A BCR-ABL Mutant Lacking Direct Binding Sites for the GRB2, CBL and CRKL Adapter Proteins Fails to Induce Leukemia in Mice

The BCR-ABL tyrosine kinase is the defining feature of chronic myeloid leukemia (CML) and its kinase activity is required for induction of this disease. Current thinking holds that BCR-ABL forms a multi-protein complex that incorporates several substrates and adaptor proteins and is stabilized by multiple direct and indirect interactions. Signaling output from this highly redundant network leads to cellular transformation. Proteins known to be associated with BCR-ABL in this complex include: GRB2, c-CBL, p62^DOK, and CRKL. These proteins in turn, link BCR-ABL to various signaling pathways indicated in cellular transformation. In this study we show that a triple mutant of BCR-ABL with mutations of the direct binding sites for GRB2, CBL, p62^DOK and CRKL, is defective for transformation of primary hematopoietic cells in vitro and in a murine CML model, while it retains the capacity to induce IL-3 independence in 32D cells. Compared to BCR-ABL, the triple mutant''s ability to activate the MAP kinase and PI3-kinase pathways is severely compromised, while STAT5 phosphorylation is maintained, suggesting that the former are crucial for the transformation of primary cells, but dispensable for transformation of factor dependent cell lines. Our data suggest that inhibition of BCR-ABL-induced leukemia by disrupting protein interactions could be possible, but would require blocking of multiple sites.     

Identification of domains of the insulin-like growth factor I receptor that are required for protection from apoptosis.

Using a series of insulin-like growth factor I (IGF-I) receptor mutants, we have attempted to define domains required for transmitting the antiapoptotic signal from the receptor and to compare these domains with those required for mitogenesis or transformation. In FL5.12 cells transfected with wild-type IGF-I receptors, IGF-I affords protection from interleukin 3 withdrawal but is not mitogenic. An IGF-I receptor lacking a functional ATP binding site provided no protection from apoptosis. However, receptors mutated at tyrosine residue 950 or in the tyrosine cluster (1131, 1135, and 1136) within the kinase domain remained capable of suppressing apoptosis, although such mutations are known to inactivate transforming and mitogenic functions. In the C terminus of the IGF-I receptor, two mutations, one at tyrosine 1251 and one which replaced residues histidine 1293 and lysine 1294, abolished the antiapoptotic function, whereas mutation of the four serines at 1280 to 1283 did not. Interestingly, receptors truncated at the C terminus had enhanced antiapoptotic function. In Rat-1/ c-MycER fibroblasts, the Y950F mutant and the tyrosine cluster mutant could still provide protection from c-Myc-induced apoptosis, whereas mutant Y1250/1251F could not. These studies demonstrate that the domains of the IGF-I receptor required for its antiapoptotic function are distinct from those required for its proliferation or transformation functions and suggest that domains of the receptor required for inhibition of apoptosis are necessary but not sufficient for transformation.     

A novel pathway from phosphorylation of tyrosine residues 239/240 of Shc, contributing to suppress apoptosis by IL-3.

Interleukin 3 (IL-3) not only induces DNA synthesis of haematopoietic cells but also maintains their viability by suppressing apoptosis. IL-3 stimulates tyrosine phosphorylation of the Shc adaptor protein and thereby formation of a complex of Shc with Grb2 at phosphorylated tyrosine (Y) residue 317-Shc. This pathway is implicated in Ras/mitogen-activated protein kinase (MAPK) activation towards c-fos gene expression. We examined the possible involvement of Shc in the antiapoptotic activity of IL-3. Conditional overexpression of the Shc SH2 domain, a dominant-negative mutant of Shc, was found to induce apoptosis of IL-3-dependent Ba/F3 cells along with a reduction of c-myc gene expression. Apoptosis was rescued by the exogenously introduced c-myc gene. Since we identify novel tyrosine phosphorylation sites of Shc: Y239 and Y240, their role on cell survival was tested by mutational analysis. Ba/F3 cells expressing mutant Shc Y317F, which is unable to stimulate efficiently the Ras pathway, still showed resistance to apoptosis. However, cells expressing Shc Y239/240F, which is able to stimulate the Ras pathway, were sensitive to apoptosis. In these cells, induction of the c-myc gene was reduced. These findings suggest that a new signalling pathway for cell survival is generated from Y239/240 of Shc to the nuclei involving c-myc gene expression.     

Tyr-716 in the platelet-derived growth factor beta-receptor kinase insert is involved in GRB2 binding and Ras activation.

Ligand stimulation of the platelet-derived growth factor (PDGF) beta-receptor leads to activation of its intrinsic tyrosine kinase and autophosphorylation of the intracellular part of the receptor. The autophosphorylated tyrosine residues mediate interactions with downstream signal transduction molecules and thereby initiate different signalling pathways. A pathway leading to activation of the GTP-binding protein Ras involves the adaptor molecule GRB2. Here we show that Tyr-716, a novel autophosphorylation site in the PDGF beta-receptor kinase insert, mediates direct binding of GRB2 in vitro and in vivo. In a panel of mutant PDGF beta-receptors, in which Tyr-716 and the previously known autophosphorylation sites were individually mutated, only PDGFR beta Y716F failed to bind GRB2. Furthermore, a synthetic phosphorylated peptide containing Tyr-716 bound GRB2, and this peptide specifically interrupted the interaction between GRB2 and the wild-type receptor. In addition, the Y716(P) peptide significantly decreased the amount of GTP bound to Ras in response to PDGF in permeabilized fibroblasts as well as in porcine aortic endothelial cells expressing transfected PDGF beta-receptors. The mutant PDGFR beta Y716F still mediated activation of mitogen-activated protein kinases and an increased DNA synthesis in response to PDGF, indicating that multiple signal transduction pathways transduce mitogenic signals from the activated PDGF beta-receptor.     

Biological function of PDGF-induced PI-3 kinase activity: its role in alpha PDGF receptor-mediated mitogenic signaling

The tyrosine phosphorylation sites in the human alpha PDGF receptor (alpha PDGFR) required for association with PI-3 kinase have been identified as tyrosines 731 and 742. Mutation of either tyrosine substantially reduced PDGF-induced PI-3 kinase activity but did not impair the receptor-mediated mitogenic response. We sought to determine whether PDGF-induced PI-3 kinase activity could be further ablated so as to exclude a low threshold requirement for PDGFR signal transduction. Thus, we mutated both tyrosine 731 and 742 and expressed the double mutant (Y731F/Y742F) in 32D hematopoietic cells. In such transfectants, PDGF induced no detectable receptor-associated or anti-P- Tyr recoverable PI-3 kinase activity. Under the same conditions, neither mobility shift of raf-1 nor tyrosine phosphorylation of either PLC gamma or MAP kinase was impaired. 32D transfectants expressing the double mutant showed wild-type alpha PDGFR levels of mitogenic and chemotactic responses to PDGF. To examine the effect of the double mutation in cells that normally respond to PDGF, we generated chimeras in which the cytoplasmic domains of wild-type alpha PDGFR, Y731F, and Y731F/Y742F were linked to the extracellular domain of colony- stimulating factor-1 (CSF-1) receptor (fms). After introduction of the chimeric receptors into mouse NIH/3T3 fibroblasts, the ability of CSF-1 to stimulate growth of these transfectants was examined. Our data show that all these chimeric receptors exhibited similar abilities to mediate CSF-1-stimulated cell growth. These findings lead us to conclude that PDGF-induced PI-3 kinase activity is not required for PDGF-stimulated mitogenic pathway in both NIH/3T3 fibroblasts and 32D hematopoietic cells.     

TAT-CC fusion protein depresses the oncogenicity of BCR-ABL in vitro and in vivo through interrupting its oligomerization

Chronic myeloid leukemia (CML) is a clonal hematologic malignancy characterized by the BCR-ABL protein. BCR-ABL is a constitutively active tyrosine kinase and plays a critical role in the pathogenesis of CML. Imatinib mesylate, a selective tyrosine kinase inhibitor, is effective in CML, but drug resistance and relapse occur. The coiled-coil (CC) domain located in BCR_1–72 mediates BCR-ABL tetramerization, which is essential for the activation of tyrosine kinase and transformation potential of BCR-ABL. CC domain is supposed to be a therapeutic target for CML. We purified a TAT-CC protein competively binding with the endogenous CC domain to reduce BCR-ABL kinase activity. We found that TAT-CC co-located and interacted with BCR-ABL in Ba/F3-p210 and K562 cells. It induced apoptosis and inhibited proliferation in these cells. It increased the sensitivity of these cells to imatinib and reduced the phosphorylation of BCR-ABL, CRKL and STAT5. We confirmed that TAT-CC could attenuate the oncogenicity of Ba/F3-p210 cells and diminish the volume of K562 solid tumor in mice. We conclude targeting the CC may provide a complementary therapy to inhibit BCR-ABL oncogenicity.     

Transformation and pp60v-src autophosphorylation correlate with SHC-GRB2 complex formation in rat and chicken cells expressing host-range and kinase-active, transformation-defective alleles of v-src.

The biochemical properties of several pp60v-src substrates believed to participate in src-mediated transformation were examined in cells expressing a kinase-active, transformation-defective v-src allele (v-src-F172 delta/Y416F) and its parental allele, v-src-F172 delta, a host-range--dependent allele that transforms chicken cells to a fusiform morphology, but does not transform rat cells. Because pp60v-src-F172 delta is dependent on autophosphorylation for transforming ability, these alleles provide a unique opportunity to examine the role of pp60v-src autophosphorylation in regulating substrate interactions. Increased pp125FAK tyrosine phosphorylation and high levels of pp60v-src-associated phosphotidylinositol-3' kinase activity were detected specifically in chicken cells exhibiting round, refractile transformation but not in cells transformed to a fusiform morphology. Increased pp125FAK kinase activity, but not increased pp125FAK tyrosine-phosphorylation correlated with pp60v-src autophosphorylation and increased anchorage-independent growth. Thus, pp125FAK and PI3'K may participate in morphological transformation by v-src. Furthermore, association of phosphorylated SHC with the adapter GRB2 correlated with increased anchorage-independent growth (and autophosphorylation) in both rat and chicken cells independent of the morphological phenotype induced. Therefore, host-range dependence for transformation may be regulated through association of SHC with GRB2, thus implicating SHC as a crucial substrate for src-dependent transformation.     

Distinct tyrosine autophosphorylation sites negatively and positively modulate neu-mediated transformation.

A number of cytoplasmic signaling molecules are thought to mediate mitogenic signaling from the activated Neu receptor tyrosine kinase through binding specific phosphotyrosine residues located within the intracellular portion of Neu/c-ErbB-2. An activated neu oncogene containing tyrosine-to-phenylalanine substitutions at each of the known autophosphorylation sites was generated and assessed for its specific transforming potential in Rat1 and NIH 3T3 fibroblasts. Mutation of these sites resulted in a dramatic impairment of the transforming potential of neu. To assess the role of these tyrosine phosphorylation sites in cellular transformation, the transforming potential of a series of mutants in which individual tyrosine residues were restored to this transformation-debilitated neu mutant was evaluated. Reversion of any one of four mutated sites to tyrosine residues restored wild-type transforming activity. While each of these transforming mutants displayed Ras-dependent signaling, the transforming activity of two of these mutants was correlated with their ability to bind either the GRB2 or SHC adapter molecules that couple receptor tyrosine kinases to the Ras signaling pathway. By contrast, restoration of a tyrosine residue located at position 1028 completely suppressed the basal transforming activity of this mutated neu molecule or other transforming neu molecules which possessed single tyrosine residues. These data argue that the transforming potential of activated neu is mediated both by positive and negative regulatory tyrosine phosphorylation sites.     

Complementation of growth factor receptor-dependent mitogenic signaling by a truncated type I phosphatidylinositol 4-phosphate 5-kinase.

Substitution of phenylalanine for tyrosine at codon 809 (Y809F) of the human colony-stimulating factor 1 (CSF-1) receptor (CSF-1R) impairs ligand-stimulated tyrosine kinase activity, prevents induction of c-MYC and cyclin D1 genes, and blocks CSF-1-dependent progression through the G1 phase of the cell cycle. We devised an unbiased genetic screen to isolate genes that restore the ability of CSF-1 to stimulate growth in cells that express mutant CSF-1R (Y809F). This screen led us to identify a truncated form of the murine type Ibeta phosphatidylinositol 4-phosphate 5-kinase (mPIP5K-Ibeta). This truncated protein lacks residues 1 to 238 of mPIP5K-Ibeta and is catalytically inactive. When we transfected cells expressing CSF-1R (Y809F) with mPIP5K-Ibeta (delta1-238), CSF-1-dependent induction of c-MYC and cyclin D1 was restored and ligand-dependent cell proliferation was sustained. CSF-1 normally triggers the rapid disappearance of CSF-1R (Y809F) from the cell surface; however, transfection of cells with mPIP5K-Ibeta (delta1-238) stabilized CSF-1R (Y809F) expression on the cell surface, resulting in elevated levels of ligand-activated CSF-1R (Y809F). These results suggest a role for PIP5K-Ibeta in receptor endocytosis and that the truncated enzyme compensated for a mitogenically defective CSF-1R by interfering with this process.     

A novel apoptotic pathway induced by nerve growth factor-mediated TrkA activation in medulloblastoma

Nerve growth factor (NGF) induces apoptosis in a human medulloblastoma cell line (MED283) engineered to express TrkA (MED283-TrkA) (Muragaki, Y., Chou, T. T., Kaplan, D. R., Trojanowski, J. Q., and Lee, V. M. (1997) J. Neurosci. 17, 530-542). To dissect the molecular signaling pathway that mediates this novel effect, specific receptor mutations in Trk have been employed. We showed that phosphorylation of tyrosine 490 is required for activation of phosphoinositide 3-OH kinase, whereas phosphorylation of tyrosine 785 is required for activation of phospholipase C-gamma. TrkA-mediated apoptosis was abolished when either the ATP-binding site or both tyrosines 490 and 785 were mutated. Because tyrosines 490 and 785 mediate redundant signaling through the Ras-extracellular signal-regulated kinase (Ras-ERK) pathway, we examined the role of Ras-ERK signaling in NGF-induced apoptosis. We found that MED283-TrkA cells expressing a dominant negative Ras inhibitor (N17Ras) failed to undergo ERK activation and apoptosis following NGF treatment, whereas the ERK kinase (mitogen-activated protein kinase kinase) inhibitors PD98059 and U0126 eliminated ERK activation but had no effect on apoptosis. We infer from these data that NGF-induced apoptosis is mediated by a novel Ras and/or Raf signaling pathway.     

Cyclin D1 is required for transformation by activated Neu and is induced through an E2F-dependent signaling pathway

The neu (c-erbB-2) proto-oncogene encodes a tyrosine kinase receptor that is overexpressed in 20 to 30% of human breast tumors. Herein, cyclin D1 protein levels were increased in mammary tumors induced by overexpression of wild-type Neu or activating mutants of Neu in transgenic mice and in MCF7 cells overexpressing transforming Neu. Analyses of 12 Neu mutants in MCF7 cells indicated important roles for specific C-terminal autophosphorylation sites and the extracellular domain in cyclin D1 promoter activation. Induction of cyclin D1 by NeuT involved Ras, Rac, Rho, extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38, but not phosphatidylinositol 3-kinase. NeuT induction of the cyclin D1 promoter required the E2F and Sp1 DNA binding sites and was inhibited by dominant negative E2F-1 or DP-1. Neu-induced transformation was inhibited by a cyclin D1 antisense or dominant negative E2F-1 construct in Rat-1 cells. Growth of NeuT-transformed mammary adenocarcinoma cells in nude mice was blocked by the cyclin D1 antisense construct. These results demonstrate that E2F-1 mediates a Neu-signaling cascade to cyclin D1 and identify cyclin D1 as a critical downstream target of neu-induced transformation.     

ALK-activating homologous mutations in LTK induce cellular transformation

Leukocyte tyrosine kinase (LTK) is a receptor tyrosine kinase reported to be overexpressed in human leukemia. Though much regarding the function of LTK remains unknown, it shares a high degree of similarity with anaplastic lymphoma kinase (ALK), which is found mutated in human cancer. In order to determine if LTK has transforming potential, we created two LTK mutants, F568L and R669Q, that correspond to two well-characterized activating mutations of ALK (F1174L and R1275Q). LTK-F568L, but not wildtype LTK or LTK-R669Q, transformed hematopoietic cells to cytokine independence. LTK-F568L exhibited a stronger ability to induce loss of contact inhibition and anchorage-independent growth of epithelial cells compared to LTK-R669Q, while wildtype LTK was non-transforming in the same cells. Likewise, LTK-F568L induced greater neurite outgrowth of PC12 cells than R669Q, while wildtype LTK could not. Correlating with transforming activity, LTK-F568L displayed significantly enhanced tyrosine phosphorylation compared to wildtype LTK and LTK-R668Q and induced activation of various signaling proteins including Shc, ERK and the JAK/STAT pathway. Expression of wildtype LTK or LTK-R669Q generally led to weaker activation of signaling proteins than expression of LTK-F568L, or no activation at all. Thus, mutating LTK at residue F568, and to a lesser extent at R669, activates the receptor tyrosine kinase, inducing cell signaling that results in transforming properties. These studies suggest that aberrant activation of LTK may contribute to neoplastic cell growth.     

A single autophosphorylation site confers oncogenicity to the Neu/ErbB-2 receptor and enables coupling to the MAP kinase pathway.

The transforming potential of the Neu/ErbB-2 receptor tyrosine kinase undergoes inactivation by deletion of the non-catalytic C-terminal tail, which contains five autophosphorylation sites. To determine which site is essential for oncogenicity, we tailed the C-terminally-deleted mutant with individual autophosphorylation sites. Complete restoration of the transforming action in vitro and in vivo was conferred by a stretch of 12 amino acids that contained the most C-terminal tyrosine autophosphorylation site (Y1253). Reconstitution of transformation was specific to this amino acid sequence because none of the other autophosphorylation sites, when grafted individually, caused transformation, and replacement of the tyrosine with a phenylalanine residue significantly reduced the oncogenic potential of both the full-length and the tailed proteins. When present alone the most C-terminal sequence enabled coupling to a biochemical pathway that includes Ras, MAP kinase and transactivation of Jun. These results indicate that the multiplicity of autophosphorylation sites on a receptor tyrosine kinase is not essential for transformability, and implicate the MAP kinase pathway in transduction of the oncogenic signal of Neu/ErbB-2.     

Identification and characterization of a nuclear interacting partner of anaplastic lymphoma kinase (NIPA)

Anaplastic large-cell lymphoma is a subtype of non-Hodgkin lymphomas characterized by the expression of CD30. More than half of these lymphomas carry a chromosomal translocation t(2;5) leading to expression of the oncogenic tyrosine kinase nucleophosmin-anaplastic lymphoma kinase (NPM-ALK). NPM-ALK is capable of transforming fibroblasts and lymphocytes in vitro and of causing lymphomas in mice. Previously, we and others demonstrated phospholipase C-gamma and phosphatidylinositol 3-kinase as crucial downstream signaling mediators of NPM-ALK-induced oncogenicity. In this study, we used an ALK fusion protein as bait in a yeast two-hybrid screen identifying NIPA (nuclear interacting partner of ALK) as a novel downstream target of NPM-ALK. NIPA encodes a 60-kDa protein that is expressed in a broad range of human tissues and contains a classical nuclear translocation signal in its C terminus, which directs its nuclear localization. NIPA interacts with NPM-ALK and other ALK fusions in a tyrosine kinase-dependent manner and is phosphorylated in NPM-ALK-expressing cells on tyrosine and serine residues with serine 354 as a major phosphorylation site. Overexpression of NIPA in Ba/F3 cells was able to protect from apoptosis induced by IL-3 withdrawal. Mutations of the nuclear translocation signal or the Ser-354 phosphorylation site impaired the antiapoptotic function of NIPA. In NPM-ALK-transformed Ba/F3 cells, apoptosis triggered by wortmannin treatment was enhanced by overexpression of putative dominant-negative NIPA mutants. These results implicate an antiapoptotic role for NIPA in NPM-ALK-mediated signaling events.     

A juxtamembrane tyrosine in the colony stimulating factor-1 receptor regulates ligand-induced Src association, receptor kinase function, and down-regulation

Recent literature implicates a regulatory function of the juxtamembrane domain (JMD) in receptor tyrosine kinases. Mutations in the JMD of c-Kit and Flt3 are associated with gastrointestinal stromal tumors and acute myeloid leukemias, respectively. Additionally, autophosphorylated Tyr559 in the JMD of the colony stimulating factor-1 (CSF-1) receptor (CSF-1R) binds to Src family kinases (SFKs). To investigate SFK function in CSF-1 signaling we established stable 32D myeloid cell lines expressing CSF-1Rs with mutated SFK binding sites (Tyr559-TFI). Whereas binding to I562S was not significantly perturbed, Y559F and Y559D exhibited markedly decreased CSF-1-dependent SFK association. All JMD mutants retained intrinsic kinase activity, but Y559F, and less so Y559D, showed dramatically reduced CSF-1-induced autophosphorylation. CSF-1-mediated wild-type (WT)-CSF-1R phosphorylation was not markedly affected by SFK inhibition, indicating that lack of SFK binding is not responsible for diminished Y559F phosphorylation. Unexpectedly, cells expressing Y559F were hyperproliferative in response to CSF-1. Hyperproliferation correlated with prolonged activation of Akt, ERK, and Stat5 in the Y559F mutant. Consistent with a defect in receptor negative regulation, c-Cbl tyrosine phosphorylation and CSF-1R/c-Cbl co-association were almost undetectable in the Y559F mutant. Furthermore, Y559F underwent reduced multiubiquitination and delayed receptor internalization and degradation. In conclusion, we propose that Tyr559 is a switch residue that functions in kinase regulation, signal transduction and, indirectly, receptor down-regulation. These findings may have implications for the oncogenic conversion of c-Kit and Flt3 with JMD mutations.     

Src-family tyrosine kinase fyn phosphorylates phosphatidylinositol 3-kinase enhancer-activating Akt, preventing its apoptotic cleavage and promoting cell survival

Phosphatidylinositol 3-kinase enhancer-activating Akt (PIKE-A) binds Akt and upregulates its kinase activity, preventing apoptosis. PIKE-A can be potently phosphorylated on tyrosine residues 682 and 774, leading to its resistance to caspase cleavage. However, the upstream tyrosine kinases responsible for PIKE-A phosphorylation and subsequent physiological significance remain unknown. Here, we show that PIKE-A can be cleaved by the active apoptosome at both D474 and D592 residues. Employing fyn-deficient mouse embryonic fibroblast cells and tissues, we demonstrate that fyn is essential for phosphorylating PIKE-A and protects it from apoptotic cleavage. Active but not kinase-dead fyn interacts with PIKE-A and phosphorylates it on both Y682 and Y774 residues. Tyrosine phosphorylation in PIKE-A is required for its association with active fyn but not for Akt. Mutation of D into A in PIKE-A protects it from caspase cleavage and promotes cell survival. Thus, this finding provides a molecular mechanism accounting for the antiapoptotic action of src-family tyrosine kinase.