The involvement of Lyn and the SH2-domain-containing inositol 5'-phosphatase 1 (SHIP1) in the negative regulation of M-CSF-induced cellular signaling events

Human monocytes have an average lifespan of 24–72 hours. In the absence of growth factors, such as Macrophage Colony-Stimulating Factor (M-CSF), monocytes undergo apoptosis. Upon encountering M-CSF, monocytes undergo a series of cellular signaling events, including phosphorylation of the M-CSF-Receptor (M-CSF-R) and the activation of Akt. However, the regulation of these events is not completely understood.; Akt activation has been linked to increased levels of PIP3 that occur as a result of cellular activation. The 5′-Inositol phosphatase SHIP1 regulates Akt activity by converting PIP3 to PIP2. Therefore, I hypothesized that SHIP1 was involved in the regulation of M-CSF-R-induced Akt activation.; To investigate this hypothesis, I used the human monocytic cell-line THP-1 and found that SHIP1 bound indirectly to tyrosine-699 of the M-CSF-R via the SH3 domain of the adaptor protein Grb2. SHIP1 also became tyrosine phosphorylated following M-CSF activation and occurred in a Src-family kinase dependent manner. Interestingly, SHIP1 bound the Src-kinase Lyn following M-CSF activation, indicating that the phosphorylation of SHIP1 and the binding of SHIP1 to Lyn was M-CSF-dependent.; Utilizing GST fusion proteins, I show that the SHIP1-SH2 domain bound Lyn only after M-CSF activation of THP-1 cells. Furthermore, the associations of SHIP1 and Lyn and the SHIP1-SH2 domain and Lyn are independent of the kinase activity of Lyn, as PP2 did not inhibit these protein interactions. I also performed transient transfections of THP-1 cells with active SHIP1, which reduced NF-κB-dependent transcriptional activation of a reporter gene, and the SHIP1-SH2 domain, which increased the activation of the reporter gene. This further substantiated the role of SHIP1 and its SH2 domain in regulating Akt activity in monocytes.; After surveying the amino acid sequence of Lyn, I noticed an ITIM-like motif surrounding tyrosine-438. By utilizing in vitro affinity binding experiments, I provide evidence that the SH2 domain of SHIP1 bound to a phosphopeptide encompassing this novel ITIM-like domain in Lyn.; Finally, macrophages isolated from both SHIP1 and Lyn-deficient mice exhibited prolonged phosphorylation and activation of Akt after M-CSF stimulation. These data provide evidence of the involvement of SHIP1 and Lyn in the regulation of M-CSF-R-induced Akt activation.