Characteristic of chronic myelogenous leukemia (CML) is the presence of the chimeric p210(bcr-abl) protein possessing elevated protein tyrosine kinase activity relative to normal c-abl tyrosine kinase. Hematopoietic progenitors isolated from CML patients in the chronic phase contain a constitutively tyrosine-phosphorylated protein that migrates at 62 kDa by SDS-PAGE and associates with the p120 ras GTPase-activating protein (GAP). We have purified p62(dok) from a hematopoietic cell line expressing p210(bcr-abl). p62(dok) is a novel protein with features of a signaling molecule. Association of p62(dok) with GAP correlates with its tyrosine phosphorylation. p62(dok) is rapidly tyrosine-phosphorylated upon activation of the c-Kit receptor, implicating it as a component of a signal transduction pathway downstream of receptor tyrosine kinases.

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.

bcr/abl has elevated tyropathways that we are now trying to define. Constitutive downstream activation of critical pathways in susceptible early prosine kinase activity that is essential for its transforming activity. We recently reported a prominent 62 kDa GAP-associagenitors that normally require KL or other factors for activation could explain most if not all features of the disease. ted P-tyr protein and five additional consistent but less prominent P-tyr proteins as well as five more minor P-tyr proteins Keywords: CML; discordant maturation; cytokine signaling pathways; kit ligand/c-kit; p210 bcr/abl tyrosine kinase activity; constitutive that are constitutively tyrosine phosphorylated in primary primitive lineage negative (lin−) chronic phase CML blasts but tyrosine phosphorylation not in comparable primary lin− normal blasts. The GAP-associated p62 protein has now been purified, sequenced and its gene has been cloned; it is a previously unidentified protein

The chronological history of the important discoveries leading to our present understanding of the essential clinical, biological, biochemical, and molecular features of chronic myelogenous leukemia (CML) are first reviewed, focusing in particular on abnormalities that are responsible for the massive myeloid expansion. CML is an excellent target for the development of selective treatment because of its highly consistent genetic abnormality and qualitatively different fusion gene product, p210(bcr-abl). It is likely that the multiple signaling pathways dysregulated by p210(bcr-abl) are sufficient to explain all the initial manifestations of the chronic phase of the disease, although understanding of the circuitry is still very incomplete. Evidence is presented that the signaling pathways that are constitutively activated in CML stem cells and primitive progenitors cooperate with cytokines to increase the proportion of stem cells that are activated and thereby increase recruitment into the committed progenitor cell pool, and that this increased activation is probably the primary cause of the massive myeloid expansion in CML. The cooperative interactions between Bcr-Abl and cytokine-activated pathways interfere with the synergistic interactions between multiple cytokines that are normally required for the activation of stem cells, while at the same time causing numerous subtle biochemical and functional abnormalities in the later progenitors and precursor cells. The committed CML progenitors have discordant maturation and reduced proliferative capacity compared to normal committed progenitors, and like them, are destined to die after a limited number of divisions. Thus, the primary goal of any curative strategy must be to eliminate all Philadelphia positive (Ph+) primitive cells that are capable of symmetric division and thereby able to expand the Ph+ stem cell pool and recreate the disease. Several highly potent and moderately selective inhibitors of Bcr-Abl kinase have recently been discovered that are capable of killing the majority of actively proliferating early CML progenitors with minimal effects on normal progenitors. However, like their normal counterparts, most of the CML primitive stem cells are quiescent at any given time and are relatively invulnerable to the Bcr-Abl kinase inhibitors as well as other drugs. We propose that survival of dormant Ph+ stem cells may be the most important reason for the inability to cure the disease during initial treatment, while resistance to the inhibitors and other drugs becomes increasingly important later. An outline of a possible curative strategy is presented that attempts to take advantage of the subtle differences in the proliferative behavior of normal and Ph+ stem cells and the newly discovered selective inhibitors of Bcr-Abl. Leukemia (2003) 17, 1211-1262. doi:10.1038/sj.leu.2402912

Dok-1 is an adaptor protein that is a substrate forIn patients with chronic myelogenous leukemia, the c-abl gene is translocated from chromosome 9 to chromosome 22 to produce a hybrid bcr-abl gene (1-3). The fusion protein that results from this translocation, p210Bcr-Abl, encodes a tyrosine kinase whose activation results in phosphorylation of a number of cellular proteins including SHIP1, SHIP2, Cbl, Lyn, SHC, and Dok-1 (4). These proteins are also tyrosine-phosphorylated when cells are stimulated with c-Kit ligand (KL), 1 a growth factor that is critical for normal hematopoiesis. KL binding to the c-Kit receptor results in dimerization and autophosphorylation of c-Kit and phosphorylation of PI 3-kinase, Tec, phospholipase C␥, and Vav in addition to the signaling proteins listed above (5-7).We have been studying the role of Dok-1 in normal and malignant cell signaling. Dok-1 is tyrosine-phosphorylated in response to a variety of growth factors including platelet-derived growth factor, insulin-like growth factor, vascular endothelial growth factor, granulocyte-macrophage colony-stimulated factor, interleukin-3 (IL-3), and KL. Cloning of the Dok-1 cDNA revealed that the overall structure of Dok-1 is similar to insulin receptor substrate-1 (IRS-1), which harbors an N-terminal pleckstrin homology (PH) domain, a central phosphotyrosine binding domain, and a C-terminal tail containing multiple tyrosine phosphorylation sites (8, 9). The PH domain of Dok-1 is thought to mediate protein interaction with the plasma membrane possibly by binding to phospholipids. The phosphotyrosine binding domain of Dok-1 is thought to mediate protein-protein interactions by binding to phosphotyrosine-containing motifs with the sequence of NPXpY (10, 11). The multiple tyrosine residues at the C-terminal region are phosphorylated in response to various growth factors. When phosphorylated they act as docking sites for SH2-containing proteins such as p120RasGAP and NCK (12).Analysis of Dok-1 Ϫ/Ϫ mice reveals that Dok-1 is a negative regulator of cell proliferation. Cells derived from Dok Ϫ/Ϫ mice hyperproliferate in response to a number of cytokines and growth factors including KL (17,21). However, the mechanism responsible for the hyperproliferative effect has not yet been elucidated. Moreover, the kinase(s) that phosphorylates Dok-1 in c-Kit-mediated signaling has not been identified. Lyn (a Src family kinase) and Tec (a Tec family kinase) have been reported to be activated upon c-Kit activation (5-7). Tec forms a trimolecular complex with Dok-1 and Lyn in KL-stimulated cells, and activation of Tec and phosphorylation of Dok-1 have been shown to require PI 3-kinase activity (13). Other studies have documented a role for Src family kinases in Dok-1 phosphorylation. For example, Lck is required for CD2-mediated phosphorylation of Dok-1 in JcaM1.6 cells, and Src, Fyn, and Lck can phosphorylate Dok-1 in .In this study, we examine the mechanisms involved in tyrosine phosphorylation, membrane recruitment, and signal transduction by Dok-1 during...