Direct binding of pRb/E2F-2 to GATA-1 regulates maturation and terminal cell division during erythropoiesis.

How cell proliferation subsides as cells terminally differentiate remains largely enigmatic, although this phenomenon is central to the existence of multicellular organisms. Here, we show that GATA-1, the master transcription factor of erythropoiesis, forms a tricomplex with the retinoblastoma protein (pRb) and E2F-2. This interaction requires a LXCXE motif that is evolutionary conserved among GATA-1 orthologs yet absent from the other GATA family members. GATA-1/pRb/E2F-2 complex formation stalls cell proliferation and steers erythroid precursors towards terminal differentiation. This process can be disrupted in vitro by FOG-1, which displaces pRb/E2F-2 from GATA-1. A GATA-1 mutant unable to bind pRb fails to inhibit cell proliferation and results in mouse embryonic lethality by anemia. These findings clarify the previously suspected cell-autonomous role of pRb during erythropoiesis and may provide a unifying molecular mechanism for several mouse phenotypes and human diseases associated with GATA-1 mutations.

Characterization of VIK-1: a new Vav-interacting Kruppel-like protein.

Binding partners of the Src homology domains of Vav-1 were characterized by a two-hybrid screening of a Jurkat cell cDNA library. One of the isolated clones encoded a new protein named VIK that belongs to the Kruppel-like zinc-finger gene family. Genome mapping showed that a single gene positioned at chromosome 7q22.1 generated three possible isoforms containing alternative domains such as proline-rich and Kruppel-associated box A or B repressor domains. The isolated isoform, VIK-1, did not contain such motifs but presented six tandemly arranged zinc-fingers and consensus Kruppel H-C links. VIK-1 interacted both with Vav-1 and cyclin-dependent kinase 4 through two independent domains and corresponded to a Vav C-Src homology domain (SH)3 partner able to shuttle between the nucleus and the cytoplasm exhibiting functional nuclear addressing and export sequences. The results indicated a restricted expression of the protein during the G1 phase and its overexpression resulted in an inhibition of the cell-cycle progression that was reversed in the presence of Vav 1. Thus, this ubiquitous factor provides a first link between Vav-1 and the cell-cycle machinery.

SIAH-1 interacts with CtIP and promotes its degradation by the proteasome pathway.

SIAH-1 and SIAH-2 are the human members of an evolutionary highly conserved E3 ligase family. SIAH-1 is a p53 and p21(Waf-1/Cip-1) induced gene during apoptosis and tumor suppression. In stable-transfected clones of MCF-7 cells, SIAH-1 overexpression was associated with apoptosis, mitotic alterations and p21(Waf-1/Cip-1) induction of expression. Using a two-hybrid screening, we identified here the transcriptional corepressor CtBP-interacting protein (CtIP) as a SIAH-1-interacting protein. CtIP has been proposed as a regulator of p21(Waf-1/Cip-1) gene transcription through a protein complex involving BRCA1. We demonstrate that SIAH-1 associates with CtIP both in vitro and in vivo. This interaction led to CtIP degradation by the ubiquitin-proteasome pathway. As expected, SIAH-1 induced p21(Waf-1/Cip-1) transcription in Jurkat-T cell. Surprisingly, a SIAH protein deleted of its RING finger, SIAH-1DeltaN, which is able to interact with CtIP but does not promote its degradation, also induced transcription from the p21(Waf-1) promoter in a similar extent as did SIAH-1. Our results suggest that p21(Waf-1/Cip-1) induction by SIAH-1 could not be mediated by CtIP degradation.

[Oncogenes and leukemia: history and perspectives]. / Oncogènes et leucémies: historique et perspectives.

Oncogenes involved in the development of hematological malignancies were first discovered through the study of experimental leukemias induced in animals by retroviruses. The discovery that some of these genes were located at the breakpoints of chromosome rearrangements in human malignancies, such as the MYC gene in Burkitt's lymphoma and the ABL gene in chronic myeloid leukemia (CML) has suggested that chromosome abnormalities were causally implicated in the pathogenesis of human diseases. Numerous nonrandom somatically acquired chromosomal translocations or inversions have been identified in human leukemias. The molecular cloning of the genes located at the breakpoints of these rearrangements allowed to identify more than 100 new oncogenes, the products of which affect normal programs of cell proliferation, differentiation and survival. Chromosome translocations can lead to the deregulated expression of a normal gene product, but in most cases of leukemia, chromosome rearrangements result in the expression of a chimeric fusion protein. Oncogene products associated with acute leukemias are often transcription factors while tyrosine kinases and antiapoptotic proteins are more commonly activated or overexpressed in chronic leukemias and in lymphomas. Recent data indicated that gene rearrangements were not the sole gene alterations occurring in human leukemia since point mutations could also affect the function of transcription factors playing a key role in hematopoiesis such as C/EBP alpha, GATA1 and AML1. But the most exciting finding was the discovery of activating point mutations in tyrosine kinase receptors such as FLT3 and c-KIT in acute leukemia. Treatment of leukemia could therefore benefit from new therapeutic approaches targeting the function of specific oncogene products as already demonstrated for CML and acute promyelocytic leukemia.

Critical role for PI 3-kinase in the control of erythropoietin-induced erythroid progenitor proliferation.

The production of red blood cells is tightly regulated by erythropoietin (Epo). The phosphoinositide 3-kinase (PI 3-kinase) pathway was previously shown to be activated in response to Epo. We studied the role of this pathway in the control of Epo-induced survival and proliferation of primary human erythroid progenitors. We show that phosphoinositide 3 (PI 3)-kinase associates with 4 tyrosine-phosphorylated proteins in primary human erythroid progenitors, namely insulin receptor substrate-2 (IRS2), Src homology 2 domain-containing inositol 5'-phosphatase (SHIP), Grb2-associated binder-1 (Gab1), and the Epo receptor (EpoR). Using different in vitro systems, we demonstrate that 3 alternative pathways independently lead to Epo-induced activation of PI 3-kinase and phosphorylation of its downstream effectors, Akt, FKHRL1, and P70S6 kinase: through direct association of PI 3-kinase with the last tyrosine residue (Tyr479) of the Epo receptor (EpoR), through recruitment and phosphorylation of Gab proteins via either Tyr343 or Tyr401 of the EpoR, or through phosphorylation of IRS2 adaptor protein. The mitogen-activated protein (MAP) kinase pathway was also activated by Epo in erythroid progenitors, but we found that this process is independent of PI 3-kinase activation. In erythroid progenitors, the functional role of PI 3-kinase was both to prevent apoptosis and to stimulate cell proliferation in response to Epo stimulation. Finally, our results show that PI 3-kinase-mediated proliferation of erythroid progenitors in response to Epo occurs mainly through modulation of the E3 ligase SCF(SKP2), which, in turn, down-regulates p27(Kip1) cyclin-dependent kinase (CDK) inhibitor via proteasome degradation.

Vav1 is a component of transcriptionally active complexes.

The importance of the hematopoietic protooncogene Vav1 in immune cell function is widely recognized, although its regulatory mechanisms are not completely understood. Here, we examined whether Vav1 has a nuclear function, as past studies have reported its nuclear localization. Our findings provide a definitive demonstration of Vav1 nuclear localization in a receptor stimulation-dependent manner and reveal a critical role for the COOH-terminal Src homology 3 (SH3) domain and a nuclear localization sequence within the pleckstrin homology domain. Analysis of DNA-bound transcription factor complexes revealed nuclear Vav1 as an integral component of transcriptionally active nuclear factor of activated T cells (NFAT)- and nuclear factor (NF)kappaB-like complexes, and the COOH-terminal SH3 domain as being critical in their formation. Thus, we describe a novel nuclear role for Vav1 as a component and facilitator of NFAT and NFkappaB-like transcriptional activity.

In vitro proliferation and differentiation of erythroid progenitors from patients with myelodysplastic syndromes: evidence for Fas-dependent apoptosis.

Erythropoiesis results from the proliferation and differentiation of pluripotent stem cells into immature erythroid progenitors (ie, erythroid burst-forming units (BFU-Es), whose growth, survival, and terminal differentiation depends on erythropoietin (Epo). Ineffective erythropoiesis is a common feature of myelodysplastic syndromes (MDS). We used a 2-step liquid-culture procedure to study erythropoiesis in MDS. CD34(+) cells from the marrow of patients with MDS were cultured for 10 days in serum-containing medium with Epo, stem cell factor, insulin-like growth factor 1, and steroid hormones until they reached the proerythroblast stage. The cells were then placed in medium containing Epo and insulin for terminal erythroid differentiation. Numbers of both MDS and normal control cells increased 10(3) fold by day 15. However, in semisolid culture, cells from patients with refractory anemia (RA) with ringed sideroblasts and RA or RA with excess of blasts produced significantly fewer BFU-Es than cells from controls. Fluorescence in situ hybridization analysis of interphase nuclei from patients with chromosomal defects indicated that abnormal clones were expanded in vitro. Epo-signaling pathways (STAT5, Akt, and ERK 1/2) were normally activated in MDS erythroid progenitors. In contrast, apoptosis was significantly increased in MDS cells once they differentiated, whereas it remained low in normal cells. Fas was overexpressed on freshly isolated MDS CD34(+) cells and on MDS erythroid cells throughout the culture. Apoptosis coincided with overproduction of Fas ligand during the differentiation stage and was inhibited by Fas-Fc chimeric protein. Thus, MDS CD34(+)-derived erythroid progenitors proliferated normally in our 2-step liquid culture with Epo but underwent abnormal Fas-dependent apoptosis during differentiation that could be responsible for the impaired erythropoiesis.

Cloning and characterization of a family of proteins associated with Mpl.

Thrombopoietin (TPO) controls the formation of megakaryocytes and platelets from hematopoietic stem cells via activation of the c-Mpl receptor and multiple downstream signal transduction pathways. We used two-hybrid screening to identify new proteins that interacted with the cytoplasmic domain of Mpl, and we found a new family of proteins designated A2D (for Ataxin-2 Domain protein). The A2D are 130-kDa proteins that have three regions similar to those of Ataxin-2, the gene product causing familial type 2 spinocerebellar ataxia. A2D has several isoforms with different C-terminal domains, all produced from a single gene by alternative splicing. Northern blotting indicated that the A2D gene is widely expressed in immortalized cell lines and hematopoietic and fetal tissues. A2D proteins were constitutively associated with Mpl in vivo in human hematopoietic UT7 cells. TPO also caused the release of A2D from the activated receptor, and the phosphorylation of A2D on tyrosines residues was dependent on the Mpl C-terminal domain. Finally, A2D bound to the unstimulated erythropoietin receptor, whereas erythropoietin caused dissociation from the erythropoietin receptor, suggesting that A2D proteins are new components of the cytokine signaling system.