Grb10 is a dual regulator of receptor tyrosine kinase signaling

The adaptor protein Grb10 is a close homolog of Grb7 and Grb14. These proteins are characterized by an N-terminal proline-rich region, a Ras–GTPase binding domain, a PH domain, an SH2 domain and a BPS domain in between the PH and SH2 domains. Human Grb10 gene encodes three splice variants. These variants show differences in functionality. Grb10 associates with multiple proteins including tyrosine kinases in a tyrosine phosphorylation dependent or independent manner. Association with multiple proteins allows Grb10 to regulate different signaling pathways resulting in different biological consequences.     

Ischemic injury to kidney induces glomerular podocyte effacement and dissociation of slit diaphragm proteins Neph1 and ZO-1

Glomerular injury is often characterized by the effacement of podocytes, loss of slit diaphragms, and proteinuria. Renal ischemia or the loss of blood flow to the kidneys has been widely associated with tubular and endothelial injury but rarely has been shown to induce podocyte damage and disruption of the slit diaphragm. In this study, we have used an in vivo rat ischemic model to demonstrate that renal ischemia induces podocyte effacement with loss of slit diaphragm and proteinuria. Biochemical analysis of the ischemic glomerulus shows that ischemia induces rapid loss of interaction between slit diaphragm junctional proteins Neph1 and ZO-1. To further understand the effect of ischemia on molecular interactions between slit diaphragm proteins, a cell culture model was employed to study the binding between Neph1 and ZO-1. Under physiologic conditions, Neph1 co-localized with ZO-1 at cell-cell contacts in cultured human podocytes. Induction of injury by ATP depletion resulted in rapid loss of Neph1 and ZO-1 binding and redistribution of Neph1 and ZO-1 proteins from cell membrane to the cytoplasm. Recovery resulted in increased Neph1 tyrosine phosphorylation, restoring Neph1 and ZO-1 binding and their localization at the cell membrane. We further demonstrate that tyrosine phosphorylation of Neph1 mediated by Fyn results in significantly increased Neph1 and ZO-1 binding, suggesting a critical role for Neph1 tyrosine phosphorylation in reorganizing the Neph1-ZO-1 complex. This study documents that renal ischemia induces dynamic changes in the molecular interactions between slit diaphragm proteins, leading to podocyte damage and proteinuria.     

Fyn binds to and phosphorylates the kidney slit diaphragm component Nephrin

Recent investigations have focused on characterizing the molecular components of the podocyte intercellular junction, because several of these components, including Nephrin, are functionally necessary for development of normal podocyte structure and filter integrity. Accumulating evidence suggests that the Nephrin-associated protein complex is a signaling nexus. As such, Nephrin-dependent signaling might be mediated in part through Nephrin phosphorylation. Described are biochemical and mouse genetics experiments demonstrating that membrane-associated Nephrin is tyrosine-phosphorylated by the Src family kinase Fyn. Nephrin fractionated in detergent-resistant glomerular membrane fractions with Fyn and Yes. Fyn directly bound Nephrin via its SH3 domain, and Fyn directly phosphorylated Nephrin. Glomeruli in which Fyn, Yes, or Fyn and Yes were genetically deleted in mice were characterized to explore the relationship between these kinases and Nephrin. Fyn deletion resulted in coarsening of podocyte foot processes and marked attenuation of Nephrin phosphorylation in isolated glomerular detergent-resistant membrane fractions. Yes deletion had no identifiable effect on podocyte morphology but dramatically increased Nephrin phosphorylating activity. Similar to Fyn deletion, simultaneous deletion of Fyn and Yes reduced Nephrin phosphorylating activity. These results demonstrate that endogenous Fyn catalyzes Nephrin phosphorylation in podocyte detergent-resistant membrane fractions. Although Yes appears to effect the regulation of Nephrin phosphorylation, the mechanism by which this occurs requires investigation.     

Motor protein Myo1c is a podocyte protein that facilitates the transport of slit diaphragm protein Neph1 to the podocyte membrane

The podocyte proteins Neph1 and nephrin organize a signaling complex at the podocyte cell membrane that forms the structural framework for a functional glomerular filtration barrier. Mechanisms regulating the movement of these proteins to and from the membrane are currently unknown. This study identifies a novel interaction between Neph1 and the motor protein Myo1c, where Myo1c plays an active role in targeting Neph1 to the podocyte cell membrane. Using in vivo and in vitro experiments, we provide data supporting a direct interaction between Neph1 and Myo1c which is dynamic and actin dependent. Unlike wild-type Myo1c, the membrane localization of Neph1 was significantly reduced in podocytes expressing dominant negative Myo1c. In addition, Neph1 failed to localize at the podocyte cell membrane and cell junctions in Myo1c-depleted podocytes. We further demonstrate that similarly to Neph1, Myo1c also binds nephrin and reduces its localization at the podocyte cell membrane. A functional analysis of Myo1c knockdown cells showed defects in cell migration, as determined by a wound assay. In addition, the ability to form tight junctions was impaired in Myo1c knockdown cells, as determined by transepithelial electric resistance (TER) and bovine serum albumin (BSA) permeability assays. These results identify a novel Myo1c-dependent molecular mechanism that mediates the dynamic organization of Neph1 and nephrin at the slit diaphragm and is critical for podocyte function.     

Gi-mediated activation of the Ras/MAP kinase pathway involves a 100 kDa tyrosine-phosphorylated Grb2 SH3 binding protein, but not Src nor Shc.

Mitogenic G protein-coupled receptors, such as those for lysophosphatidic acid (LPA) and thrombin, activate the Ras/MAP kinase pathway via pertussis toxin (PTX)-sensitive Gi, tyrosine kinase activity and recruitment of Grb2, which targets guanine nucleotide exchange activity to Ras. Little is known about the tyrosine phosphorylations involved, although Src activation and Shc phosphorylation are thought to be critical. We find that agonist-induced Src activation in Rat-1 cells is not mediated by Gi and shows no correlation with Ras/MAP kinase activation. Furthermore, LPA-induced tyrosine phosphorylation of Shc is PTX-insensitive and Ca2+-dependent in COS cells, but undetectable in Rat-1 cells. Expression of dominant-negative Src or Shc does not affect MAP kinase activation by LPA. Thus, Gi-mediated Ras/MAP kinase activation in fibroblasts and COS cells involves neither Src nor Shc. Instead, we detect a 100 kDa tyrosine-phosphorylated protein (p100) that binds to the C-terminal SH3 domain of Grb2 in a strictly Gi- and agonist-dependent manner. Tyrosine kinase inhibitors and wortmannin, a phosphatidylinositol (PI) 3-kinase inhibitor, prevent p100-Grb2 complex formation and MAP kinase activation by LPA. Our results suggest that the p100-Grb2 complex, together with an upstream non-Src tyrosine kinase and PI 3-kinase, couples Gi to Ras/MAP kinase activation, while Src and Shc act in a different pathway.     

Regulation of the bovine kidney microsomal chloride channel p64 by p59fyn, a Src family tyrosine kinase

p64 is a chloride channel of intracellular membranes which is present in regulated secretory vesicles. Mechanisms by which the p64 channel could be regulated are largely unknown. p59(fyn) is a non-receptor tyrosine kinase of the Src family that has been implicated in a variety of intracellular signaling events. The N-terminal portion of p64 has several potential binding sites for Src family SH2 domains. In this paper, we demonstrate that p64 becomes tyrosine phosphorylated when co-expressed with p59(fyn) in HeLa cells. We show that co-expression of p64 with p59(fyn) renders p64 a ligand for the SH2 domain of p59(fyn) and this SH2 binding is eliminated by treating p64 with alkaline phosphatase. Using site-directed mutagenesis, we find that tyrosine 33 in the p64 sequence is necessary for SH2 binding. We also characterized p64-p59(fyn) interactions using native material from bovine kidney. We found that a small fraction of native kidney p64 can bind Fyn SH2 in vitro. Immunoprecipitation of p64 from solubilized kidney membranes yields a kinase activity with the same mobility by SDS-polyacrylamide gel electrophoresis as authentic bovine p59(fyn). Finally, we demonstrate that co-expression of p64 and p59(fyn) in HeLa cells results in enhanced p64-associated chloride channel activity.     

Direct binding of the signaling adapter protein Grb2 to the activation loop tyrosines on the nerve growth factor receptor tyrosine kinase, TrkA

We demonstrate that the signaling adapter, Grb2, binds directly to the neurotrophin receptor tyrosine kinase, TrkA. Grb2 binding to TrkA is independent of Shc, FRS-2, phospholipase Cgamma-1, rAPS, and SH2B and is observed in in vitro binding assays, yeast two-hybrid assays, and in co-immunoprecipitation assays. Grb2 binding to TrkA is mediated by the central SH2 domain, requires a kinase-active TrkA, and is phosphotyrosine-dependent. By analyzing a series of rat TrkA mutants, we demonstrate that Grb2 binds to the carboxyl-terminal residue, Tyr(794), as well as to the activation loop tyrosines, Tyr(683) and Tyr(684). By using acidic amino acid substitutions of the activation loop tyrosines on TrkA, we can stimulate constitutive kinase activity and TrkA-Shc interactions but, importantly, abolish TrkA/Grb2 binding. Thus, in addition to providing the first evidence of direct Grb2 binding to the neurotrophin receptor, TrkA, these data provide the first direct evidence that the activation loop tyrosines of a receptor tyrosine kinase, in addition to their essential role in kinase activation, also serve a direct role in the recruitment of intracellular signaling molecules.     

Etk, a Btk family tyrosine kinase, mediates cellular transformation by linking Src to STAT3 activation

Etk (also called Bmx) is a member of the Btk tyrosine kinase family and is expressed in a variety of hematopoietic, epithelial, and endothelial cells. We have explored biological functions, regulators, and effectors of Etk. Coexpression of v-Src and Etk led to a transphosphorylation on tyrosine 566 of Etk and subsequent autophosphorylation. These events correlated with a substantial increase in the kinase activity of Etk. STAT3, which was previously shown to be activated by Etk, associated with Etk in vivo. To investigate whether Etk could mediate v-Src-induced activation of STAT3 and cell transformation, we overexpressed a dominant-negative mutant of Etk in an immortalized, untransformed rat liver epithelial cell line, WB, which contains endogenous Etk. Dominant-negative inactivation of Etk not only blocked v-Src-induced tyrosine phosphorylation and activation of STAT3 but also caused a great reduction in the transforming activity of v-Src. In NIH3T3 cells, although Etk did not itself induce transformation, it effectively enhanced the transforming ability of a partially active c-Src mutant (c-Src378G). Furthermore, Etk activated STAT3-mediated gene expression in synergy with this Src mutant. Our findings thus indicate that Etk is a critical mediator of Src-induced cell transformation and STAT3 activation. The role of STAT3 in Etk-mediated transformation was also examined. Expression of Etk in a human hepatoma cell line Hep3B resulted in a significant increase in its transforming ability, and this effect was abrogated by dominant-negative inhibition of STAT3. These data strongly suggest that Etk links Src to STAT3 activation. Furthermore, Src-Etk-STAT3 is an important pathway in cellular transformation.     

Phosphorylation of cyclin dependent kinase 4 on tyrosine 17 is mediated by Src family kinases

Cyclin dependent kinase 4 is a key regulator of the cell cycle and its activity is frequently deregulated in cancer. The activity of cyclin dependent kinase 4 is controlled by multiple mechanisms, including phosphorylation of tyrosine 17. This site is equivalent to tyrosine 15 of cyclin dependent kinase 1, which undergoes inhibitory phosphorylation by WEE1 and MYT1; however, the kinases that phosphorylate cyclin dependent kinase 4 on tyrosine 17 are still unknown. In the present study, we generated a phosphospecific antibody to the tyrosine 17-phosphorylated form of cyclin dependent kinase 4, and showed that this site is phosphorylated to a low level in asynchronously proliferating HCT116 cells. We purified tyrosine 17 kinases from HeLa cells and found that the Src family non-receptor tyrosine kinase C-YES contributes a large fraction of the tyrosine 17 kinase activity in HeLa lysates. C-YES also phosphorylated cyclin dependent kinase 4 when transfected into HCT116 cells, and treatment of cells with Src family kinase inhibitors blocked the tyrosine 17 phosphorylation of cyclin dependent kinase 4. Taken together, the results obtained in the present study provide the first evidence that Src family kinases, but not WEE1 or MYT1, phosphorylate cyclin dependent kinase 4 on tyrosine 17, and help to resolve how the phosphorylation of this site is regulated.     

Phosphorylated Grb14 is an endogenous inhibitor of retinal protein tyrosine phosphatase 1B, and light-dependent activation of Src phosphorylates Grb14

Growth factor receptor-bound protein 14 (Grb14) is an adapter protein implicated in receptor tyrosine kinase signaling. Grb14(-/-) studies highlight both the positive and negative roles of Grb14 in receptor tyrosine kinase signaling in a tissue-specific manner. In this study, we made a novel finding that Grb14 inhibits the activity of PTP1B, the major negative regulator of insulin receptor (IR) signaling, in a phosphorylation-regulated manner. Phosphorylation of Tyr-347 in the BPS domain of Grb14 is critical for interaction with PTP1B, resulting in the competitive inhibition of PTP1B activity. We also found that rhodopsin-regulated Src kinase activation in retina leads to the phosphorylation of Grb14. Further, ablation of Grb14 resulted in significantly elevated retinal PTP1B activity in vivo. PTP1B is known to be regulated by oxidation, glutathionylation, phosphorylation, and SUMOlyation, and our study for the first time demonstrates the inhibition of PTP1B activity in vivo by protein molecule Grb14 in a tissue-specific manner.     

Phospholipase Cgamma2 modulates integrin signaling in the osteoclast by affecting the localization and activation of Src kinase

Integrin engagement induces a cascade of signaling pathways that include tyrosine phosphorylation of numerous proteins that lead to modulation of the actin cytoskeleton. Src is a major intracellular mediator of integrin-dependent functions, but the mechanism(s) by which Src is regulated in response to integrin signals is not fully understood. Here, we demonstrate an important role for phospholipase C gamma 2 (PLCgamma2) in Src activation in the osteoclast. Through analysis of primary cells from PLCgamma2(-/-) mice, PLCgamma2 was found to be an important regulator of alpha(v)beta(3) integrin-mediated bone osteoclast cell adhesion, migration, and bone resorption. Adhesion-induced PYK2 and Src phosphorylation is decreased in the absence of PLCgamma2, and the interaction of Src with beta(3) integrin and PYK2 is dramatically reduced. Importantly, PLCgamma2 was found to be required for proper localization of Src to the sealing actin ring, and this function required both its catalytic activity and adapter domains. Based on these results, we propose that PLCgamma2 influences Src activation by mediating the localization of Src to the integrin complex and thereby regulating integrin-mediated functions in the osteoclast.     

A non-catalytic function of the Src family tyrosine kinases controls prolactin-induced Jak2 signaling

The cytokine prolactin (PRL) plays important roles in the proliferation and differentiation of the mammary gland and it has been implicated in tumorigenesis. The prolactin receptor (PRLR) is devoid of catalytic activity and its mitogenic response is controlled by cytoplasmic tyrosine kinases of the Src (SFK) and Jak families. How PRLR uses these kinases for signaling is not well understood. Previous studies indicated that PRLR-induced Jak2 activation does not require SFK catalytic activity in favor of separate signaling operating on this cellular response. Here we show that, nevertheless, PRLR requires Src-SH2 and -SH3 domains for Jak2 signaling. In W53 lymphoid cells, conditional expression of two c-Src non-catalytic mutants, either SrcK295M/Y527F or Src?K, whose SH3 and SH2 domains are exposed, controls Jak2/Stat5 activation by recruiting Jak2, avoiding its activation by endogenous active SFK. In contrast, the kinase inactive SrcK295M mutant, with inaccessible SH3 and SH2 domains, does not. Furthermore, all three mutants attenuate PRLR-induced Akt and p70S6K activation. Accordingly, PRLR-induced Jak2/Stat5 signaling is inhibited in MCF7 breast cancer cells by Src depletion, expression of SrcK295M/Y527F or active Src harboring an inactive SH2 (SrcR175L) or SH3 domain (SrcW118A). Finally, Jak2/Stat5 pathway is also reduced in Src^?/? mice mammary glands. We thus conclude that, in addition to Akt and p70S6K, SFK regulate PRLR-induced Jak2 signaling through a kinase-independent mechanism.     

Evidence that a starfish egg Src family tyrosine kinase associates with PLC-gamma1 SH2 domains at fertilization

The initiation of calcium release at fertilization in the eggs of most animals relies on the production of IP3, implicating the activation of phospholipase C. Recent work has demonstrated that injection of PLC-gamma SH2 domain fusion proteins into starfish eggs specifically inhibits the initiation of calcium release in response to sperm, indicating that PLC-gamma is necessary for Ca2+ release at fertilization [Carroll et al. (1997) J. Cell Biol. 138, 1303-1311]. Here we investigate how PLC-gamma may be activated, by using the PLC-gamma SH2 domain fusion protein as an affinity matrix to identify interacting proteins. A tyrosine kinase activity and an egg protein of ca. Mr 58 K that is recognized by an antibody directed against Src family tyrosine kinases associate with PLC-gamma SH2 domains in a fertilization-dependent manner. These associations are detected by 15 s postfertilization, consistent with a function in releasing Ca2+. Calcium ionophore treatment of eggs did not cause association of the kinase activity or of the Src family protein with the PLC-gamma SH2 domains. These data identify an egg Src family tyrosine kinase as a potential upstream regulator of PLC-gamma in the activation of starfish eggs.     

TAB-1 modulates intracellular localization of p38 MAP kinase and downstream signaling

Stress-activated mitogen-activated protein (MAP) kinase p38 mediates stress signaling in mammalian cells via threonine and tyrosine phosphorylation in its conserved TGY motif by upstream MAP kinase kinases (MKKs). In addition, p38 MAP kinase can also be activated by an MKK-independent mechanism involving TAB-1 (TAK-1-binding protein)-mediated autophosphorylation. Although TAB-1-mediated p38 activation has been implicated in ischemic heart, the biological consequences and downstream signaling of TAB-1-mediated p38 activation in cardiomyocytes is largely unknown. We show here that TAB-1 expression leads to a significant induction of p38 autophosphorylation and consequent kinase activation in cultured neonatal cardiomyocytes. In contrast to MKK3-induced p38 kinase downstream effects, TAB-1-induced p38 kinase activation does not induce expression of pro-inflammatory genes, cardiac marker gene expression, or changes in cellular morphology. Rather, TAB-1 binds to p38 and prevents p38 nuclear localization. Furthermore, TAB-1 disrupts p38 interaction with MKK3 and redirects p38 localization in the cytosol. Consequently, TAB-1 expression antagonizes the downstream activity of p38 kinase induced by MKK3 and attenuates interleukin-1beta-induced inflammatory gene induction in cardiomyocytes. These data suggest that TAB-1 can mediate MKK-independent p38 kinase activation while negatively modulating MKK-dependent p38 function. Our study not only redefines the functional role of TAB-1 in p38 kinase-mediated signaling pathways but also provides the first evidence that intracellular localization of p38 kinase and complex interaction dictates its downstream effects. These results suggest a previously unknown mechanism for stress-MAP kinase regulation in mammalian cells.     

Src homology 3 binding sites in the P2Y2 nucleotide receptor interact with Src and regulate activities of Src, proline-rich tyrosine kinase 2, and growth factor receptors

Many G protein-coupled receptors activate growth factor receptors, although the mechanisms controlling this transactivation are unclear. We have identified two proline-rich, SH3 binding sites (PXXP) in the carboxyl-terminal tail of the human P2Y(2) nucleotide receptor that directly associate with the tyrosine kinase Src in protein binding assays. Furthermore, Src co-precipitated with the P2Y(2) receptor in 1321N1 astrocytoma cells stimulated with the P2Y(2) receptor agonist UTP. A mutant P2Y(2) receptor lacking the PXXP motifs was found to stimulate calcium mobilization and serine/threonine phosphorylation of the Erk1/2 mitogen-activated protein kinases, like the wild-type receptor, but was defective in its ability to stimulate tyrosine phosphorylation of Src and Src-dependent tyrosine phosphorylation of the proline-rich tyrosine kinase 2, epidermal growth factor receptor (EGFR), and platelet-derived growth factor receptor. Dual immunofluorescence labeling of the P2Y(2) receptor and the EGFR indicated that UTP caused an increase in the co-localization of these receptors in the plasma membrane that was prevented by the Src inhibitor PP2. Together, these data suggest that agonist-induced binding of Src to the SH3 binding sites in the P2Y(2) receptor facilitates Src activation, which recruits the EGFR into a protein complex with the P2Y(2) receptor and allows Src to efficiently phosphorylate the EGFR.     

Identification of PSD-93 as a substrate for the Src family tyrosine kinase Fyn

In order to study the role of tyrosine kinase signaling in the post-synaptic density (PSD), tyrosine-phosphorylated proteins associated with the PSD-95/NMDA receptor complex were analyzed. The NMDA receptor complex from the mouse brain was successfully solubilized with deoxycholate and immunopurified with anti-PSD-95 or anti-phosphotyrosine antibody. Immunoblot analyses revealed that the predominantly tyrosine-phosphorylated proteins in the NMDA receptor complex are the NR2A/B subunits and a novel 120 kDa protein. Purification and microsequencing analysis showed that the 120 kDa protein is mouse PSD-93/Chapsyn-110. Recombinant PSD-93 was phosphorylated by Fyn in vitro, and Tyr-384 was identified as a major phosphorylation site. Tyrosine phosphorylation of PSD-93 was greatly reduced in brain tissue from Fyn-deficient mice compared with wild-type mice. Furthermore, an N-terminal palmitoylation signal of PSD-93 was found to be essential for its anchoring to the membrane, where Fyn is also localized. In COS7 cells, exogenously expressed PSD-93 was phosphorylated, dependent on its membrane localization. In addition, tyrosine-phosphorylated PSD-93 was able to bind to Csk, a negative regulator of Src family kinases, in vitro as well as in a brain lysate. These results suggest that PSD-93 serves as a membrane-anchored substrate of Fyn and plays a role in the regulation of Fyn-mediated modification of NMDA receptor function.     

ErbB2, EphrinB1, Src kinase and PTPN13 signaling complex regulates MAP kinase signaling in human cancers

In non-cancerous cells, phosphorylated proteins exist transiently, becoming de-phosphorylated by specific phosphatases that terminate propagation of signaling pathways. In cancers, compromised phosphatase activity and/or expression occur and contribute to tumor phenotype. The non-receptor phosphatase, PTPN13, has recently been dubbed a putative tumor suppressor. It decreased expression in breast cancer correlates with decreased overall survival. Here we show that PTPN13 regulates a new signaling complex in breast cancer consisting of ErbB2, Src, and EphrinB1. To our knowledge, this signaling complex has not been previously described. Co-immunoprecipitation and localization studies demonstrate that EphrinB1, a PTPN13 substrate, interacts with ErbB2. In addition, the oncogenic V660E ErbB2 mutation enhances this interaction, while Src kinase mediates EphrinB1 phosphorylation and subsequent MAP Kinase signaling. Decreased PTPN13 function further enhances signaling. The association of oncogene kinases (ErbB2, Src), a signaling transmembrane ligand (EphrinB1) and a phosphatase tumor suppressor (PTPN13) suggest that EphrinB1 may be a relevant therapeutic target in breast cancers harboring ErbB2-activating mutations and decreased PTPN13 expression.     

A direct binding site for Grb2 contributes to transformation and leukemogenesis by the Tel-Abl (ETV6-Abl) tyrosine kinase

A direct binding site for the Grb2 adapter protein is required for the induction of fatal chronic myeloid leukemia (CML)-like disease in mice by Bcr-Abl. Here, we demonstrate direct binding of Grb2 to the Tel-Abl (ETV6-Abl) fusion protein, the product of complex (9;12) chromosomal translocations in human leukemia, via tyrosine 314 encoded by TEL exon 5. A Tel-Abl point mutant (Y314F) and a splice variant without TEL exon 5 sequences (Deltae5) lacked Grb2 interaction and exhibited decreased binding and phosphorylation of the scaffolding protein Gab2 and impaired activation of phosphatidylinositol 3-kinase, Akt, and extracellular signal-regulated kinase/mitogen-activated protein kinase in hematopoietic cells. Tel-Abl Y314F and Deltae5 were unable to transform fibroblasts to anchorage-independent growth and were defective for B-lymphoid transformation in vitro and lymphoid leukemogenesis in vivo. Previously, we demonstrated that full-length Tel-Abl induced two distinct myeloproliferative diseases in mice: CML-like leukemia similar to that induced by Bcr-Abl and a novel syndrome of small-bowel myeloid infiltration endotoxemia and hepatic and renal failure. Lack of the Grb2 binding site had no effect on development of small bowel syndrome but significantly attenuated the induction of CML-like disease by Tel-Abl. These results suggest that direct binding of Grb2 is a common mechanism contributing to leukemogenesis by oncogenic Abl fusion proteins.