Identification and subcellular localization of proteins that are rapidly phosphorylated in tyrosine in response to colony-stimulating factor 1.
To investigate growth factor-mediated signal transduction, we have studied phosphorylation events that take place within seconds of the binding of colony-stimulating factor 1 (CSF-1) to its cell-surface receptor. CSF-1 stimulated rapid tyrosine phosphorylation of cellular proteins in murine BAC1.2F5 macrophages at 37 degrees C and 4 degrees C. The pattern of CSF-1-stimulated tyrosine phosphorylation of at least 15 different proteins at both temperatures was similar and unchanged by treatment of the lysate with reducing agent. With the exception of the 185-kDa CSF-1 receptor, a 260-kDa protein and a 133-kDa protein, the proteins were predominantly cytoplasmic. At 37 degrees C, all the proteins were phosphorylated within 30 sec of addition of growth factor. At 4 degrees C, CSF-1 receptor sites were saturated after 2 min of incubation in the presence of high concentrations of CSF-1 and differences in the order of appearance of phosphorylated proteins were observed: 185 kDa (CSF-1 receptor) (by 2 min); 99 kDa (by 4 min); 125 kDa (by 10 min); 61 kDa (by 30 min); and 260 kDa, 84 kDa, and 41 kDa (by 180 min). In addition to stimulating the phosphorylation of these proteins in tyrosine, CSF-1 caused dephosphorylation of phosphorylated serine residues on the receptor. As neither CSF-1 nor its receptor is internalized at 4 degrees C, analysis of these early reactions and the phosphotyrosine-containing proteins in intact cells under these conditions should lead to an understanding of the early events in growth factor receptor-mediated signal transduction.
Sengupta
A
A
Department of Developmental Biology and Cancer, Albert Einstein College of Medicine, Bronx, NY 10461.
Liu
W K
WK
Yeung
Y G
YG
Yeung
D C
DC
Frackelton
A R
AR
Stanley
E R
ER
D000818:Animals; D003115:Colony-Stimulating Factors / Q000494:pharmacology*; D008264:Macrophages / Q000378:metabolism; D051379:Mice; D008970:Molecular Weight; D010766:Phosphorylation; D019000:Phosphotyrosine; D011506:Proteins / Q000032:analysis*; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D016186:Receptor, Macrophage Colony-Stimulating Factor; D015398:Signal Transduction; D013347:Subcellular Fractions / Q000032:analysis*; D013696:Temperature; D014443:Tyrosine / Q000031:analogs & derivatives / Q000032:analysis / Q000378:metabolism*
D003115:Colony-Stimulating Factors; D011506:Proteins; D011518:Proto-Oncogene Proteins; D019000:Phosphotyrosine; D014443:Tyrosine; D016186:Receptor, Macrophage Colony-Stimulating Factor