The Phosphatidylinositol-3 Kinase Signaling Pathway of Colony Stimulating Factor-1 (CSF-1)

By Lin Lin Mentor: Dr. Angel Lee Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO

Regulation of the process of cell growth and proliferation is a critical component of the biological processes in normal development. Without sufficient regulation, as a result of mutations in control mechanisms, uncontrolled cell proliferation leads to cancer. In order to better understand the normal regulatory process, Dr. Lee's lab is currently investigating the signaling pathways of growth factors that participate in the control of cell growth.

The focus of research is on the macrophage colony-stimulating factor, abbreviated MCSF or CSF-1. CSF- 1 is a hematopoietic growth factor that is primarily produced by fibroblasts and by the stromal layer in bone marrow. It stimulates the proliferation of mononuclear cells, specifically monocytes and macrophages. The corresponding receptor for CSF-1 is the CSF-1 cell-surface receptor (CSF-1R). CSF-1 binds to the ligand-binding domain of the CSF-1R and stimulates autophosphorylation of tyrosines in the cytoplasmic domain of CSF-1R, beginning a cascade of signals.

There is particular interest in one of the protein-lipid kinase enzymes activated by CSF-1, phosphatidylinositol-3 kinase (PI-3K). Pl-3K has long been known to be involved in various cellular responses to stimuli, but three recent discoveries have intensified interest in the function of PI-3K. First, Pl-3K has been shown to prevent apoptosis, or programmed cell death in several cell types. The second discovery involves a "retrovirus-encoded Pl-3K that was found to cause haemangiosarcomas in chickens and to transform fibroblasts" (Toker and Cantley, 1997). Third, "mutations in a Caenorhabditis elegans Pl-3K gene cause a threefold increase in the lifespan of the adult" (Toker, 673).

Three of the signaling pathways for CSF-1R have been previously mapped out, and the pathway beginning with the Y 721 motif has been shown to involve direct binding to PI-3K. However, when the Y721 motif was mutated and thus disabled, PI-3K, although it failed to bind to CSF-1R as expected, was still activated. A downstream molecule of PI-3K, the AKT protein, was also still activated. This implies that another pathway is also involved in the activation of PI-3 kinase. Because Src is a tyrosine kinase and has the Src Homology-2 Domain (SH2), it can bind to Y559 of CSF-1R

We are currently trying to map out the pathway beginning with Y559. It has been shown that Y559 might activate c-Src. There are three possible ways c-Src activates Pl-3K. First, Src may directly activate PI-3K. This has been shown in one specific case but the generality of this mechanism is not clear. Second, Src may activate an intermediary molecule, the Cbl protein; then Cbl, in turn, binds and activates PI-3K. The third possibility is that the Gab protein may act as an intermediary between Src and Pl-3K.

I investigated the possibility of the involvement of Cbl as the intermediary protein. The goal of my experiment was to determine whether Cbl directly activated Pl-3K and whether, in turn, Pl-3K activated AKT.

First, I worked on expressing the wildtype HA-tagged Cbl gene in the vector pSRa in 32D mammalian cells. To determine that Cbl proteins were produced from the Cbl gene in pSRa, the plasmid pSRa with the insert HAc-cbl was transformed into DH5a bacteria. High quality DNA was prepared using the Qiagen column and transfected into 32D cells. Total cell lysates were prepared and analyzed by Western blotting with aHA and aCbl antibodies.

Then I worked on determining if the Cbl protein directly bound Pl-3K by co-immunoprecipitating Cbl and the p85 subunit of Pl-3K. The p85 subunit also has a SH2 domain that has been shown to bind to Y731 in Cbl. However, I was unable to detect an interaction, possibly because immunoprecipitation conditions were not optimized. In addition to Y731, tyrosines Y700 and Y774 on Cbl are also known to become phosphorylated in response to growth factor stimuli. In order to pinpoint the point of binding between Cbl and Pl-3K, all three motifs Y700F, 731F, 774F were mutated. I also worked on subcloning the mutated c-Cbl gene (mutated at Y700F/731F/774F) from vector pAlter into vector pSRa. Unfortunately, the insert was cloned in the wrong orientation. Further research needs to be done to determine whether Cbl is the intermediary between the c-Src and PI-3K.