Identification of novel SH3 domain ligands for the Src family kinase Hck. Wiskott-Aldrich syndrome protein (WASP), WASP-interacting protein (WIP), and ELMO1.
The importance of the SH3 domain of Hck in kinase regulation, substrate phosphorylation, and ligand binding has been established. However, few in vivo ligands are known for the SH3 domain of Hck. In this study, we used mass spectrometry to identify approximately 25 potential binding partners for the SH3 domain of Hck from the monocyte cell line U937. Two major interacting proteins were the actin binding proteins Wiskott-Aldrich syndrome protein (WASP) and WASP-interacting protein (WIP). We also focused on a novel interaction between Hck and ELMO1, an 84-kDa protein that was recently identified as the mammalian ortholog of the Caenorhabditis elegans gene, ced-12. In mammalian cells, ELMO1 interacts with Dock180 as a component of the CrkII/Dock180/Rac pathway responsible for phagocytosis and cell migration. Using purified proteins, we confirmed that WASP-interacting protein and ELMO1 interact directly with the SH3 domain of Hck. We also show that Hck and ELMO1 interact in intact cells and that ELMO1 is heavily tyrosine-phosphorylated in cells that co-express Hck, suggesting that it is a substrate of Hck. The binding of ELMO1 to Hck is specifically dependent on the interaction of a polyproline motif with the SH3 domain of Hck. Our results suggest that these proteins may be novel activators/effectors of Hck.
Scott
Margaret Porter
MP
Department of Physiology and Biophysics, School of Medicine, State University of New York, Stony Brook, New York 11794-8661, USA.
Zappacosta
Francesca
F
Kim
Eun Young
EY
Annan
Roland S
RS
Miller
W Todd
WT
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism*; D003001:Cloning, Molecular; D004926:Escherichia coli; D006801:Humans; D008024:Ligands; D008322:Mammals; D009124:Muscle Proteins; D010455:Peptides / Q000494:pharmacology; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051572:Proto-Oncogene Proteins c-hck; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection; D020298:U937 Cells; D014923:Wiskott-Aldrich Syndrome / Q000378:metabolism*; D051316:Wiskott-Aldrich Syndrome Protein; D018909:src Homology Domains; D019061:src-Family Kinases / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; C465214:ELMO1 protein, human; D008024:Ligands; D009124:Muscle Proteins; C486062:NCKIPSD protein, human; D010455:Peptides; D011506:Proteins; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C495767:WAS protein, human; D051316:Wiskott-Aldrich Syndrome Protein; C011083:polyproline; D011505:Protein-Tyrosine Kinases; C496631:HCK protein, human; D051572:Proto-Oncogene Proteins c-hck; D019061:src-Family Kinases