Activation of phosphatidylinositol 3-kinase is necessary for differentiation of FDC-P1 cells following stimulation of type III receptor tyrosine kinases.

Signaling molecules that are responsible for proliferation and differentiation of hematopoietic cells following ectopic expression of receptor tyrosine kinases (RTKs) were investigated in the interleukin 3 (IL-3)-dependent hematopoietic cell line, FDC-P1. Cells were transfected with human platelet-derived growth factor receptor (PDGF-R), macrophage colony stimulating factor-1 receptor (CSF-1R), epidermal growth factor receptor (EGF-R), and chimeras consisting of the extracellular domain of EGF-R and the transmembrane and cytoplasmic domains of either HER2 (HER1-2) or c-kit (EK-R). All FDC-P1 transfectants proliferated in response to the corresponding growth factor in the absence of IL-3. However, only cells expressing PDGF-R, CSF-1R, and EK-R (type III RTKs) differentiated along the monocyte-macrophage lineage after treatment with their activating ligands. Analysis of proteins from these RTK-expressing cells revealed that a Mr 85,000 protein showed in vitro phosphorylation, and V8 protease peptide mapping showed that this protein was p85, the regulatory subunit of phosphatidylinositol 3-kinase (PI3-kinase). Accordingly, activation of PDGF-R-, CSF-1R-, and EK-R-expressing cells led to an increase in PI3-kinase activity. Expression of EK-R mutant Y721F, which lacked the known p85 binding site, blocked differentiation and activation of PI3-kinase, without affecting proliferation. Last, addition of wortmannin to cells expressing PDGF-R, CSF-1R, and EK-R blocked ligand-induced differentiation in a concentration-dependent manner, and this effect correlated with wortmannin's ability to inhibit PI3-kinase. Thus, ectopic expression of both type I and III RTKs could stimulate FDC-P1 proliferation in the absence of IL-3; however, only activation of type III RTKs led to differentiation via selective coupling to p85 and PI3-kinase activation.