A mutational analysis of phosphatidylinositol-3-kinase activation by human colony-stimulating factor-1 receptor.
Colony-stimulating factor-1 (CSF1) is a cell lineage-specific hemopoietin required for the growth, differentiation, and survival of macrophages and their precursors. The human CSF1 receptor (CSF1R) is a 150-kDa transmembrane glycoprotein whose cytoplasmic tyrosine kinase domain is split by a kinase insert (KI) region of approximately 70 amino acids. We tested the ability of CSF1R KI domain deletion mutants to stimulate phosphatidylinositol-3-kinase (PtdIns-3-kinase), an enzyme whose activity is augmented by tyrosine kinase oncogenes and receptor tyrosine kinases, and to support mitogenesis in transfected cells. Receptor immunoprecipitates from CSF1-stimulated cells contained greater than 5-fold more PtdIns-3-kinase activity compared to nonstimulated cells. High performance liquid chromatography analysis of the PtdIns-3-kinase product scraped from thin layer chromatography plates indicated that PtdIns-3-P was produced. CSF1R KI domain deletion mutants retained tyrosine kinase activity in vitro. Receptor immunoprecipitates of two partially overlapping 28 and 30 amino acid KI deletion mutants of CSF1R retained some PtdIns-3-kinase activity, in contrast to immunoprecipitates of CSF1R lacking 67 amino acids of the KI domain. Each deletion mutant stimulated CSF1-dependent DNA synthesis in transfected cells at much reduced levels compared to wild-type receptor expressing cells. These data suggest a role for the CSF1R KI domain in PtdIns-3-kinase association and for CSF1-induced thymidine incorporation into DNA.
Choudhury
G G
GG
Department of Cellular and Structural Biology, University of Texas Health Science Center, San Antonio 78284-7762.
Wang
L M
LM
Pierce
J
J
Harvey
S A
SA
Sakaguchi
A Y
AY
D019870:1-Phosphatidylinositol 4-Kinase; D000818:Animals; D002460:Cell Line; D002461:Cell Line, Transformed; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D004789:Enzyme Activation; D006801:Humans; D051379:Mice; D016297:Mutagenesis, Site-Directed; D019869:Phosphatidylinositol 3-Kinases; D010766:Phosphorylation; D010770:Phosphotransferases / Q000378:metabolism*; D011233:Precipitin Tests; D051381:Rats; D016186:Receptor, Macrophage Colony-Stimulating Factor / Q000235:genetics* / Q000378:metabolism; D014162:Transfection
D010770:Phosphotransferases; D019869:Phosphatidylinositol 3-Kinases; D019870:1-Phosphatidylinositol 4-Kinase; D016186:Receptor, Macrophage Colony-Stimulating Factor