Proteomic analysis of macrophage differentiation. p46/52(Shc) Tyrosine phosphorylation is required for CSF-1-mediated macrophage differentiation.
Macrophage colony stimulating factor (M-CSF or CSF-1) acts to regulate the development and function of cells of the macrophage lineage. Murine myeloid FDC-P1 cells transfected with the CSF-1 receptor (FD/WT) adopt a macrophage-like morphology when cultured in CSF-1. This process is abrogated in FDC-P1 cells transfected with the CSF-1 receptor with a tyrosine to phenyalanine substitution at position 807 (FD/807), suggesting that a molecular interaction critical to differentiation signaling is lost (Bourette, R. P., Myles, G. M., Carlberg, K., Chen, A. R., and Rohrschneider, L. R. (1995) Cell Growth Differ. 6, 631--645). A detailed examination of lysates of CSF-1-treated FD/807 cells by two-dimensional SDS-polyacrylamide gel electrophoresis (PAGE) revealed a number of proteins whose degree of tyrosine phosphorylation was modulated by the Y807F mutation. Included in this category were three phosphorylated proteins that co-migrated with p46/52(Shc). Immunoprecipitation, Western blotting, and in vitro binding studies suggest that they are indeed p46/52(Shc). A key regulator of differentiation in a number of cell systems, ERK was observed to exhibit an activity that correlated with the relative degree of differentiation induced by CSF-1 in the two cell types. Transfection of cells with a non-tyrosine-phosphorylatable form of p46/52(Shc) prevented the normally observed CSF-1-mediated macrophage differentiation as determined by adoption of macrophage-like morphology and expression of the monocyte/macrophage lineage cell surface marker, Mac-1. These results are the first to suggest that p46/52(Shc) may play a role in CSF-1-induced macrophage differentiation. Additionally, a number of proteins were identified by two-dimensional SDS-PAGE whose degree of tyrosine phosphorylation is also modulated by the Y807F substitution. This group of molecules may contain novel signaling molecules important in macrophage differentiation.
Csar
X F
XF
Arthritis and Inflammation Research Centre, University of Melbourne, Department of Medicine, Royal Melbourne Hospital, Parkville, Victoria, Australia 3050. xfc@unimelb.edu.au
Wilson
N J
NJ
McMahon
K A
KA
Marks
D C
DC
Beecroft
T L
TL
Ward
A C
AC
Whitty
G A
GA
Kanangasundarum
V
V
Hamilton
J A
JA
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology; D019070:Cell Lineage / Q000502:physiology; D016173:Macrophage Colony-Stimulating Factor / Q000494:pharmacology; D008264:Macrophages / Q000166:cytology / Q000502:physiology*; D051379:Mice; D010766:Phosphorylation; D011506:Proteins / Q000502:physiology*; D055765:Shc Signaling Adaptor Proteins; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D014443:Tyrosine
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D011506:Proteins; D055765:Shc Signaling Adaptor Proteins; C528034:Shc1 protein, mouse; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D014443:Tyrosine; D016173:Macrophage Colony-Stimulating Factor