Purification and identification of protein-tyrosine kinase-binding proteins using synthetic phosphopeptides as affinity reagents.
Protein-tyrosine kinases are known regulators of cell division that have been implicated in the onset of a variety of malignancies. They act through cellular signaling proteins that bind to specific autophosphorylation sites. To find out whether these autophosphorylation sites can be used to identify downstream signaling proteins, synthetic peptides based on an autophosphorylation site in the colony-stimulating factor-1 (CSF-1) receptor were linked to agarose beads and incubated with lysates from macrophages. Bound proteins were analyzed by MS, leading to the identification of both known and novel CSF-1 receptor-interacting proteins. The approach presented here can be applied to phosphorylation sites in a wide variety of proteins. It will lead to the identification of novel protein-protein interactions and provide new insights into the mechanics of signal transduction. Novel protein-protein interactions may provide useful targets for the development of drugs that interfere with the activation of signaling cascades used by protein-tyrosine kinases to turn on cell division.
Wilhelmsen
Kevin
K
Department of Chemistry and Biochemistry, University of California San Diego, La Jolla, CA 92093-0601, USA.
Copp
Jeremy
J
Glenn
Gary
G
Hoffman
Ross C
RC
Tucker
Philip
P
van der Geer
Peter
P
D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000302:isolation & purification*; D002460:Cell Line; D017124:Conserved Sequence; D006801:Humans; D066298:In Vitro Techniques; D007202:Indicators and Reagents; D051379:Mice; D008969:Molecular Sequence Data; D010748:Phosphopeptides; D021382:Protein Sorting Signals; D011505:Protein-Tyrosine Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016186:Receptor, Macrophage Colony-Stimulating Factor / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014443:Tyrosine / Q000737:chemistry
D000345:Affinity Labels; D002352:Carrier Proteins; D007202:Indicators and Reagents; D010748:Phosphopeptides; D021382:Protein Sorting Signals; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D016186:Receptor, Macrophage Colony-Stimulating Factor