Molecular Cloning and Characterization of p56dok-2 Defines a New Family of RasGAP-binding Proteins

Antonio Di Cristofano,Nick Carpino,Nicolas Dunant,Gayle Friedland,Ryuji Kobayashi,Annabel Strife,David Wisniewski,Bayard Clarkson,Pier Paolo Pandolfi,Marilyn D Resh

doi:10.1074/jbc.273.9.4827

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.

Highlights

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF034970 and AF035117
Our strategy was motivated by the recent identification of another Tyr(P) protein, p62dok, in p210bcr-abl expressing cells. p62dok is a substrate for Bcr-Abl and binds to p120RasGAP [7, 8]
The identification of p56dok-2 in this manuscript has served to define a new family of Dok proteins

Summary

Introduction

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF034970 and AF035117. In CML, Bcr sequences are fused to the second exon of Abl, resulting in activation of the chimeric p210bcr-abl tyrosine kinase. Several tyrosine phosphorylated proteins were apparent in the early blast subpopulations derived from the marrows of CML patients but not normal donors. One of these Tyr(P) proteins, p62dok, was purified, and its gene was cloned [7]. P62dok binds to RasGAP and exhibits additional features of a signaling protein, including an N-terminal PH domain and clusters of PXXP motifs [7, 8] These studies detected a second Tyr(P) protein, p56, which exhibited increased Tyr(P) levels in primary CML cells and in CML cell lines [6]. We describe the purification, cDNA cloning, and characterization of p56 and show that it is a member of a Dok family of RasGAP binding proteins

Methods

Results

Conclusion