CHARACTERIZATION OF SIGNALS LINKING THE MACROPHAGE COLONY-STIMULATING FACTOR RECEPTOR TO THE FOCAL ADHESION KINASE

The survival, proliferation and differentiation of mononuclear phagocytes are primarily regulated by macrophage colony-stimulating factor (MCSF). MCSF also regulates macrophage morphology and induces chemotaxis. The effects of MCSF are mediated through its specific binding to a transmembrane protein tyrosine kinase receptor (MCSFR). p125FAK is a unique protein tyrosine kinase which is phosphorylated and activated following the engagement of integrins with the extracellular matrix. This study was undertaken to characterize the mechanisms responsible for p125FAK phosphorylation following MCSF treatment. We found that in the murine macrophage cell line BAC1.2F5 the tyrosine phosphorylation of p125FAK induced by MCSF treatment is rapid (peak after 3-5 minutes) and transient (back to basal level after 30 minutes). By means of antibodies reacting specifically against single p125FAK phospho-tyrosine residues, we found that all the known p125FAK tyrosine residues are phosphorylated following MCSF treatment. In particular, the phosphorylation of Y397 and Y577, which are related to p125FAK activity, is prolonged for at least 30 min, while tyrosine phosphorylation of other residues involved in signaling dowstream p125FAK is more transient. Furthermore, MCSF determined p125FAK redistribution from the cytosol to the membrane and cytoskeleton fractions. Since MCSFR also redistributes to the latter fraction under the same conditions, we looked for an interaction between the two proteins, and we found that they co-immunoprecipitate. We also found that Src is involved in the MCSF-induced p125FAK phosphorylation. Indeed, the activated form of Src is also redistributed to the cytoskeleton following MCSF, and pretreatment with PP2, a specific Src inhibitor, abrogates MCSF-induced p125FAK phosphorylation. p125FAK phosphorylation does not occur in the presence of cytocalasin- D, which interferes with cytoskeleton organization. Finally, we found that the MAPK ERK is also involved in p125FAK phosphorylation, which was partially abrogated by the pretreatment with PD98059, a specific ERK inhibitor. Our results suggest the following scenario: MCSF-induced redistribution of p125FAK to the cytoskeleton induces p125FAK autophosphorylation in Y397, the first step of p125FAK activation. Src, which is also redistributed to the cytoskeleton following MCSF treatment, phosphorylates p125FAK further, which is therefore fully activated. p125FAK is then able to trigger the known downstream pathways involved in the control of cell morphology and motility. These results contribute to the understanding of the mechanism of p125FAK phosphorylation elicited by tyrosine kinase receptors in general (and MCSFR in particular), which is less characterized with respect to that elicited by integrin clustering.