Contribution of the membrane-distal tyrosine in intracellular signaling by the granulocyte colony-stimulating factor receptor.
We have evaluated the contribution of intracellular tyrosine residues of the granulocyte colony-stimulating factor receptor (GCSF-R) to its signaling and cellular outcomes. We began with stable BaF3 cell lines overexpressing wild-type or mutant GCSF-Rs. When all four intracellular tyrosines of the GCSF-R were replaced with phenylalanine (FFFF GCSF-R), cell proliferation and survival were compromised. Replacement of only the membrane-distal tyrosine (YYYF GCSF-R) also showed reduced survival following a GCSF withdrawal/replacement protocol, suggesting a role for this tyrosine. Proliferation by FFFY GCSF-R cells was attenuated by approximately 70%. In evaluating the biochemical steps involved in signaling, we then showed that the membrane-distal tyrosine was necessary and sufficient for c-Jun N-terminal kinase (JNK) activation. With the use of a cell-permeable JNK-inhibitory peptide, JNK was implicated in the proliferation of the FFFY GCSF-R mutant. To further define the events linking the membrane-distal tyrosine and JNK activation, the Src homology 2 domains of Shc, Grb2, and 3BP2 were shown to bind the full-length GCSF-R and a phosphopeptide encompassing the membrane-distal tyrosine. When binding to variant phosphopeptides based on this membrane-distal tyrosine was tested, altering the amino acids immediately following the phosphotyrosine could selectively abolish the interaction with Shc or Grb2, or the binding to both Grb2 and 3BP2. When these changes were introduced into the full-length GCSF-R and new cell lines created, only the mutant that did not interact with Grb2 and 3BP2 did not activate JNK. Our results suggest that direct binding of Shc by the GCSF-R is not essential for JNK activation.
Kendrick
Tulene S
TS
Biochemistry and Molecular Biology, School of Biomedical and Chemical Sciences, University of Western Australia, Crawley, Western Australia 6009, Australia.
Lipscombe
Richard J
RJ
Rausch
Oliver
O
Nicholson
Sandra E
SE
Layton
Judith E
JE
Goldie-Cregan
Lauren C
LC
Bogoyevitch
Marie A
MA
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D002455:Cell Division; D002460:Cell Line; D002470:Cell Survival; D004789:Enzyme Activation; D016179:Granulocyte Colony-Stimulating Factor / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D010446:Peptide Fragments; D010649:Phenylalanine; D010748:Phosphopeptides / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D016188:Receptors, Granulocyte Colony-Stimulating Factor / Q000502:physiology*; D011994:Recombinant Proteins / Q000494:pharmacology; D015290:Second Messenger Systems / Q000187:drug effects / Q000502:physiology*; D014443:Tyrosine
D010446:Peptide Fragments; D010748:Phosphopeptides; D016188:Receptors, Granulocyte Colony-Stimulating Factor; D011994:Recombinant Proteins; D016179:Granulocyte Colony-Stimulating Factor; D014443:Tyrosine; D010649:Phenylalanine; D020928:Mitogen-Activated Protein Kinases