Identification and functional characterization of an Src homology domain 3 domain-binding site on Cbl

Archana Sanjay, Tsuyoshi Miyazaki, Cecile Itzstein, Enkhtsetseg Purev, William C Horne, Roland Baron

Research output: Contribution to journalArticle

17 Citations (Scopus)

Abstract

Cbl is an adaptor protein and ubiquitin ligase that binds and is phosphorylated by the nonreceptor tyrosine kinase Src. We previously showed that the primary interaction between Src and Cbl is mediated by the Src homology domain 3 (SH3) of Src binding to proline-rich sequences of Cbl. The peptide Cbl RDLPPPPPPDRP(540-551), which corresponds to residues 540-551 of Cbl, inhibited the binding of a GST-Src SH3 fusion protein to Cbl, whereas RDLAPPAPPPDR(540-551) did not, suggesting that Src binds to this site on Cbl in a class I orientation. Mutating prolines 543-548 reduced Src binding to the Cbl 479-636 fragment significantly more than mutating the prolines in the PPVPPR(494-499) motif, which was previously reported to bind Src SH3. Mutating Cbl prolines 543-548 to alanines substantially reduced Src binding to Cbl, Src-induced phosphorylation of Cbl, and the inhibition of Src kinase activity by Cbl. Expressing the mutated Cbl in osteoclasts induced a moderate reduction in bone-resorbing activity and increased amounts of Src protein. In contrast, disabling the tyrosine kinase-binding domain of full-length Cbl by mutating glycine 306 to glutamic acid, and thereby preventing the previously described binding of the tyrosine kinase-binding domain to the Src phosphotyrosine 416, had no effect on Cbl phosphorylation, the inhibition of Src activity by full-length Cbl, or bone resorption. These data indicate that the Cbl RDLPPPP(540-546) sequence is a functionally important binding site for Src.
Original languageEnglish
Pages (from-to)5442-5456
Number of pages15
JournalFEBS Journal
Volume273
Issue number23
Early online date9 Nov 2006
DOIs
Publication statusPublished - Dec 2006

Fingerprint

src Homology Domains
Proline
Binding Sites
src-Family Kinases
Protein-Tyrosine Kinases
Phosphorylation
Phosphotyrosine
Ubiquitin-Protein Ligases
Osteoclasts
Bone Resorption
Alanine
Glycine
Glutamic Acid
Proteins
Peptides

Keywords

  • Amino Acid Motifs
  • Binding Sites
  • Cells, Cultured
  • Mutation
  • Osteoclasts
  • Peptides
  • Phosphorylation
  • Proline
  • Proto-Oncogene Proteins c-cbl
  • Recombinant Fusion Proteins
  • Up-Regulation
  • src Homology Domains

Cite this

Sanjay, A., Miyazaki, T., Itzstein, C., Purev, E., Horne, W. C., & Baron, R. (2006). Identification and functional characterization of an Src homology domain 3 domain-binding site on Cbl. FEBS Journal, 273(23), 5442-5456. https://doi.org/10.1111/j.1742-4658.2006.05535.x

Identification and functional characterization of an Src homology domain 3 domain-binding site on Cbl. / Sanjay, Archana; Miyazaki, Tsuyoshi; Itzstein, Cecile; Purev, Enkhtsetseg; Horne, William C; Baron, Roland.

In: FEBS Journal, Vol. 273, No. 23, 12.2006, p. 5442-5456.

Research output: Contribution to journalArticle

Sanjay, A, Miyazaki, T, Itzstein, C, Purev, E, Horne, WC & Baron, R 2006, 'Identification and functional characterization of an Src homology domain 3 domain-binding site on Cbl', FEBS Journal, vol. 273, no. 23, pp. 5442-5456. https://doi.org/10.1111/j.1742-4658.2006.05535.x
Sanjay, Archana ; Miyazaki, Tsuyoshi ; Itzstein, Cecile ; Purev, Enkhtsetseg ; Horne, William C ; Baron, Roland. / Identification and functional characterization of an Src homology domain 3 domain-binding site on Cbl. In: FEBS Journal. 2006 ; Vol. 273, No. 23. pp. 5442-5456.
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AU - Sanjay, Archana

AU - Miyazaki, Tsuyoshi

AU - Itzstein, Cecile

AU - Purev, Enkhtsetseg

AU - Horne, William C

AU - Baron, Roland

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N2 - Cbl is an adaptor protein and ubiquitin ligase that binds and is phosphorylated by the nonreceptor tyrosine kinase Src. We previously showed that the primary interaction between Src and Cbl is mediated by the Src homology domain 3 (SH3) of Src binding to proline-rich sequences of Cbl. The peptide Cbl RDLPPPPPPDRP(540-551), which corresponds to residues 540-551 of Cbl, inhibited the binding of a GST-Src SH3 fusion protein to Cbl, whereas RDLAPPAPPPDR(540-551) did not, suggesting that Src binds to this site on Cbl in a class I orientation. Mutating prolines 543-548 reduced Src binding to the Cbl 479-636 fragment significantly more than mutating the prolines in the PPVPPR(494-499) motif, which was previously reported to bind Src SH3. Mutating Cbl prolines 543-548 to alanines substantially reduced Src binding to Cbl, Src-induced phosphorylation of Cbl, and the inhibition of Src kinase activity by Cbl. Expressing the mutated Cbl in osteoclasts induced a moderate reduction in bone-resorbing activity and increased amounts of Src protein. In contrast, disabling the tyrosine kinase-binding domain of full-length Cbl by mutating glycine 306 to glutamic acid, and thereby preventing the previously described binding of the tyrosine kinase-binding domain to the Src phosphotyrosine 416, had no effect on Cbl phosphorylation, the inhibition of Src activity by full-length Cbl, or bone resorption. These data indicate that the Cbl RDLPPPP(540-546) sequence is a functionally important binding site for Src.

AB - Cbl is an adaptor protein and ubiquitin ligase that binds and is phosphorylated by the nonreceptor tyrosine kinase Src. We previously showed that the primary interaction between Src and Cbl is mediated by the Src homology domain 3 (SH3) of Src binding to proline-rich sequences of Cbl. The peptide Cbl RDLPPPPPPDRP(540-551), which corresponds to residues 540-551 of Cbl, inhibited the binding of a GST-Src SH3 fusion protein to Cbl, whereas RDLAPPAPPPDR(540-551) did not, suggesting that Src binds to this site on Cbl in a class I orientation. Mutating prolines 543-548 reduced Src binding to the Cbl 479-636 fragment significantly more than mutating the prolines in the PPVPPR(494-499) motif, which was previously reported to bind Src SH3. Mutating Cbl prolines 543-548 to alanines substantially reduced Src binding to Cbl, Src-induced phosphorylation of Cbl, and the inhibition of Src kinase activity by Cbl. Expressing the mutated Cbl in osteoclasts induced a moderate reduction in bone-resorbing activity and increased amounts of Src protein. In contrast, disabling the tyrosine kinase-binding domain of full-length Cbl by mutating glycine 306 to glutamic acid, and thereby preventing the previously described binding of the tyrosine kinase-binding domain to the Src phosphotyrosine 416, had no effect on Cbl phosphorylation, the inhibition of Src activity by full-length Cbl, or bone resorption. These data indicate that the Cbl RDLPPPP(540-546) sequence is a functionally important binding site for Src.

KW - Amino Acid Motifs

KW - Binding Sites

KW - Cells, Cultured

KW - Mutation

KW - Osteoclasts

KW - Peptides

KW - Phosphorylation

KW - Proline

KW - Proto-Oncogene Proteins c-cbl

KW - Recombinant Fusion Proteins

KW - Up-Regulation

KW - src Homology Domains

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DO - 10.1111/j.1742-4658.2006.05535.x

M3 - Article

C2 - 17094785

VL - 273

SP - 5442

EP - 5456

JO - FEBS Journal

JF - FEBS Journal

SN - 1742-464X

IS - 23

ER -