The adapter protein CrkL associates with CD34.
CD34 is a cell-surface transmembrane protein expressed specifically at the stem/progenitor stage of lymphohematopoietic development that appears to regulate adhesion. To elucidate intracellular signals modified by CD34, we designed and constructed glutathione-S-transferase (GST)- fusion proteins of the intracellular domain of full-length CD34 (GST-CD34i(full)). Precipitation of cell lysates using GST-CD34i(full) identified proteins of molecular mass 39, 36, and 33 kd that constitutively associated with CD34 and a 45-kd protein that associated with CD34 after adhesion. By Western analysis, we identified the 39-kd protein as CrkL. In vivo, CrkL was coimmunoprecipitated with CD34 using CD34 antibodies, confirming the association between CrkL and CD34. CD34 peptide inhibition assays demonstrated that CrkL interacts at a membrane-proximal region of the CD34 tail. To identify the CrkL domain responsible for interaction with CD34, we generated GST-fusion constructs of adapter proteins including GST-CrkL3' (C-terminal SH3) and GST-CrkL5' (N-terminal SH2SH3). Of these fusion proteins, only GST-CrkL3' could precipitate endogenously expressed CD34, suggesting that CD34 binds the C-terminal SH3 domain of CrkL. Interestingly, there appears to be differential specificity between CrkL and CrkII for CD34, because GST-CD34i(full) did not precipitate CrkII, a highly homologous Crk family member. Furthermore, GST-CD34i(full) did not bind c-Abl, c-Cbl, C3G, or paxillin proteins that are known to associate with CrkL, suggesting that CD34 directly interacts with the CrkL protein. CD34i(full) association with Grb or Shc adapter proteins was not detected. Our investigations shed new light on signaling pathways of CD34 by demonstrating that CD34 couples to the hematopoietic adapter protein CrkL. (Blood. 2001;97:3768-3775)
Felschow
D M
DM
Johns Hopkins Oncology Center, Johns Hopkins University School of Medicine, Bunting-Blaustein Cancer Research Building, 1650 Orleans St., Baltimore, MD, USA.
McVeigh
M L
ML
Hoehn
G T
GT
Civin
C I
CI
Fackler
M J
MJ
D048868:Adaptor Proteins, Signal Transducing; D000906:Antibodies / Q000494:pharmacology; D018952:Antigens, CD34 / Q000276:immunology / Q000378:metabolism* / Q000494:pharmacology; D001665:Binding Sites; D002448:Cell Adhesion / Q000187:drug effects; D004347:Drug Interactions; D005978:Glutathione; D006412:Hematopoietic Stem Cells / Q000166:cytology; D006801:Humans; D009687:Nuclear Proteins / Q000378:metabolism* / Q000502:physiology; D010750:Phosphoproteins / Q000502:physiology; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction; D014407:Tumor Cells, Cultured; D018909:src Homology Domains / Q000502:physiology
D048868:Adaptor Proteins, Signal Transducing; D000906:Antibodies; D018952:Antigens, CD34; C082869:CRKL protein; D009687:Nuclear Proteins; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; D005978:Glutathione