Isoform-specific insertion near the Grb2-binding domain modulates the intrinsic guanine nucleotide exchange activity of hSos1

Two human hSos1 isoforms (Isf I and Isf II; Rojas et al., Oncogene 12, 2291-2300, 1996) defined by the presence of a distinct 15 amino acid stretch in one of them, were compared biologically and biochemically using representative NIH3T3 transfectants overexpressing either one. We showed that hSos1-Isf II is significantly more effective than hSos1-Isf I to induce proliferation or malignant transformation of rodent fibroblasts when transfected alone or in conjunction with normal H-Ras (Gly12). The hSos1-Isf II-Ras cotransfectants consistently exhibited higher saturation density, lower cell-doubling times, increased focus-forming activity and higher ability to grow on semisolid medium and at low serum concentration than their hSos1-Isf I-Ras counterparts. Furthermore, the ratio of GTP/GDP bound to cellular p21ras was consistently higher in the hSos1-Isf II-transfected clones, both under basal and stimulated conditions. However, no significant differences were detected in vivo between Isf I- and Isf II-transfected clones regarding the amount, stability and subcellular localization of Sos1-Grb2 complex, or the level of hSos1 phosphorylation upon cellular stimulation. Interestingly, direct Ras guanine nucleotide exchange activity assays in cellular lysates showed that Isf II transfectants consistently exhibited about threefold higher activity than Isf I transfectants under basal, unstimulated conditions. Microinjection into Xenopus oocytes of purified peptides corresponding to the C-terminal region of both isoforms (encompassing the 15 amino acid insertion area and the first Grb2-binding motif) showed that only the Isf II peptide, but not its corresponding Isf I peptide, was able to induce measurable rates of meiotic maturation, and synergyzed with insulin, but not progesterone, in induction of GVBD. Our results suggest that the increased biological potency displayed by hSos1-Isf II is due to higher intrinsic guanine nucleotide exchange activity conferred upon this isoform by the 15 a.a. insertion located in proximity to its Grb2 binding region.     

The C-terminal SH3 domain of the adapter protein Grb2 binds with high affinity to sequences in Gab1 and SLP-76 which lack the SH3-typical P-x-x-P core motif

The adapter Grb2 is an important mediator of normal cell proliferation and oncogenic signal transduction events. It consists of a central SH2 domain flanked by two SH3 domains. While the binding specificities of the Grb2 SH2 and N-terminal SH3 domain [Grb2 SH3(N)] have been studied in detail, binding properties of the Grb2 SH3(C) domain remained poorly defined. Gab1, a receptor tyrosine kinase substrate which associates with Grb2 and the c-Met receptor, was previously shown to bind Grb2 via a region which lacks a Grb2 SH3(N)-typical motif (P-x-x-P-x-R). Precipitation experiments with the domains of Grb2 show now that Gab1 can bind stably to the Grb2 SH3(C) domain. For further analyses, Gab1 mutants were generated by PCR to test in vivo residues thought to be crucial for Grb2 SH3(C) binding. The Grb2 SH3(C) binding region of Gab1 has significant homology to a region of the adapter protein SLP-76. Peptides corresponding to epitopes SLP-76, Gab1, SoS and other proteins with related sequences, as well as mutant peptides were synthesized and analysed by tryptophan-fluorescence spectrometry and by in vitro competition experiments. These experiments define a 13 amino acid sequence with the unusual consensus motif P-x-x-x-R-x-x-K-P as required for a stable binding to the SH3(C) domain of Grb2. Additional analyses point to a distinct binding specificity of the Grb2-homologous adapter protein Mona (Gads), indicating that the proteins of the Grb2 adapter family may have partially overlapping, yet distinct protein binding properties.     

GRB2-mediated recruitment of GAB2, but not GAB1, to SF-STK supports the expansion of Friend virus-infected erythroid progenitor cells

Friend virus induces the development of erythroleukemia in mice through the interaction of a viral glycoprotein, gp55, with a truncated form of the Stk receptor tyrosine kinase, short form-Stk (Sf-Stk), and the EpoR. We have shown previously that the ability of Sf-Stk to participate in the transformation of Friend virus-infected cells requires the kinase activity and Grb2-binding site of Sf-Stk. Here we show that Grb2 heterozygous mice exhibit decreased susceptibility to Friend erythroleukemia and that expansion of erythroid progenitors in response to infection requires the C-terminal SH3 domain of Grb2. A fusion protein in which the Grb2-binding site in Sf-Stk is replaced by Gab2, supports the growth of progenitors from mice lacking Sf-Stk, whereas a Sf-Stk/Gab1 fusion protein does not. Gab2 is expressed in spleens from Friend virus-infected mice, co-immunoprecipitates with Sf-Stk and is tyrosine phosphorylated in the presence of Sf-Stk. Mice with a targeted deletion in Gab2 are less susceptible to Friend erythroleukemia and the expansion of erythroid progenitor cells in response to infection can be rescued by expression of Gab2, but not Gab1. Taken together, these data indicate that a Sf-Stk/Grb2/Gab2 complex mediates the growth of primary erythroid progenitor cells in response to Friend virus.     

Domain-specific function of ShcC docking protein in neuroblastoma cells

ShcC is a family member of the Shc docking proteins that possess two different phosphotyrosine-binding motifs and conduct signals as Grb2-binding substrates of various receptor tyrosine kinases. We have recently shown that some neuroblastoma cell lines, such as NB-39-nu cells, express a protein complex of hyperphosphorylated ShcC and anaplastic lymphoma kinase (ALK), which is self-activated by gene amplification. Here, we demonstrate that the expression of a mutant ShcC lacking Grb2-binding sites, 3YF-ShcC, significantly impaired the survival, differentiation and motility of NB-39-nu cells by blocking the ERK and Akt pathways. On the other hand, cells overexpressing ShcC or 3YF-ShcC, but not a mutant ShcC that lacks SH2, showed decreased anchorage independency and in vivo tumorigenicity, suggesting a novel ShcC-specific suppressive effect through its SH2 domain on cell transformation. Notably, overexpression of ShcC suppressed the sustained phosphorylation of Src family kinase after cell detachment, which might be independent of phosphorylation of Grb2-binding site. It was indicated that the Src/Fyn-Cas pathway is modulated as a target of these suppressive effects by ShcC. Reciprocal change of ShcC expression and phosphorylation observed in malignant neuroblastoma cell lines might be explained by these phosphotyrosine-dependent and -independent functions of ShcC.     

Regulation of growth factor signaling by FRS2 family docking/scaffold adaptor proteins

The FRS2 family of adaptor/scaffold proteins has two members, FRS2α and FRS2β. Both proteins contain N-terminal myristylation sites for localization on the plasma membrane and a PTB domain for binding to limited species of receptor tyrosine kinases (RTKs), including the FGF receptor, the neurotophin receptor, RET, and ALK. Activation of these RTKs allows FRS2 proteins to become phosphorylated of tyrosine residues and then bind to Grb2 and Shp2, a SH2 domain-containing adaptor and a tyrosine phosphatase, respectively. Subsequently, Shp2 activates a Ras/ERK pathway and Grb2 activates a Ras/ERK, phosphatidyl inositol (PI)-3 kinase and ubiquitination/degradation pathways by binding to SOS, Gab1, and Cbl via the SH3 domains of Grb2. FRS2α acts as 'a conning center' in FGF signaling mainly because it induces sustained levels of activation of ERK via Shp2-binding sites and Grb2-binding sites, though the contribution of the former is greater. Indeed, FRS2α knockout mice and mice with mutated Shp2-binding sites exhibit a variety of phenotypes due to defects in FGF signaling in vivo . Although FRS2β binds to the EGF receptor, it does not induce tyrosine phosphorylation on the receptor. Instead, it inhibits EGF signaling, resulting in inhibition of EGF-induced cell proliferation and cell transformation. Based on these findings, the involvement of FRS2 proteins in tumorigenesis should be studied extensively to be validated as candidate biomarkers for the effectiveness of treatments targeting RTKs such as the FGF receptor and EGF receptor. ( Cancer Sci 2008; 99: 1319–1325)     

Evidence for SH3 domain directed binding and phosphorylation of Sam68 by Src.

Sam68 is a 68 kDa protein that associates with and is phosphorylated by the c-Src kinase at mitosis. It contains a KH domain implicated in RNA binding and several proline-rich motifs that resemble known SH3 binding sites. The SH3 domains of c-Src, phosphatidylinositol 3-OH kinase, phospholipase C-gamma and Grb2 protein (containing two SH3 domains), but not other SH3 domains tested, were capable of binding Sam68 in vitro. Synthetic peptides corresponding to the proline motifs of Sam68 inhibited with different efficiencies the binding of SH3 domains to Sam68 suggesting that the proline motifs of Sam68 function as specific SH3 domain binding sites. Mutation of Sam68 SH3 binding sites further indicated that the SRC SH3 domain mediates binding of Src to unphosphorylated Sam68. Phosphorylation of Sam68 by Src kinase was inhibited when the Src SH3 binding site of Sam68 was mutated or when corresponding peptides were added to in vitro kinase reactions indicating that binding of the Src SH3 domain to a specific site near the amino-terminus of Sam68 (including residues 38 - 45: PPLPHRSR) facilitates phosphorylation of Sam68 by the Src kinase domain. Sam68-based proline peptides had no effect on the phosphorylation of another in vitro substrate of Src, enolase. These results suggest that Src effectively mounts Sam68 through its SH3 domain, possibly as a mechanism to position the kinase domain close to substrate tyrosine residues in the carboxyl-half of the protein.     

Activation of the focal adhesion kinase signaling pathway by structural alterations in the carboxyl-terminal region of c-Crk II

The Crk II adaptor protein encodes an SH2/SH3-domain containing adaptor protein with an SH2-SH3-SH3 domain structure that transmits signals from tyrosine kinases. The two SH3 domains are separated by a 54 amino acid linker region, whose length is highly conserved in xenopus, chicken, and mamalian Crk II proteins. To gain a better understanding into the role of the C-terminal region of Crk, we generated a series of C-terminal SH3 domain and SH3 linker mutants and examined their role in tyrosine kinase pathways. Expression of point mutations in the C-terminal SH3 domain (W276K Crk), at the tyrosine phosphorylation site (Y222F Crk II), or truncation of the entire C-terminus (Crk I or Crk Delta242), all increased c-Abl binding to the N-terminal SH3 domain of Crk and, where relevant, increased Tyr(222) phosphorylation. Deletion analysis of c-Crk II also revealed the presence of a C-terminal segment important for trans-activation of FAK. Such mutants, Crk Delta255 or Crk Delta242 Extended Linker (Crk Delta242([EL])), characterized by a disruption in the SH3 linker/C-terminal SH3 boundary, induced robust hyperphosphorylation of focal adhesion kinase (FAK) on Tyr(397), hyperphosphorylation of focal adhesion proteins p130(cas) and paxillin and increased focal adhesion formation in NIH3T3 cells. The effects of Crk Delta242([EL]) could be abrogated by co-expression of dominant negative c-Src or the protein tyrosine phosphatase PTP-PEST, but not by dominant negative Abl. Our results suggest that the C-terminal region of Crk contains negative regulatory elements important for both Abl and FAK dependent signal pathways, and offers a paradigm for an autoinhibitory region in the SH3 linker/C-terminal SH3 domain.     

Meltrin alpha cytoplasmic domain interacts with SH3 domains of Src and Grb2 and is phosphorylated by v-Src

Meltrin alpha/ADAM12 is a member of the ADAM/MDC family proteins characterized by the presence of metalloprotease and disintegrin domains. This protein also contains a single transmembrane domain and a relatively long cytoplasmic domain containing several proline-rich sequences. These sequences are compatible with the consensus sequences for binding the Src homology 3 (SH3) domains. To determine whether the proline-rich sequences interact with SH3 domains in several proteins, binding of recombinant SH3 domains to the meltrin alpha cytoplasmic domain was analysed by pull-down assays. The SH3 domains of Src and Yes bound strongly, but that of Abl or phosphatidylinositol 3-kinase p85 subunit did not. Full-length Grb2/Ash bound strongly, whereas its N-terminal SH3 domain alone did less strongly. Src and Grb2 in bovine brain extracts also bound to meltrin alpha cytoplasmic domain on affinity resin. Furthermore, immunoprecipitation with a monoclonal antibody to meltrin alpha resulted in coprecipitation of Src and Grb2 with meltrin alpha in cell extracts, suggesting that Src and Grb2 are associated in vivo with meltrin alpha cytoplasmic domain. This notion was also supported by the findings that exogenously expressed meltrin cytoplasmic domain coexisted with Src and Grb2 on the membrane ruffles. The C-terminal Tyr901 of meltrin alpha was phosphorylated both in vitro and in cultured cells by v-Src. These results may imply that meltrin alpha cytoplasmic domain is involved in a signal transduction for some biological function through the interaction with SH3-containing proteins.     

Multiple Grb2-protein complexes in human cancer cells

Grb2 is an SH2/SH3 domain-containing adaptor protein that links receptor tyrosine kinases to the ras signaling pathway. The Grb2-SH2 domain binds phosphotyrosine sequences on activated tyrosine kinases, and one target of the SH3 domains is the ras-nucleotide-exchange factor Sos1. We have examined Grb2-protein interactions in human cancer cells that over-express the receptor tyrosine kinase erbB2. Our results show that the 2 Grb2-SH3 domains complex with Sos1, dynamin and at least 4 other proteins (p228, p140, p55, p28) in these cells. The 2 Grb2-SH3 domains bind these proteins differently, with the N-terminal SH3 domain interacting preferentially with p228, Sos1, p140 and dynamin. The C-terminal SH3 domain has higher affinity toward p28. The Grb2-SH3 domain interactions appear to be similar in erbB2 over-expressing breast, ovarian and lung cancer cells. Also, the major tyrosine-phosphorylated proteins that associate with Grb2 in erbB2 over-expressing cancer cells appear to be erbB2 and Shc. The multiple Grb2-SH3 domain interactions in these cells may mediate novel cellular functions. Int. J. Cancer, 70:208–213, 1977. © 1997 Wiley-Liss Inc.     

Gab1 but not Grb2 mediates tumor progression in Met overexpressing colorectal cancer cells

Hepatocyte growth factor receptor (Met) plays an important role in the progression of multiple cancer types. The overexpression of Met in DLD-1 colon carcinoma cells with kirsten rat sarcoma oncogene homolog (KRAS) oncogene activation resulted in enhanced subcutaneous and orthotopic tumor growth rate and increased metastatic potential. To elucidate the mechanism of this effect, we stably expressed kinase-inactive Met(K1110A), Src homology 2 (SH2)-binding domain-inactive Met(Y1349/1356F), growth factor receptor-bound protein 2 (Grb2) non-binding Met(N1358H) and mutant receptors with ability to selectively recruit signaling proteins Grb2, src homology domain c-terminal adaptor homolog (Shc), phospholipase c-gamma (PLCgamma) and p85 phosphatidyl inositol 3 kinase. As subcutaneous implants, DLD-1 cells that expressed the majority of these receptor constructs failed to recapitulate the tumor growth-enhancing effect of the wild-type Met receptor. The Grb2- and Shc-recruiting Met mutants demonstrated slight but consistent tumor-suppressive activity, whereas the expression of N1358H mutant stimulated tumor growth rate comparable with the wild-type receptor. This suggests that direct Grb2/Shc binding does not contribute to the tumor progression activity of Met receptor. The tumors expressing Grb2- and Shc-recruiting Met receptors demonstrated a marked loss in Grb2-associated adaptor protein 1 (Gab1) protein levels, which was not observed in the cell lines, consistent with a post-translationally regulated process. Moreover, a moderate level of Gab1 overexpression stimulated tumor growth. The findings suggest a delicate balance for intact Y1349/1356 SH2-binding domain to mediate the tumor progression activity of the coactivated Met-rat sarcoma oncogene homolog (RAS) pathways. Selectivity for specific adaptor protein involvement may be the key that determines the tissue- and cell-type specificity of Met-mediated tumorigenicity in human cancers.     

The elucidation of novel SH2 binding sites on PLD2

Our laboratory has recently reported that the enzyme phospholipase D2 (PLD2) exists as a ternary complex with PTP1b and the growth factor receptor bound protein 2 (Grb2). Here, we establish the mechanistic underpinnings of the PLD2/Grb2 association. We have identified residues Y(169) and Y(179) in the PLD2 protein as being essential for the Grb2 interaction. We present evidence indicating that Y(169) and Y(179) are located within two consensus sites in PLD2 that mediate an SH2 interaction with Grb2. This was demonstrated with an SH2-deficient GSTGrb2 R86K mutant that failed to pull-down PLD2 in vitro. In order to elucidate the functions of the two neighboring tyrosines, we created a new class of deletion and point mutants in PLD2. Phenylalanine replacement of Y(169) (PLD2 Y169F) or Y(179) (PLD2 Y179F) reduced Grb2 binding while simultaneous mutation completely abolished it. The role of the two binding sites on PLD2 was found to be functionally nonequivalent: Y(169) serves to modulate the activity of the enzyme, whereas Y(179) regulates total tyrosine phosphorylation of the protein. Interestingly, binding of Grb2 to PLD2 occurs irrespectively of lipase activity, since Grb2 binds to catalytically inactive PLD2 mutants. Finally, PLD2 residues Y(169) and Y(179) are necessary for the recruitment of Sos, but only overexpression of the PLD2 Y179F mutant resulted in increased Ras activity, p44/42(Erk) phosphorylation and enhanced DNA synthesis. Since Y(169) remains able to modulate enzyme activity and is capable of binding to Grb2 in the PLD2 Y179F mutant, we propose that Y(169) is kept under negative regulation by Y(179). When this is released, Y(169) mediates cellular proliferation through the Ras/MAPK pathway.     

Key role of Shc signaling in the transforming pathway triggered by Ret/ptc2 oncoprotein.

The RET/PTC oncogenes, generated by chromosomal rearrangements in papillary thyroid carcinomas, are constitutively activated versions of protoRET, a gene encoding two protein isoforms of a transmembrane tyrosine kinase receptor. By using Ret/ptc2 short isoform (iso9), we have previously demonstrated that Tyr586 (Tyr1062 of protoRet) is the docking site for both the PTB and the SH2 domains of Shc. To determine the relevance of this interaction for the transforming activity of Ret/ptc oncogenes, we have generated and characterized novel Ret/ptc mutants unable to activate Shc: Ret/ptc2 long isoform (iso51)-Y586F and both isoforms of Ret/ptc2-N583A. These mutants neither activate Shc nor transform NIH3T3 cells. Since Tyr1062 shows features of a multifunctional docking site, we have used a Shc mutant (Shc Y317F) to directly assess Shc role. We have demonstrated that in our cell system Shc Y317F behaves like a dominant interfering mutant on the activation of the Grb2-Sos pathway by endogenous Shc triggered by Ret/ptc2. A strong reduction of the transforming activity of Ret/ptc2 in presence of this mutant was also demonstrated. Our data suggest that Shc activation play a key role in the transforming pathways triggered by Ret/ptc oncoproteins. Moreover, we have shown that coexpression of the Shc-Y317F mutant with Ret/ptc2 specifically causes apoptosis, and that the surviving cells lose the long-term expression of one of the two genes.     

Gab-family adapter molecules in signal transduction of cytokine and growth factor receptors, and T and B cell antigen receptors

Gab1 and Gab2 (Grb2 associated binder 1 and 2) are scaffolding adapter molecules that display sequence similarity with Drosophila DOS (daughter of sevenless), which is a potential substrate for the protein tyrosine phosphatase, Corkscrew, Both Gab1 and Gab2, like DOS, have a pleckstrin homology domain and potential binding sites for SH2 and SH3 domains. Gab1 and Gab2 are phosphorylated on tyrosine upon the stimulation of various cytokines, growth factors, and antigen receptors, and interact with signaling molecules, such as Grb2, SHP-2, and PI-3 kinase. Overexpression of Gab1 or Gab2 mimics or enhances growth factor or cytokine-mediated biological processes and activates ERK MAP kinase. These data imply that Gab1 and Gab2 act downstream of a broad range of cytokine and growth factor receptors, as well as T and B antigen receptors, and link these receptors to ERK MAP kinase and biological actions.     

Concomitant activation of pathways downstream of Grb2 and PI 3-kinase is required for MET-mediated metastasis

The Met tyrosine kinase - the HGF receptor - induces cell transformation and metastasis when constitutively activated. Met signaling is mediated by phosphorylation of two carboxy-terminal tyrosines which act as docking sites for a number of SH2-containing molecules. These include Grb2 and p85 which couple the receptor, respectively, with Ras and PI 3-kinase. We previously showed that a Met mutant designed to obtain preferential coupling with Grb2 (Met2xGrb2) is permissive for motility, increases transformation, but - surprisingly - is impaired in causing invasion and metastasis. In this work we used Met mutants optimized for binding either p85 alone (Met2xPI3K) or p85 and Grb2 (MetPI3K/Grb2) to evaluate the relative importance of Ras and PI 3-kinase as downstream effectors of Met. Met2xPI3K was competent in eliciting motility, but not transformation, invasion, or metastasis. Conversely, MetP13K/Grb2 induced motility, transformation, invasion and metastasis as efficiently as wild type Met. Furthermore, the expression of constitutively active PI 3-kinase in cells transformed by the Met2xGrb2 mutant, fully rescued their ability to invade and metastasize. These data point to a central role for PI 3-kinase in Met-mediated invasiveness, and indicate that simultaneous activation of Ras and PI 3-kinase is required to unleash the Met metastatic potential.     

Growth of chronic myeloid leukemia cells is inhibited by infection with Ad-SH2-HA adenovirus that disrupts Grb2-Bcr-Abl complexes

The persistence of Bcr-Abl-positive cells in patients on imatinib therapy indicates that inhibition of the Bcr-Abl kinase activity alone might not be sufficient to eradicate the leukemia cells. Many downstream effectors of Bcr-Abl have been described, including activation of both the Grb2-SoS-Ras-MAPK and Grb2-Gab2-PI3K-Akt pathways. The Bcr-Abl-Grb2 interaction, which is mediated by the direct interaction of the Grb2 SH2 domain with the phospho-Bcr-Abl Y177, is required for activation of these signaling pathways. Therefore, disrupting their interaction represents a potential therapeutic strategy for inhibiting the oncogenic downstream signals of Bcr-Abl. Adenovirus Ad-SH2-HA expressing the Grb2 SH2 domain was constructed and applied in this study. As expected, Ad-SH2-HA efficiently infected CML cells and functioned by binding to the phospho-Bcr-Abl Y177 site, competitively disrupting the Grb2 SH2-phospho-Bcr-Abl Y177 complex. They induced potent anti-proliferation and apoptosis-inducing effects in CML cell lines. Moreover, the Ras, MAPK and Akt activities were significantly reduced in the Ad-SH2-HA treated cells. These were not observed with the point-mutated control adenovirus Ad-Sm-HA with abolished phospho-Bcr-Abl Y177 binding sites. These data indicate that, in addition to the direct targeting of Bcr-Abl, selective inhibition of its downstream signaling pathways may be a therapeutic option for CML, and the Ad-SH2-HA-mediated killing strategy could be explored as a promising anti-leukemia agent in CML.     

c-Abl stabilizes p73 by a phosphorylation-augmented interaction

The proapoptotic function of c-Abl is in part mediated by its functional interaction with p73, a p53 homologue. Although it has been shown that c-Abl-mediated p73 activation in response to genotoxic stress is associated with an increase of p73 protein levels, the underlying mechanism remains unclear. We show here that c-Abl increases the cellular p73 abundance through a mode of posttranslational regulation. Analogous to its functional activation of p73, the kinase activity is essential for c-Abl to up-regulate p73 protein levels. Analysis of phosphorylation-resistant mutants of p73 reveals that the effect of c-Abl is mediated by its direct phosphorylation on the p73 protein. Consequence to the phosphorylation is a marked increase of the association between c-Abl and p73 via the binding of tyrosine-phosphorylated p73 to the c-Abl Src homology 2 (SH2) domain. Of functional importance of this phosphorylation-induced interaction in p73 stabilization is the demonstration that expression of a c-Abl SH2 domain peptide, which impedes phosphorylation-dependent association, results in an almost complete abrogation of c-Abl-dependent p73 accumulation. Importantly, expression of the c-Abl SH2 domain peptide also leads to an efficient inhibition of cisplatin-induced accumulation of endogenous p73, highlighting the biological significance. In keeping with its retained phosphorylation sites, the NH(2)-terminal truncated (Delta N) isoforms of p73, which are antiapoptotic, are also phosphorylated and stabilized by c-Abl, suggesting a possibility that c-Abl contributes to either pro- or antiapoptotic process depending on the expression profile of p73 isoforms.     

The adaptor Grb7 is a novel calmodulin-binding protein: functional implications of the interaction of calmodulin with Grb7

We demonstrate using Ca2+-dependent calmodulin (CaM)-affinity chromatography and overlay with biotinylated CaM that the adaptor proteins growth factor receptor bound (Grb)7 and Grb7V (a naturally occurring variant lacking the Src homology 2 (SH2) domain) are CaM-binding proteins. Deletion of an amphiphilic basic amino-acid sequence (residues 243-256) predicted to form an alpha-helix located in the proximal region of its pleckstrin homology (PH) domain demonstrates the location of the CaM-binding domain. This site is identical in human and rodents Grb7, and shares great homology with similar regions of Grb10 and Grb14, and the Mig10 protein from Caenorhabditis elegans. We show that Grb7 and Grb7V are present in the cytosol and bound to membranes, while the deletion mutants (Grb7Delta and Grb7VDelta) have less capacity to be associated to membranes. Grb7Delta maintains in part the capacity to bind phosphoinositides, and CaM competes for phosphoinositide binding. Activation of ErbB2 by heregulin beta1 decreases the pool of Grb7 associated to membranes. The cell-permeable CaM antagonist W7 (N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide), but not the CaM-dependent protein kinase II inhibitor KN93, prevents this effect. Highly specific cell-permeable CaM inhibitory peptides decrease the association of Grb7 to membranes. This suggests that CaM regulates the intracellular mobilization of Grb7 in living cells. Direct interaction between enhanced yellow fluorescent protein (EYFP)-Grb7 and enhanced cyan fluorescent protein (ECFP)-CaM chimeras at the plasma membrane of living cells was demonstrated by fluorescence resonance energy transfer (FRET). The FRET signal dramatically decreased in cells loaded with a cell-permeable Ca2+ chelator, and was significantly attenuated when enhanced yellow fluorescent protein-Grb7 chimera (EYFP-Grb7)Delta instead of EYFP-Grb7 was used. Finally, we show that conditioned media from cells transiently transfected with Grb7Delta and Grb7VDelta lost its angiogenic activity, in contrast to those from cells transiently transfected with their wild-type counterparts.     

C/EBPalphap30, a myeloid leukemia oncoprotein, limits G-CSF receptor expression but not terminal granulopoiesis via site-selective inhibition of C/EBP DNA binding

Heterozygous mutations of the CEBPA gene are present in 5% of acute myeloid leukemia (AML) cases and often lead to the expression of an N-terminally truncated, 30 kDa isoform, C/EBPalphap30, from an internal translation start site. We have assessed the effect of C/EBPalphap30 on granulopoiesis utilizing C/EBPalphap30-ER, containing the estradiol receptor ligand-binding domain. In contrast to C/EBPalpha-ER, C/EBPalphap30-ER did not induce 32Dcl3 myeloid cell differentiation in IL-3. However, both isoforms, when expressed at high levels, were capable of inhibiting E2F activity in 32Dcl3 cells and of slowing their G1 to S progression. C/EBPalphap30 repressed expression of the endogenous G-CSF receptor several-fold. To facilitate investigation of the effect of C/EBPalphap30-ER on granulopoiesis downstream of G-CSF signalling, we coexpressed exogenous G-CSF receptor. C/EBPalphap30-ER/GR cells expressed several granulocytic markers in G-CSF and demonstrated nuclear maturation. Rat C/EBPalpha-ER and C/EBPalphap30-ER, expressed in 293T cells, bound the C/EBP site from the NE gene with similar affinity, as did human C/EBPalpha and C/EBPalphap30. In contrast, C/EBPalphap30 bound the C/EBP sites in the PU.1 or GR gene with 3-6-fold reduced affinity. Thus, the selective inhibition of GR expression by C/EBPalphap30-ER is due in part to its variable affinity for C/EBP sites. Variation in affinity for selected cis elements among isoforms may affect the biology of basic region-leucine zipper (bZIP) proteins.     

The interaction of the Bcr-Abl tyrosine kinase with the Src kinase Hck is mediated by multiple binding domains.

Bcr-Abl is found in more than 95% of cases with CML. The mechanism of Bcr-Abl-induced transformation is not fully understood. Bcr-Abl is a constitutively active tyrosine kinase with transforming capacity for hematopoietic cells. We demonstrated recently that the Src kinase Hck interacts directly with Bcr-Abl by a kinase-independent mechanism. Moreover, the inhibition of the Hck kinase seems to block some of the transforming effects of Bcr-Abl. To identify the binding domains mediating this interaction of Hck with Bcr-Abl, we co-expressed different plasmid and baculovirus vectors containing mutants or single domains of Bcr-Abl and/or Hck in COS7 and Sf9 cells. At least four independent binding regions for Hck were identified in Bcr-Abl, one in Bcr, one in the region comprising the SH3 and SH2 domain of Abl, one in the SH1 domain of Abl, and one in the C-terminal domain of Abl. In the Hck kinase, deletion of the SH2 and/or the SH3 region abolished binding to Bcr-Abl. In contrast, deletion of the Hck SH1 domain enhanced binding of Hck to Abl and Bcr-Abl. In conclusion, the results indicate that the interaction of Bcr-Abl with Hck is mediated by a novel, complex mechanism that involves multiple domains of Bcr-Abl and the SH2 and SH3 domains of Hck.