title: Phosphoproteomic profiling of mouse primary HSPCs reveals new regulators of HSPC mobilization.
abstract: Protein phosphorylation is a central mechanism of signal transduction that both positively and negatively regulates protein function. Large-scale studies of the dynamic phosphorylation states of cell signaling systems have been applied extensively in cell lines and whole tissues to reveal critical regulatory networks, and candidate-based evaluations of phosphorylation in rare cell populations have also been informative. However, application of comprehensive profiling technologies to adult stem cell and progenitor populations has been challenging, due in large part to the scarcity of such cells in adult tissues. Here, we combine multicolor flow cytometry with highly efficient 3-dimensional high performance liquid chromatography/mass spectrometry to enable quantitative phosphoproteomic analysis from 200 000 highly purified primary mouse hematopoietic stem and progenitor cells (HSPCs). Using this platform, we identify ARHGAP25 as a novel regulator of HSPC mobilization and demonstrate that ARHGAP25 phosphorylation at serine 363 is an important modulator of its function. Our approach provides a robust platform for large-scale phosphoproteomic analyses performed with limited numbers of rare progenitor cells. Data from our study comprises a new resource for understanding the molecular signaling networks that underlie hematopoietic stem cell mobilization.
authors.affiliation: Joslin Diabetes Center, Boston, MA; Harvard Stem Cell Institute, Harvard University, Cambridge, MA; Dana-Farber Boston Children's Cancer and Blood Disorders Center, Boston, MA;
authors.forename: Leo D
authors.identifier: 0000-0002-2945-9005
authors.initials: LD
authors.lastname: Wang
authors.affiliation: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA; Department of Cancer Biology and Blais Proteomics Center, Dana-Farber Cancer Institute, Boston, MA;
authors.forename: Scott B
authors.initials: SB
authors.lastname: Ficarro
authors.affiliation: Department of Biostatistics, Harvard T. H. Chan School of Public Health, Harvard University, Boston, MA;
authors.forename: John N
authors.initials: JN
authors.lastname: Hutchinson
authors.affiliation: Department of Physiology, Semmelweis University, Budapest, Hungary; and.
authors.forename: Roland
authors.identifier: 0000-0001-6825-7142
authors.initials: R
authors.lastname: Csepanyi-Komi
authors.affiliation: Joslin Diabetes Center, Boston, MA; Harvard Stem Cell Institute, Harvard University, Cambridge, MA;
authors.forename: Phi T
authors.initials: PT
authors.lastname: Nguyen
authors.affiliation: Department of Physiology, Semmelweis University, Budapest, Hungary; and.
authors.forename: Eva
authors.initials: E
authors.lastname: Wisniewski
authors.affiliation: Joslin Diabetes Center, Boston, MA; Harvard Stem Cell Institute, Harvard University, Cambridge, MA;
authors.forename: Jessica
authors.initials: J
authors.lastname: Sullivan
authors.affiliation: Department of Biostatistics, Harvard T. H. Chan School of Public Health, Harvard University, Boston, MA;
authors.forename: Oliver
authors.identifier: 0000-0002-7738-1513
authors.initials: O
authors.lastname: Hofmann
authors.affiliation: Department of Physiology, Semmelweis University, Budapest, Hungary; and.
authors.forename: Erzsebet
authors.identifier: 0000-0001-6374-729X
authors.initials: E
authors.lastname: Ligeti
authors.affiliation: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA; Department of Cancer Biology and Blais Proteomics Center, Dana-Farber Cancer Institute, Boston, MA;
authors.forename: Jarrod A
authors.initials: JA
authors.lastname: Marto
authors.affiliation: Joslin Diabetes Center, Boston, MA; Harvard Stem Cell Institute, Harvard University, Cambridge, MA; Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA.
authors.forename: Amy J
authors.initials: AJ
authors.lastname: Wagers
mesh_terms: D000818:Animals; D016026:Bone Marrow Transplantation; D049109:Cell Proliferation; D054377:Chemokine CXCL12 / Q000378:metabolism*; D005260:Female; D005434:Flow Cytometry; D020690:GTPase-Activating Proteins / Q000502:physiology*; D016179:Granulocyte Colony-Stimulating Factor / Q000378:metabolism; D019650:Hematopoietic Stem Cell Mobilization; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000378:metabolism; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D040901:Proteomics; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D015398:Signal Transduction
chemical_list: C497036:ARHGAP25 protein, mouse; D054377:Chemokine CXCL12; D020690:GTPase-Activating Proteins; D010750:Phosphoproteins; D016179:Granulocyte Colony-Stimulating Factor; D051057:Proto-Oncogene Proteins c-akt