Molecular Cloning and Characterization of p56 Defines a New Family of RasGAP-binding Proteins

Abstract

Chronic myelogenous leukemia (CML) is a disease characterized by the presence of p210bcr-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, p56dok-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 p120RasGAP . 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.

DOK4 and DOK5: new dok-related genes expressed in human T cells

“…11,14 These studies have led to the identification of an amino acid sequence in the PTB domain displaying a high similarity between the first three Dok family members: Dok-1 (human DOK1 and mouse Dok1 genes), Dok-2/FRIP/DokR (human DOK2 and mouse Dok2 genes) and also Dok3/DokL (mouse Dokl-pending gene). This sequence has been defined as a Dok homology (DKH) sequence motif (WPxxxLRxxGx DxxxFxFExGR, where x is any amino acid residue).…”

“…This sequence has been defined as a Dok homology (DKH) sequence motif (WPxxxLRxxGx DxxxFxFExGR, where x is any amino acid residue). 11,14 To identify potential new members of the Dok adapter family, we performed public database similarity searches using this DKH motif and also the sequence of human DOK1 as probes. Based on the screening with the DKH motif, we identified several candidates containing this signature as it has been shown previously.…”

“…The sequences of DOK1/2 in human and Dok1/2/3 in mice have been reported previously. 3,7,[11][12][13]15,16 Recently, Dok4/5 genes have been cloned from mouse tissues. 21 The amino acid sequences from human DOK3/4/5 genes have been deduced (see Material and Methods).…”

“…3,[6][7][8] The structure of Dok-1 presents some similarities with other adapter molecules such as Gab proteins or insulin receptor substrate (IRS) proteins. 9,10 Two proteins sharing extensive sequence homology with the amino-terminal part (PH and PTB domains) of Dok-1 have been cloned: Dok-2 (also known as FRIP or Dok-R) [11][12][13][14] and Dok-3 (also known as Dok-L). 15,16 These proteins are essentially expressed in hematopoietic tissues.…”

“…A first screening has been performed with a sequence containing a high homology between Dok-1, Dok-2 and also Dok-3. 11,14 Among putative proteins displaying a Dok homology (DKH) sequence motif, a search has been performed for proteins containing a PH domain followed by a PTB domain.…”