The RING finger domain of Cbl is essential for negative regulation of the Syk tyrosine kinase

Satoshi Ota, Kaoru Hazeki, Navin Rao, Mark L. Lupher, Christopher E. Andoniou, Brian Druker, Hamid Band

Research output: Contribution to journalArticle

79 Citations (Scopus)

Abstract

The proto-oncogene product Cbl has emerged as a negative regulator of a number of protein-tyrosine kinases, including the ZAP-70/Syk tyrosine kinases that are critical for signaling in hematopoietic cells. The evolutionarily conserved N-terminal tyrosine kinase-binding domain is required for Cbl to associate with ZAP-70/Syk and for their subsequent negative regulation. However, the role of the remaining C-terminal regions of Cbl remains unclear. Here, we used a COS-7 cell reconstitution system to address this question. Analysis of a series of C-terminally truncated Cbl mutants revealed that the N-terminal half of the protein, including the TKB and RING finger domains, was sufficient to mediate negative regulation of Syk. Further truncations, which delete the RING finger domain, abrogated the negative regulatory effects of Cbl on Syk. Point mutations of conserved cysteine residues or a histidine in the RING finger domain, which are required for zinc binding, abrogated the ability of Cbl to negatively regulate Syk in COS-7 cells and Ramos B lymphocytic cells. In addition, Syk-dependent transactivation of a serum response element-luciferase reporter in transfected 293T cells was reduced by wild type Cbl; mutations of the RING finger domain or its deletion abrogated this effect. These results establish the RING finger domain as an essential element in Cbl-mediated negative regulation of a tyrosine kinase and reveal that the evolutionarily conserved N-terminal half of the protein is sufficient for this function.

Original languageEnglish (US)
Pages (from-to)414-422
Number of pages9
JournalJournal of Biological Chemistry
Volume275
Issue number1
DOIs
StatePublished - Jan 7 2000
Externally publishedYes

Fingerprint

RING Finger Domains
Protein-Tyrosine Kinases
COS Cells
Serum Response Element
ZAP-70 Protein-Tyrosine Kinase
Oncogene Proteins
Luciferases
Histidine
Proto-Oncogenes
HEK293 Cells
Cysteine
Zinc
Proteins
Point Mutation
Transcriptional Activation
Syk Kinase
Mutation

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

The RING finger domain of Cbl is essential for negative regulation of the Syk tyrosine kinase. / Ota, Satoshi; Hazeki, Kaoru; Rao, Navin; Lupher, Mark L.; Andoniou, Christopher E.; Druker, Brian; Band, Hamid.

In: Journal of Biological Chemistry, Vol. 275, No. 1, 07.01.2000, p. 414-422.

Research output: Contribution to journalArticle

Ota, Satoshi ; Hazeki, Kaoru ; Rao, Navin ; Lupher, Mark L. ; Andoniou, Christopher E. ; Druker, Brian ; Band, Hamid. / The RING finger domain of Cbl is essential for negative regulation of the Syk tyrosine kinase. In: Journal of Biological Chemistry. 2000 ; Vol. 275, No. 1. pp. 414-422.
@article{6379913e0b634b86a434d0bd61d15699,
title = "The RING finger domain of Cbl is essential for negative regulation of the Syk tyrosine kinase",
abstract = "The proto-oncogene product Cbl has emerged as a negative regulator of a number of protein-tyrosine kinases, including the ZAP-70/Syk tyrosine kinases that are critical for signaling in hematopoietic cells. The evolutionarily conserved N-terminal tyrosine kinase-binding domain is required for Cbl to associate with ZAP-70/Syk and for their subsequent negative regulation. However, the role of the remaining C-terminal regions of Cbl remains unclear. Here, we used a COS-7 cell reconstitution system to address this question. Analysis of a series of C-terminally truncated Cbl mutants revealed that the N-terminal half of the protein, including the TKB and RING finger domains, was sufficient to mediate negative regulation of Syk. Further truncations, which delete the RING finger domain, abrogated the negative regulatory effects of Cbl on Syk. Point mutations of conserved cysteine residues or a histidine in the RING finger domain, which are required for zinc binding, abrogated the ability of Cbl to negatively regulate Syk in COS-7 cells and Ramos B lymphocytic cells. In addition, Syk-dependent transactivation of a serum response element-luciferase reporter in transfected 293T cells was reduced by wild type Cbl; mutations of the RING finger domain or its deletion abrogated this effect. These results establish the RING finger domain as an essential element in Cbl-mediated negative regulation of a tyrosine kinase and reveal that the evolutionarily conserved N-terminal half of the protein is sufficient for this function.",
author = "Satoshi Ota and Kaoru Hazeki and Navin Rao and Lupher, {Mark L.} and Andoniou, {Christopher E.} and Brian Druker and Hamid Band",
year = "2000",
month = "1",
day = "7",
doi = "10.1074/jbc.275.1.414",
language = "English (US)",
volume = "275",
pages = "414--422",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "1",

}

TY - JOUR

T1 - The RING finger domain of Cbl is essential for negative regulation of the Syk tyrosine kinase

AU - Ota, Satoshi

AU - Hazeki, Kaoru

AU - Rao, Navin

AU - Lupher, Mark L.

AU - Andoniou, Christopher E.

AU - Druker, Brian

AU - Band, Hamid

PY - 2000/1/7

Y1 - 2000/1/7

N2 - The proto-oncogene product Cbl has emerged as a negative regulator of a number of protein-tyrosine kinases, including the ZAP-70/Syk tyrosine kinases that are critical for signaling in hematopoietic cells. The evolutionarily conserved N-terminal tyrosine kinase-binding domain is required for Cbl to associate with ZAP-70/Syk and for their subsequent negative regulation. However, the role of the remaining C-terminal regions of Cbl remains unclear. Here, we used a COS-7 cell reconstitution system to address this question. Analysis of a series of C-terminally truncated Cbl mutants revealed that the N-terminal half of the protein, including the TKB and RING finger domains, was sufficient to mediate negative regulation of Syk. Further truncations, which delete the RING finger domain, abrogated the negative regulatory effects of Cbl on Syk. Point mutations of conserved cysteine residues or a histidine in the RING finger domain, which are required for zinc binding, abrogated the ability of Cbl to negatively regulate Syk in COS-7 cells and Ramos B lymphocytic cells. In addition, Syk-dependent transactivation of a serum response element-luciferase reporter in transfected 293T cells was reduced by wild type Cbl; mutations of the RING finger domain or its deletion abrogated this effect. These results establish the RING finger domain as an essential element in Cbl-mediated negative regulation of a tyrosine kinase and reveal that the evolutionarily conserved N-terminal half of the protein is sufficient for this function.

AB - The proto-oncogene product Cbl has emerged as a negative regulator of a number of protein-tyrosine kinases, including the ZAP-70/Syk tyrosine kinases that are critical for signaling in hematopoietic cells. The evolutionarily conserved N-terminal tyrosine kinase-binding domain is required for Cbl to associate with ZAP-70/Syk and for their subsequent negative regulation. However, the role of the remaining C-terminal regions of Cbl remains unclear. Here, we used a COS-7 cell reconstitution system to address this question. Analysis of a series of C-terminally truncated Cbl mutants revealed that the N-terminal half of the protein, including the TKB and RING finger domains, was sufficient to mediate negative regulation of Syk. Further truncations, which delete the RING finger domain, abrogated the negative regulatory effects of Cbl on Syk. Point mutations of conserved cysteine residues or a histidine in the RING finger domain, which are required for zinc binding, abrogated the ability of Cbl to negatively regulate Syk in COS-7 cells and Ramos B lymphocytic cells. In addition, Syk-dependent transactivation of a serum response element-luciferase reporter in transfected 293T cells was reduced by wild type Cbl; mutations of the RING finger domain or its deletion abrogated this effect. These results establish the RING finger domain as an essential element in Cbl-mediated negative regulation of a tyrosine kinase and reveal that the evolutionarily conserved N-terminal half of the protein is sufficient for this function.

UR - http://www.scopus.com/inward/record.url?scp=0034614386&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0034614386&partnerID=8YFLogxK

U2 - 10.1074/jbc.275.1.414

DO - 10.1074/jbc.275.1.414

M3 - Article

VL - 275

SP - 414

EP - 422

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 1

ER -