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Molecular cloning and characterization of p56dok-2 defines a new family of RasGAP-binding proteins
A Di Cristofano1, N Carpino, N Dunant
1Department of Human Genetics and Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA.
Abstract:
Chronic myelogenous leukemia (CML) is a disease characterized by the presence of p210(bcr-abl), a chimeric protein with tyrosine kinase activity. Substrates for p210(bcr-abl) are likely to be involved in the pathogenesis of CML. Here we describe the purification, cDNA cloning, and characterization of a 56-kDa tyrosine phosphorylated protein, p56(dok-2) (Dok-2), from p210(bcr-abl) expressing cells. The human dok-2 cDNA encodes a 412-amino acid protein with a predicted N-terminal pleckstrin homology domain as well as several other features of a signaling molecule, including 13 potential tyrosine phosphorylation sites, six PXXP motifs, and the ability to bind to p120(RasGAP). Dok-2 was shown to be 35% identical to p62(dok-1), a recently identified RasGAP binding protein from CML cells, and analysis of the expressed sequence tag data base revealed the presence of at least four additional proteins containing a Dok homology sequence motif. Dok mRNAs were primarily expressed in tissues of hematopoietic origin. These findings strongly suggest that a family of Dok-related proteins exists that bind to RasGAP and may mediate the effects of p210(bcr-abl) in CML.
Insights
Researchers identified a new protein, Dok-2, in chronic myelogenous leukemia (CML) cells. Dok-2 binds to RasGAP and may play a role in CML pathogenesis, suggesting a family of Dok proteins involved in the disease.
Area of Science:
- Molecular Biology
- Oncology
- Biochemistry
Background:
- Chronic myelogenous leukemia (CML) is associated with the p210(bcr-abl) tyrosine kinase.
- Understanding p210(bcr-abl) substrates is crucial for CML pathogenesis research.
Purpose of the Study:
- To identify and characterize novel substrates of p210(bcr-abl) in CML.
- To investigate the role of these substrates in CML development.
Main Methods:
- Purification and cDNA cloning of a 56-kDa tyrosine phosphorylated protein from p210(bcr-abl) expressing cells.
- Characterization of the protein's domains, phosphorylation sites, and binding partners.
- Bioinformatic analysis of related protein families and tissue expression.
Main Results:
- A novel protein, p56(dok-2) (Dok-2), was purified and cloned.
- Dok-2 possesses a pleckstrin homology domain, multiple tyrosine phosphorylation sites, and binds to p120(RasGAP).
- Dok-2 shares homology with Dok-1 and is part of a larger family of RasGAP-binding proteins primarily expressed in hematopoietic tissues.
Conclusions:
- A family of Dok-related proteins that bind RasGAP has been identified.
- These Dok proteins may mediate the effects of p210(bcr-abl) in chronic myelogenous leukemia.
- Dok-2 represents a potential new target for CML research and therapy.