of its activity were reduced in M1 cells transfected with the CSF-1R with a tyrosine-to-phenylalanine mutation at position 559 (M1/559 cells), compared with the corresponding responses in CSF-1-treated M1/WT cells. This evidence for an involvement of a reduction in PP2A activity in the differentiation process was supported by the restoration of the defect in the CSF-1-mediated differentiation of M1/559 cells by the addition of the PP2A inhibitor, okadaic acid. It was also found that the degree of activation of extracellular-signal-regulated kinase (ERK) activities by CSF-1 was reduced in M1/559 cells, suggesting their involvement in the differentiation process. These data suggest that PP2A and ERK form part of the Src-dependent signal-transduction cascade governing CSF-1-mediated macrophage differentiation in M1 cells.

A novel 110 kDa form of myosin XVIIIA (MysPDZ) is tyrosine-phosphorylated after colony-stimulating factor-1 receptor signalling.

Macrophage colony-stimulating factor (M-CSF or CSF-1) controls the development of macrophage lineage cells via activation of its tyrosine kinase receptor, c-Fms. After adding CSF-1 to M1 myeloid cells expressing CSF-1R (CSF-1 receptor), tyrosine phosphorylation of many cellular proteins occurs, which might be linked to subsequent macrophage differentiation. The biological significance and characterization of such proteins were explored by a dual strategy comprising two-dimensional SDS/PAGE analysis of cell lysates of CSF-1-treated M1 cells expressing the wild-type or a mutated receptor, together with an enrichment strategy involving a tyrosine-phosphorylated receptor construct. In the present study, we report the identification by MS of a novel, low-abundance, 110 kDa form of myosin XVIIIA (MysPDZ, myosin containing PDZ domain), which appears to be preferentially tyrosine-phosphorylated after CSF-1R activation when compared with other known isoforms. Receptor mutation studies indicate that CSF-1R-dependent tyrosine phosphorylation of p110myosin XVIIIA requires Tyr-559 in the cytoplasmic domain of the receptor and is therefore Src-family kinase-dependent. Gelsolin, Erp61 protein disulphide-isomerase and possibly non-muscle myosin IIA were also tyrosine-phosphorylated under similar conditions. Similar to the more abundant p190 isoform, p110 myosin XVIIIA lacks a PDZ domain and, in addition, it may lack motor activity. The phosphorylation of p110 myosin XVIIIA by CSF-1 may alter its cellular localization or target its association with other proteins.