Cbl-mediated negative regulation of the Syk tyrosine kinase. A critical role for Cbl phosphotyrosine-binding domain binding to Syk phosphotyrosine 323

Mark L. Lupher, Navin Rao, Nancy L. Lill, Christopher E. Andoniou, Sachiko Miyake, Edward A. Clark, Brian Druker, Hamid Band

Research output: Contribution to journalArticle

150 Citations (Scopus)

Abstract

The proto-oncogene product Cbl has emerged as a potential negative regulator of the Syk tyrosine kinase; however, the nature of physical interactions between Cbl and Syk that are critical for this negative regulation remains unclear. Here we show that the phosphotyrosine-binding (PTB) domain within the N-terminal transforming region of Cbl (Cbl-N) binds to phosphorylated Tyr323 in the linker region between the Src homology 2 and kinase domains of Syk, confirming recent results by another laboratory using the yeast two-hybrid approach (Deckert, M., Elly, C., Altman, A., and Liu, Y. C. (1998) J. Biol. Chem. 273, 8867-8874). A PTB domain-inactivating point mutation (G306E), corresponding to a loss-of-function mutation in the Caenorhabditis elegans Cbl homologue SLI-1, severely compromised Cbl-N/Syk binding in vitro and Cbl/Syk association in transfected COS-7 cells. Using heterologous expression in COS-7 cells, we investigated the role of Cbl PTB domain binding to Syk Tyr323 in the negative regulation of Syk. Co- expression of Cbl with Syk in COS-7 cells led to a dose-dependent decrease in the autophosphorylated pool of Syk and in phosphorylation of an in vivo substrate, CD8-ζ. Unexpectedly, these effects were largely due to the loss of Syk protein. Both the decrease in Syk and CD8-ζ phosphorylation and reduction in Syk protein levels were blocked by either G306E mutation in Cbl or by Y323F mutation in Syk. These results demonstrate a critical role for the Cbl PTB domain in the recruitment of Cbl to Syk and in Cbl-mediated negative regulation of Syk.

Original languageEnglish (US)
Pages (from-to)35273-35281
Number of pages9
JournalJournal of Biological Chemistry
Volume273
Issue number52
DOIs
StatePublished - Dec 25 1998

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Phosphotyrosine
Protein-Tyrosine Kinases
COS Cells
Phosphorylation
Mutation
Proto-Oncogenes
Oncogene Proteins
Caenorhabditis elegans
Point Mutation
Yeast
Proteins
Phosphotransferases
Yeasts
Association reactions
Syk Kinase
Substrates

ASJC Scopus subject areas

  • Biochemistry

Cite this

Cbl-mediated negative regulation of the Syk tyrosine kinase. A critical role for Cbl phosphotyrosine-binding domain binding to Syk phosphotyrosine 323. / Lupher, Mark L.; Rao, Navin; Lill, Nancy L.; Andoniou, Christopher E.; Miyake, Sachiko; Clark, Edward A.; Druker, Brian; Band, Hamid.

In: Journal of Biological Chemistry, Vol. 273, No. 52, 25.12.1998, p. 35273-35281.

Research output: Contribution to journalArticle

Lupher, Mark L. ; Rao, Navin ; Lill, Nancy L. ; Andoniou, Christopher E. ; Miyake, Sachiko ; Clark, Edward A. ; Druker, Brian ; Band, Hamid. / Cbl-mediated negative regulation of the Syk tyrosine kinase. A critical role for Cbl phosphotyrosine-binding domain binding to Syk phosphotyrosine 323. In: Journal of Biological Chemistry. 1998 ; Vol. 273, No. 52. pp. 35273-35281.
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abstract = "The proto-oncogene product Cbl has emerged as a potential negative regulator of the Syk tyrosine kinase; however, the nature of physical interactions between Cbl and Syk that are critical for this negative regulation remains unclear. Here we show that the phosphotyrosine-binding (PTB) domain within the N-terminal transforming region of Cbl (Cbl-N) binds to phosphorylated Tyr323 in the linker region between the Src homology 2 and kinase domains of Syk, confirming recent results by another laboratory using the yeast two-hybrid approach (Deckert, M., Elly, C., Altman, A., and Liu, Y. C. (1998) J. Biol. Chem. 273, 8867-8874). A PTB domain-inactivating point mutation (G306E), corresponding to a loss-of-function mutation in the Caenorhabditis elegans Cbl homologue SLI-1, severely compromised Cbl-N/Syk binding in vitro and Cbl/Syk association in transfected COS-7 cells. Using heterologous expression in COS-7 cells, we investigated the role of Cbl PTB domain binding to Syk Tyr323 in the negative regulation of Syk. Co- expression of Cbl with Syk in COS-7 cells led to a dose-dependent decrease in the autophosphorylated pool of Syk and in phosphorylation of an in vivo substrate, CD8-ζ. Unexpectedly, these effects were largely due to the loss of Syk protein. Both the decrease in Syk and CD8-ζ phosphorylation and reduction in Syk protein levels were blocked by either G306E mutation in Cbl or by Y323F mutation in Syk. These results demonstrate a critical role for the Cbl PTB domain in the recruitment of Cbl to Syk and in Cbl-mediated negative regulation of Syk.",
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