The inositol 5'-phosphatase SHIP-1 and the Src kinase Lyn negatively regulate macrophage colony-stimulating factor-induced Akt activity.
Upon encountering macrophage colony-stimulating factor (M-CSF), human monocytes undergo a series of cellular signaling events leading to an increase in Akt activity. However, the regulation of these events is not completely understood. Because the inositol 5'-phosphatase SHIP-1 is an important regulator of intracellular levels of phosphatidylinositol 3,4,5-trisphosphate, an important second messenger necessary for Akt activation, we hypothesized that SHIP-1 was involved in the regulation of M-CSF receptor (M-CSF-R)-induced Akt activation. In the human monocytic cell line, THP-1, SHIP-1 became tyrosine-phosphorylated following M-CSF activation in a Src family kinase-dependent manner. Transfection of 3T3-Fms cells, which express the human M-CSF-R, with wild-type SHIP-1 showed that SHIP-1 was necessary for the negative regulation of M-CSF-induced Akt activation. In THP-1 cells, SHIP-1 bound Lyn, independent of the kinase activity of Lyn, following M-CSF activation. Utilizing a glutathione S-transferase fusion protein, we found that SHIP-1 bound to Lyn via the SHIP-1 Src homology 2 domain. Furthermore, transfection of THP-1 cells with a wild-type SHIP-1 construct reduced NF-kappaB-dependent transcriptional activation of a reporter gene, whereas a SHIP-1 Src homology 2 domain construct resulted in an increase in NF-kappaB activation. Additionally, in 3T3-Fms cells, Lyn enhanced the ability of SHIP-1 to regulate Akt activation by stabilizing SHIP-1 at the cellular membrane. Finally, macrophages isolated from both SHIP-1- and Lyn-deficient mice exhibited enhanced Akt phosphorylation following M-CSF stimulation. These data provide the first evidence of the involvement of both SHIP-1 and Lyn in the negative regulation of M-CSF-R-induced Akt activation.
Baran
Christopher P
CP
Dorothy M. Davis Heart and Lung Research Institute, Ohio State University, Columbus 43210, USA.
Tridandapani
Susheela
S
Helgason
Cheryl D
CD
Humphries
R Keith
RK
Krystal
Gerald
G
Marsh
Clay B
CB
D016475:3T3 Cells; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D015971:Gene Expression Regulation, Enzymologic; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D016173:Macrophage Colony-Stimulating Factor / Q000378:metabolism*; D008264:Macrophages / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D009000:Monocytes / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism; D000072183:Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism / Q000502:physiology*; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D017346:Protein Serine-Threonine Kinases; D017434:Protein Structure, Tertiary; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction; D013997:Time Factors; D014162:Transfection; D019061:src-Family Kinases / Q000502:physiology*
D016328:NF-kappa B; D018129:Phosphatidylinositol Phosphates; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; C060974:phosphatidylinositol 3,4,5-triphosphate; D016173:Macrophage Colony-Stimulating Factor; D005982:Glutathione Transferase; C060902:lyn protein-tyrosine kinase; D019061:src-Family Kinases; C494918:AKT1 protein, human; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D010744:Phosphoric Monoester Hydrolases; C098706:INPPL1 protein, human; D000072183:Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases