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J. Biol. Chem., Vol. 279, Issue 1, 326-340, January 2, 2004

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Contribution of the Membrane-distal Tyrosine in Intracellular Signaling by the Granulocyte Colony-stimulating Factor Receptor^*

From the ^aBiochemistry and Molecular Biology, School of Biomedical and Chemical Sciences, University of Western Australia, Crawley, Western Australia 6009, Australia, the ^bProtein Facility, University of Western Australia, Crawley, Western Australia 6009, Australia, the ^dInstitute of Cancer Research, Chester Beatty Laboratories, 237 Fulham Road, London SW3 6JB, United Kingdom, the ^fLudwig Institute for Cancer Research, Melbourne Tumour Biology Branch, P. O. Royal Melbourne Hospital, Victoria 3050, Australia, and the ^hWestern Australian Institute for Medical Research, Perth, Western Australia 6000, Australia

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 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 membranedistal 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.

Received for publication, September 12, 2003 , and in revised form, October 8, 2003.

^* This work was supported by the Cancer Foundation of Western Australia and the Australian Research Council Small Grants Scheme. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^c Current address: Proteomics International, Perth, Western Australia 6000, Australia.

^e Current address: Celltech R&D, Great Abington, Cambridge CB1 6GS, United Kingdom.

^g Current address: Walter and Eliza Hall Institute of Medical Research, Royal Melbourne Hospital, Victoria 3050, Australia.

ⁱ To whom correspondence should be addressed: Cell Signaling Laboratory, Biochemistry and Molecular Biology (M310), School of Biomedical and Chemical Sciences, University of Western Australia, 35 Stirling Hwy., Crawley, Western Australia 6009, Australia. Tel.: 61-8-6488-1348; Fax: 61-8-6488-1148; E-mail: marieb{at}cyllene.uwa.edu.au.

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