5-Lipoxygenase contains a functional Src homology 3-binding motif that interacts with the Src homology 3 domain of Grb2 and cytoskeletal proteins.
A short, proline-rich region spanning residues 566-577 in human 5-lipoxygenase is a binding site for the Src homology 3 (SH3) domain of growth factor receptor-bound protein 2 (Grb2), an "adaptor" protein for tyrosine kinase-mediated cell signaling. Purified 5-lipoxygenase bound to glutathione S-transferase fusion products of Grb2 and a truncated version of Grb2 containing its SH3 domain. A peptide corresponding to the proline-rich, SH3-binding motif inhibited formation of the 5-lipoxygenase.Grb2 complex in vitro. The peptide also inhibited the redistribution of 5-lipoxygenase from the cytosol to the membrane in intact or permeabilized neutrophils activated by calcium ionophore A23187. 5-Lipoxygenase did not bind to the SH3 domains of other signaling proteins, such as GTPase-activating protein and phospholipase C gamma; however, it bound to certain cytoskeletal proteins including alpha-actinin and actin. 5-Lipoxygenase contains a consensus guanine nucleotide-binding site at residues 296-299, and guanine nucleotides inhibit 5-lipoxygenase activity in vitro. Our results suggest that 5-lipoxygenase may have a previously unrecognized role in tyrosine kinase signaling, distinct from its catalysis of lipid mediator formation. Our results also clarify the molecular basis for compartmentalization and translocation of 5-lipoxygenase in myeloid cells, implying that it binds to proteins other than its activating protein.
Lepley
R A
RA
Department of Pharmacology, University of Colorado Health Science Center, Denver 80262.
Fitzpatrick
F A
FA
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000818:Animals; D001094:Arachidonate 5-Lipoxygenase / Q000187:drug effects / Q000378:metabolism*; D001692:Biological Transport; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D066246:ErbB Receptors / Q000378:metabolism*; D051380:GRB2 Adaptor Protein; D005982:Glutathione Transferase / Q000378:metabolism; D006150:Guanine Nucleotides / Q000494:pharmacology; D006801:Humans; D008969:Molecular Sequence Data; D009504:Neutrophils / Q000201:enzymology; D015688:Oncogene Protein pp60(v-src) / Q000378:metabolism*; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016415:Sequence Alignment
D048868:Adaptor Proteins, Signal Transducing; D003598:Cytoskeletal Proteins; D051380:GRB2 Adaptor Protein; C495948:GRB2 protein, human; D006150:Guanine Nucleotides; D011506:Proteins; D011993:Recombinant Fusion Proteins; D001094:Arachidonate 5-Lipoxygenase; D005982:Glutathione Transferase; D066246:ErbB Receptors; D015688:Oncogene Protein pp60(v-src)