8p12 stem cell myeloproliferative disorder: the FOP-fibroblast growth factor receptor 1 fusion protein of the t(6;8) translocation induces cell survival mediated by mitogen-activated protein kinase and phosphatidylinositol 3-kinase/Akt/mTOR pathways.

The FOP-fibroblast growth factor receptor 1 (FGFR1) fusion protein is expressed as a consequence of a t(6;8) (q27;p12) translocation associated with a stem cell myeloproliferative disorder with lymphoma, myeloid hyperplasia and eosinophilia. In the present report, we show that the fusion of the leucine-rich N-terminal region of FOP to the catalytic domain of FGFR1 results in conversion of murine hematopoietic cell line Ba/F3 to factor-independent cell survival via an antiapoptotic effect. This survival effect is dependent upon the constitutive tyrosine phosphorylation of FOP-FGFR1. Phosphorylation of STAT1 and of STAT3, but not STAT5, is observed in cells expressing FOP-FGFR1. The survival function of FOP-FGFR1 is abrogated by mutation of the phospholipase C gamma binding site. Mitogen-activated protein kinase (MAPK) is also activated in FOP-FGFR1-expressing cells and confers cytokine-independent survival to hematopoietic cells. These results demonstrate that FOP-FGFR1 is capable of protecting cells from apoptosis by using the same effectors as the wild-type FGFR1. Furthermore, we show that FOP-FGFR1 phosphorylates phosphatidylinositol 3 (PI3)-kinase and AKT and that specific inhibitors of PI3-kinase impair its ability to promote cell survival. In addition, FOP-FGFR1-expressing cells show constitutive phosphorylation of the positive regulator of translation p70S6 kinase; this phosphorylation is inhibited by PI3-kinase and mTOR (mammalian target of rapamycin) inhibitors. These results indicate that translation control is important to mediate the cell survival effect induced by FOP-FGFR1. Finally, FOP-FGFR1 protects cells from apoptosis by survival signals including BCL2 overexpression and inactivation of caspase-9 activity. Elucidation of signaling events downstream of FOP-FGFR1 constitutive activation provides insight into the mechanism of leukemogenesis mediated by this oncogenic fusion protein.

Patched1 interacts with cyclin B1 to regulate cell cycle progression.

The initiation of mitosis requires the activation of M-phase promoting factor (MPF). MPF activation and its subcellular localization are dependent on the phosphorylation state of its components, cdc2 and cyclin B1. In a two-hybrid screen using a bait protein to mimic phosphorylated cyclin B1, we identified a novel interaction between cyclin B1 and patched1 (ptc1), a tumor suppressor associated with basal cell carcinoma (BCC). Ptc1 interacted specifically with constitutively phosphorylated cyclin B1 derivatives and was able to alter their normal subcellular localization. Furthermore, addition of the ptc1 ligand, sonic hedgehog (shh), disrupts this interaction and allows cyclin B1 to localize to the nucleus. Expression of ptc1 in 293T cells was inhibitory to cell proliferation; this inhibition could be relieved by coexpression of a cyclin B1 derivative that constitutively localizes to the nucleus and that could not interact with ptc1 due to phosphorylation-site mutations to ALA: In addition, we demonstrate that endogenous ptc1 and endogenous cyclin B1 interact in vivo. The findings reported here demonstrate that ptc1 participates in determining the subcellular localization of cyclin B1 and suggest a link between the tumor suppressor activity of ptc1 and the regulation of cell division. Thus, we propose that ptc1 participates in a G(2)/M checkpoint by regulating the localization of MPF.

The ERBB2/HER2 receptor differentially interacts with ERBIN and PICK1 PSD-95/DLG/ZO-1 domain proteins.

Identification of protein complexes associated with the ERBB2/HER2 receptor may help unravel the mechanisms of its activation and regulation in normal and pathological situations. Interactions between ERBB2/HER2 and Src homology 2 or phosphotyrosine binding domain signaling proteins have been extensively studied. We have identified ERBIN and PICK1 as new binding partners for ERBB2/HER2 that associate with its carboxyl-terminal sequence through a PDZ (PSD-95/DLG/ZO-1) domain. This peptide sequence acts as a dominant retention or targeting basolateral signal for receptors in epithelial cells. ERBIN belongs to the newly described LAP (LRR and PDZ) protein family, whose function is crucial in non vertebrates for epithelial homeostasis. Whereas ERBIN appears to locate ERBB2/HER2 to the basolateral epithelium, PICK1 is thought to be involved in the clustering of receptors. We show here that ERBIN and PICK1 bind to ERBB2/HER2 with different mechanisms, and we propose that these interactions are regulated in cells. Since ERBIN and PICK1 tend to oligomerize, further complexity of protein networks may participate in ERBB2/HER2 functions and specificity.

Characterization of the mouse Cblc/Cbl3 gene.

The mouse Cblc/Cbl3 gene was cloned and characterized. It comprises 12 exons and encodes a putative protein of 496 amino acid residues which shares an overall 67% identity with its human ortholog; it also shares 70% of amino acid identity with mouse CBL over their conserved SH2 and Ring finger domains. Mouse Cblc mRNA is expressed in embryo and adult tissues and has a rather ubiquitous distribution.

ERBIN: a basolateral PDZ protein that interacts with the mammalian ERBB2/HER2 receptor.

The ERBB receptors have a crucial role in morphogenesis and oncogenesis. We have identified a new PDZ protein we named ERBIN (ERBB2 interacting protein) that acts as an adaptor for the receptor ERBB2/HER2 in epithelia. ERBIN contains 16 leucine-rich repeats (LRRs) in its amino terminus and a PDZ (PSD-95/DLG/ZO-1) domain at its carboxy terminus, and belongs to a new PDZ protein family. The PDZ domain directly and specifically interacts with ERBB2/HER2. ERBIN and ERBB2/HER2 colocalize to the lateral membrane of human intestinal epithelial cells. The ERBIN-binding site in ERBB2/HER2 has a critical role in restricting this receptor to the basolateral membrane of epithelial cells, as mutation of the ERBIN-binding site leads to the mislocalization of the receptor in these cells. We suggest that ERBIN acts in the localization and signalling of ERBB2/HER2 in epithelia.

Cyclin F regulates the nuclear localization of cyclin B1 through a cyclin-cyclin interaction.

The key regulator of G(2)-M transition of the cell cycle is M-phase promoting factor (MPF), a complex composed of cdc2 and a B-type cyclin. Cyclin B1 nuclear localization involves phosphorylation within a region called the cytoplasmic retention signal, which also contains a nuclear export signal. The mechanism of MPF nuclear localization remains unclear since it contains no functional nuclear localization signal (NLS). We exploited the yeast two-hybrid screen to find protein(s) potentially mediating localization of cyclin B1 and identified a novel interaction between cyclin B1 and cyclin F. We found that cdc2, cyclin B1 and cyclin F form a complex that exhibits histone H1 kinase activity. Cyclin B1 and cyclin F also colocalize through immunofluorescence studies. Additionally, deletion analysis revealed that each putative NLS of cyclin F is functional. Taken together, the data suggest that the NLS regions of cyclin F regulate cyclin B1 localization to the nucleus. The interaction between cyclin B1 and cyclin F represents the first example of direct cyclin-cyclin binding, and elucidates a novel mechanism that regulates MPF localization and function.

Characterization of FIM-FGFR1, the fusion product of the myeloproliferative disorder-associated t(8;13) translocation.

The t(8;13) translocation found in a rare type of stem cell myeloproliferative disorder generates a constitutively activated tyrosine kinase containing N-terminal sequence encoded by the FIM gene linked to the FGFR1 kinase domain. Here we have further characterized FIM and FIM-FGFR1 proteins. Firstly, we have studied their respective subcellular localization. We show that FIM has nuclear and nucleolar localization, whereas FIM-FGFR1 is mainly cytoplasmic. Within the nucleolus, FIM colocalizes with the upstream binding factor in interphasic cells, indicating that FIM may be involved in the regulation of rRNA transcription. We demonstrate that the targetting of FIM to the nucleus depends upon its C-terminal region, which is absent in the cytoplasmic FIM-FGFR1 protein. Secondly, we demonstrate that FIM-FGFR1 has constitutive dimerization capability mediated by the FIM N-terminal sequences. Finally, we show that FIM-FGFR1 promotes survival of pro-B Ba/F3 cells after interleukin-3 withdrawal, whereas ligand-activated FGFR1 induced not only cell survival but also interleukin-3 independence. Taken together, these results indicate that FIM-FGFR1 is activated by dimerization as a cytoplasmic kinase and suggest that FIM-FGFR1 partially signals through the FGFR1 pathways.

A third human CBL gene is on chromosome 19.

CBL genes encode cytoplasmic proteins involved in signal transduction downstream of a number of receptors including tyrosine kinases, cytokine receptors, and T-cell or B-cell receptors. They seem to be transducers associated with negative regulation of signals, and, as such, may be potential tumor suppressors. Using a probe derived from an expressed sequence tag, we isolated a cosmid containing part of a new CBL gene, CBLc, related to the two characterized paralogous genes CBLa and CBLb. Using the cosmid in fluorescence in situ hybridization of human metaphase chromosomes, we localized the CBLc gene to band 13.2 of chromosome 19. We show that the 19q12.2-13.3 region where CBLc is located shows paralogy with two other regions of the human genome, 3q22-q27 and 11q22-q24 where CBLb and CBLa are located, respectively. Genes from several other families are located in these regions.

Fibroblast growth factor receptor 1 is fused to FIM in stem-cell myeloproliferative disorder with t(8;13).

Chromosome 8p11-12 is the site of a recurrent breakpoint in a myeloproliferative disorder that involves lymphoid (T- or B-cell), myeloid hyperplasia and eosinophilia, and evolves toward acute leukemia. This multilineage involvement suggests the malignant transformation of a primitive hematopoietic stem cell. In this disorder, the 8p11-12 region is associated with three different partners 6q27, 9q33, and 13q12. We describe here the molecular characterization of the t(8;13) translocation that involves the FGFR1 gene from 8p12, encoding a tyrosine kinase receptor for members of the fibroblast growth factor family, and a gene from 13q12, tentatively named FIM (Fused In Myeloproliferative disorders). FIM is related to DXS6673E, a candidate gene for X-linked mental retardation in Xq13.1; this defines a gene family involved in different human pathologies. The two reciprocal fusion transcripts, FIM/FGFR1 and FGFR1/FIM are expressed in the malignant cells. The FIM/FGFR1 fusion protein contains the FIM putative zinc finger motifs and the catalytic domain of FGFR1. We show that it has a constitutive tyrosine kinase activity.

The serine/threonine phosphatase PP5 interacts with CDC16 and CDC27, two tetratricopeptide repeat-containing subunits of the anaphase-promoting complex.

The evolutionarily conserved multisubunit complex known as the cyclosome or anaphase-promoting complex is involved in catalyzing the ubiquitination of diverse substrates in M phase, allowing their destruction by the 26 S proteasome and the completion of mitosis. Three of the eight subunits of the anaphase-promoting complex (CDC16, CDC23, and CDC27) have been shown to be phosphorylated in M phase, and their phosphorylation is required for the anaphase-promoting complex to be active as a ubiquitin ligase. Several subunits of the anaphase-promoting complex contain tetratricopeptide repeats, a protein motif involved in protein/protein interactions. PP5 is a serine/threonine phosphatase that also contains four copies of the tetratricopeptide repeats motif. Here we show by a combination of two-hybrid analysis and in vitro binding that PP5 interacts with CDC16 and CDC27, two subunits of the anaphase-promoting complex. Only the NH2-terminal domain of PP5, containing all four tetratricopeptide repeats, is required for this physical interaction. Deletion analysis suggests that the site of binding to PP5 is localized to the COOH-terminal block of tetratricopeptide repeats in CDC16 and CDC27. In addition, indirect immunofluorescence showed that PP5 localizes to the mitotic spindle apparatus. The direct interaction of PP5 with CDC16 and CDC27, as well as its overlapping spindle localization in mitosis, suggests that PP5 may be involved in the regulation of the activity of the anaphase-promoting complex.

Structure and developmental expression of mouse Garp, a gene encoding a new leucine-rich repeat-containing protein.

Proteins with leucine-rich repeats (LRR) constitute a large family of molecules playing a role in protein-protein interactions and signal transduction. They are involved in various cellular processes in different species. We characterized the organization and pattern of expression of the mouse Garp gene. It is composed of two coding exons, expressed as a major 4.3 kb mRNA, and encodes a putative LRR transmembrane protein with an extracellular region almost entirely made of 20 repeats, and a short intracytoplasmic region. The mouse GARP deduced amino-acid sequence is highly similar to that of the human protein. The Garp gene is expressed in various areas in the mid-gestation developing embryo, including skin, lens fibre cells, nasal cavity, smooth and skeletal muscles, lung, and megakaryocytes of the fetal liver. In the adult it is expressed in the megakaryocytes of the spleen and in endothelial cells of the placenta. The data suggests that GARP might be involved in platelet-endothelium interactions.

The GARP gene encodes a new member of the family of leucine-rich repeat-containing proteins.

We have characterized a new human gene, named GARP, localized in the 11q14 chromosomal region. GARP comprises two coding exons, is expressed as two major transcripts of 4.4 and 2.8 kilobases, respectively, and encodes a putative transmembrane protein of 662 amino acids, the extracellular portion of which is almost entirely made of leucine-rich repeats. The molecular weight of the protein immunoprecipitated from transfected cells is 80,000. The GARP protein has structural similarities with the human GP Ib alpha and GP V platelet proteins, and with the Chaoptin, Toll, and Connectin adhesion molecules of Drosophila.

Characterization of a yeast artificial chromosome from human chromosome band 13q12 containing the FLT1 and FLT3 receptor-type tyrosine kinase genes.

A yeast artificial chromosome containing the two receptor-type tyrosine kinase genes FLT1 and FLT3 was isolated, analyzed, and compared to a genomic map in order to establish their organization and linkage. FLT1 and FLT3 are physically linked in a head-to-tail configuration and separated by about 150 kb. The region contains three CpG islands. Two of them are likely to correspond to FLT1 and FLT3, whereas the third one is suggestive of another putative, unidentified RTK gene.

The human and mouse fibroblast growth factor 6 (FGF6) genes and their products: possible implication in muscle development.

FGF6 is structurally very similar to the other members of the FGF gene family, and particularly to the FGF4 gene, which was instrumental in its isolation. Its longest open reading frame encodes a 208 amino acid residues long protein, both in man and in the mouse. It is expressed as a 4.8 kb transcript in skeletal muscle. In developing muscle, expression starts at the myotomal stage and culminates in differentiated fetal muscle masses. In culture, FGF6 protein is mitogenic and has a transforming capacity for fibroblasts. It represses the terminal differentiation of myoblasts. Action of FGF6 could be mediated by the FGFR4 receptor, which binds FGF6 and whose gene is also expressed in developing skeletal muscle.

Expression of the Fgf6 gene is restricted to developing skeletal muscle in the mouse embryo.

Fgf6, a member of the Fibroblast Growth Factor (FGF) family, is developmentally regulated and its expression is highly restricted in the adult. To gain further insight into the role of Fgf6, we studied its expression during embryogenesis using RNA in situ hybridization. Fgf6 expression is restricted to developing skeletal muscle. Fgf6 transcripts are first detected in the somites at 9.5 days post-conceptus, and expression continues in developing skeletal muscles up to at least 16.5 days post-conceptus. Fgfr4 is a putative receptor for FGF6. Its pattern of expression during myogenesis overlaps that of Fgf6, but both genes are not expressed in exactly the same population of cells. In addition, recombinant FGF6 protein is able to repress the terminal differentiation of myoblasts in culture, providing additional support to the concept that FGF6 plays an important role in myogenesis.

DNA amplification at 11q13.5-q14 in human breast cancer.

Band q13 of chromosome 11 is frequently amplified in human breast cancers, but the gene(s) responsible for the emergence of this amplicon remain(s) elusive as yet. As a tribute to the complexity of the amplification events involving 11q13 sequences in human breast cancer, we have now studied a more telomeric region at 11q13.5-q14 defined by a new transcription unit, D11S833E. We have observed that amplicons present in cell lines and primary tumors amplified for both BCL1 and D11S833E could be interrupted between these two loci. Such discontinuities were demonstrated by using a probe for the KRN1 gene, which we have localized between the BCL1/FGF4 region and D11S833E. In fact, KRN1 was not present in 4 out of 10 amplicons bearing both BCL1 and D11S833E. Furthermore, we have observed tumors in which D11S833E could be amplified in the absence of amplification of other known markers of 11q13. Therefore, D11S833E defines a new and independent amplification unit in this region.

Isolation and sequence of the murine Fgf6 cDNA.

We have studied the structure of the murine Fgf6 gene encoding a fibroblast growth factor with the purpose of looking for putative regulatory sequences in the 5' and 3' non-coding regions. The Fgf6 cDNA contains a very long 3' untranslated portion of 4015 nucleotides.

New gene in the homologous human 11q13-q14 and mouse 7F chromosomal regions.

Alterations in the chromosomal region 11q13-11q14 are involved in several pathologies in which most of the key genes remain to be identified. In an effort to isolate as many candidates as possible, we are cloning genes from this region. We report here the mapping of a new sequence from 11q13.5-11q14. This sequence, designated D11S833E, putatively encodes a new gene, provisionally named GARP. We cloned its homologous sequence in the mouse and located it on Chromosome (Chr) 7, region F. The human and mouse genes belong to a conserved group of synteny. This, together with the similar conservation of the FGF and TYR genes, indicates that the human 11q13-q14 and mouse 7E-7F regions share homology.