Colony-stimulating factor-1 stimulates the formation of multimeric cytosolic complexes of signaling proteins and cytoskeletal components in macrophages.
Stimulation of macrophages with colony-stimulating factor-1 (CSF-1) results in the protein tyrosine phosphorylation of the CSF-1 receptor (CSF-1R) and many other, primarily cytosolic, proteins. Stimulation by CSF-1 at 4 degreesC was used to facilitate the purification and identification of the proteins of the cytosolic anti-phosphotyrosine (PY)-reactive fraction (alphaPY-RF) involved in downstream signaling pathways. Confocal microscopy revealed that the PY proteins are in close proximity to the CSF-1R at the plasma membrane. The alphaPY-RF contained pre-existing complexes of PY proteins and non-PY proteins which generally increased in size and PY protein content following CSF-1 stimulation. PY proteins identified by microsequencing and Western blotting include Cbl, STAT3, STAT5a, STAT5b, SHP-1, Shc, and two novel proteins pp57 and pp37. Other proteins included cytoskeletal/contractile proteins (paxillin, vimentin, elongation factor-1alpha, F-actin, tropomyosin, and myosin regulatory light chain), Ras family signaling proteins (p85 (phosphoinositide 3-kinase), Vav, Ras-GTPase-activating protein SH3 domain-binding protein, and Grb2), DnaJ-like protein, and glyceraldehyde-3-phosphate dehydrogenase. CSF-1 induced the de novo recruitment of Cbl, STAT3, STAT5a, STAT5b, p85, SHP-1, Shc, vimentin, and Grb2 to complexes and caused pre-existing complexes involving Vav, elongation factor-1alpha, and F-actin to increase in size. These studies indicate that CSF-1-induced protein tyrosine phosphorylation is associated with the reorganization of complexes of cytoskeletal, signaling, and other proteins that mediate CSF-1-regulated motility and growth.
Yeung
Y G
YG
Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Wang
Y
Y
Einstein
D B
DB
Lee
P S
PS
Stanley
E R
ER
D000199:Actins / Q000378:metabolism; D000595:Amino Acid Sequence; D001704:Biopolymers; D015153:Blotting, Western; D002460:Cell Line; D002850:Chromatography, Gel; D015638:Cytochalasin D / Q000494:pharmacology; D003598:Cytoskeletal Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D003600:Cytosol / Q000187:drug effects* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D016173:Macrophage Colony-Stimulating Factor / Q000494:pharmacology*; D008264:Macrophages / Q000187:drug effects* / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D010766:Phosphorylation; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism; D015398:Signal Transduction
D000199:Actins; D001704:Biopolymers; D003598:Cytoskeletal Proteins; D015638:Cytochalasin D; D016173:Macrophage Colony-Stimulating Factor; D017027:Protein Tyrosine Phosphatases