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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

Baran, Christopher Phillip
http://rave.ohiolink.edu/etdc/view?acc_num=osu1050605166

Abstract Details

2003, Doctor of Philosophy, Ohio State University, Ohio State Biochemistry Program.
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-KB-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.
Clay Marsh (Advisor)
Health Sciences, Immunology
SHIP-1; Lyn; M-CSF Receptor; Akt; tyrosine-699

Recommended Citations

Citations

  • Baran, C. P. (2003). 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 [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1050605166

    APA Style (7th edition)

  • Baran, Christopher. 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. 2003. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1050605166.

    MLA Style (8th edition)

  • Baran, Christopher. "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." Doctoral dissertation, Ohio State University, 2003. http://rave.ohiolink.edu/etdc/view?acc_num=osu1050605166

    Chicago Manual of Style (17th edition)

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This open access ETD is published by The Ohio State University and OhioLINK.