S. Peter Klinken

Novel zinc finger gene implicated as myc collaborator by retrovirally accelerated lymphomagenesis in Eμ-myc transgenic mice

To search for genes that can collaborate with myc in lymphomagenesis, we exploited retroviral insertional mutagenesis in E mu-myc transgenic mice. Moloney murine leukemia virus accelerated development of B lymphoid tumors. Three quarters contained a provirus within the known pim-1 or pim-2 loci, new loci bmi-1 and emi-1, or combinations of these. bmi-1 insertions predominated, occurring in half the tumors, and resulted in elevated bmi-1 mRNA levels. Significantly, the bmi-1 gene, which is expressed in diverse normal cells, encodes a Cys/His metal-binding motif (C3HC4) that resembles those in several DNA-binding proteins and defines a new category of zinc finger gene. Thus, myc-induced lymphomagenesis can entail the concerted action of several genes, including the presumptive nuclear regulator bmi-1.

Hemopoietic lineage switch: v-raf oncogene converts Eμmyc transgenic B cells into macrophages

Hemopoietic lineage commitment can be breached by concomitant expression of the c-myc and v-raf oncogenes. Switching to the myeloid lineage occurred frequently when B lineage cells, from either lymphomas or preleukemia bone marrow cells of Emu-myc transgenic mice, were infected with a retrovirus bearing v-raf. Cloned pre-B and B cell lines changed into either mature or immature macrophages as assessed by morphology, adherence, phagocytic activity, surface markers, and lysozyme production, but retained clonotypic immunoglobulin gene rearrangements. Although expression of the Emu-myc transgene was reduced or abolished in the more differentiated lines, the lines remained tumorigenic. The converted lines produced the myeloid growth factor GM-CSF, and most had karyotypic alterations. These results suggest that constitutive myc plus raf expression can provoke genetic reprogramming in lymphocytes.

Red blood cells

The red blood cell, or erythrocyte, possesses a most unusual cell shape. The discoid form, together with a distinctive red colouration, makes it one of the most easily recognised cell types in the body. It is also probably the most studied cell.

Lyn tyrosine kinase is essential for erythropoietin- induced differentiation of J2E erythroid cells

Erythropoietin stimulates the immature erythroid J2E cell line to terminally differentiate and maintains the viability of the cells in the absence of serum. In contrast, a mutant J2E clone (J2E‐NR) fails to mature in response to erythropoietin; however, it remains viable in the presence of the hormone. We have shown previously that intracellular signalling is disrupted in the J2E‐NR cell line and that tyrosine phosphorylation is dramatically reduced after erythropoietin stimulation. In this study we investigated the defect in J2E‐NR cells that is responsible for their inability to differentiate. Screening of numerous signalling molecules revealed that the lyn tyrosine kinase appeared to be absent from J2E‐NR cells. On closer examination, both lyn mRNA and protein content were reduced >500‐fold. Consistent with a defect in lyn, amphotropic retroviral infection of J2E‐NR cells with lyn restored the ability of the cells to synthesize haemoglobin and enabled the cells to mature morphologically. Conversely, the ability of J2E cells to differentiate in response to epo was severely curtailed when antisense lyn oligonucleotides or a dominant negative lyn were introduced into the cells. However, erythropoietin‐supported viability was unaffected by reducing lyn activity. The ability of two other erythropoietin‐responsive cell lines (R11 and R24) to differentiate in response to the hormone was also reduced by dominant negative lyn. Finally, co‐immunoprecipitation and yeast two‐hybrid analyses indicated that lyn directly associated with the erythropoietin receptor complex. These data indicate for the first time an essential role for lyn in erythropoietin‐initiated differentiation of J2E cells but not in the maintenance of cell viability.

HLS7, a hemopoietic lineage switch gene homologous to the leukemia‐inducing gene MLF1

Hemopoietic lineage switching occurs when leukemic cells, apparently committed to one lineage, change and display the phenotype of another pathway. cDNA representational difference analysis was used to identify myeloid‐specific genes that may be associated with an erythroid to myeloid lineage switch involving the murine J2E erythroleukemic cell line. One of the genes isolated (HLS7) is homologous to the novel human oncogene myeloid leukemia factor 1 (MLF1) involved in the t(3;5)(q25.1;q34) translocation associated with acute myeloid leukemia. Enforced expression of HLS7 in J2E cells induced a monoblastoid phenotype, thereby recapitulating the spontaneous erythroid to myeloid lineage switch. HLS7 also inhibited erythropoietin‐ or chemically‐induced differentiation of erythroleukemic cell lines and suppressed development of erythropoietin‐responsive colonies in semi‐solid culture. However, intracellular signaling activated by erythropoietin was not impeded by ectopic expression of HLS7. In contrast, HLS7 promoted maturation of M1 monoblastoid cells and increased myeloid colony formation in vitro. These data show that HLS7 can influence erythroid/myeloid lineage switching and the development of normal hemopoietic cells.

New insights into the regulation of erythroid cells

The regulation of erythroid cells is complex and occurs at multiple levels. Erythroid precursors, once committed to this lineage, develop in association with specific macrophages within erythroblastic islands. While erythropoietin (Epo) is the principal regulator of erythroid progenitors, other cytokines and nuclear hormones also play an important role in the maturation of these cells. Signalling from the Epo‐receptor activates several pathways, including the JAK/STAT, ras/raf/MAP kinase and PI3 kinase/Akt cascades to promote cell survival, proliferation and differentiation. Transcription factors such as GATA‐1, EKLF and NF‐E2 are crucial for progression along the erythroid maturation pathway; these, and a myriad of other transcription factors, must be expressed at the correct developmental stage for normal red blood cells to be formed. IUBMB Life, 56: 177‐184, 2004

MADM, a Novel Adaptor Protein That Mediates Phosphorylation of the 14-3-3 Binding Site of Myeloid Leukemia Factor 1

A yeast two-hybrid screen was conducted to identify binding partners of Mlf1, an oncoprotein recently identified in a translocation with nucleophosmin that causes acute myeloid leukemia. Two proteins isolated in this screen were 14-3-3ζ and a novel adaptor, Madm. Mlf1 contains a classic RSXSXP sequence for 14-3-3 binding and is associated with 14-3-3ζ via this phosphorylated motif. Madm co-immunoprecipitated with Mlf1 and co-localized in the cytoplasm. In addition, Madm recruited a serine kinase, which phosphorylated both Madm and Mlf1 including the RSXSXP motif. In contrast to wild-type Mlf1, the oncogenic fusion protein nucleophosmin (NPM)-MLF1 did not bind 14-3-3ζ, had altered Madm binding, and localized exclusively in the nucleus. Ectopic expression of Madm in M1 myeloid cells suppressed cytokine-induced differentiation unlike Mlf1, which promotes maturation. Because the Mlf1 binding region of Madm and its own dimerization domain overlapped, the levels of Madm and Mlf1 may affect complex formation and regulate differentiation. In summary, this study has identified two partner proteins of Mlf1 that may influence its subcellular localization and biological function.

Crystal structures of the Lyn Protein Tyrosine Kinase Domain in Its Apo- and Inhibitor-bound state

The Src-family protein-tyrosine kinase (PTK) Lyn is the most important Src-family kinase in B cells, having both inhibitory and stimulatory activity that is dependent on the receptor, ligand, and developmental context of the B cell. An important role for Lyn has been reported in acute myeloid leukemia and chronic myeloid leukemia, as well as certain solid tumors. Although several Src-family inhibitors are available, the development of Lyn-specific inhibitors, or inhibitors with reduced off-target activity to Lyn, has been hampered by the lack of structural data on the Lyn kinase. Here we report the crystal structure of the non-liganded form of Lyn kinase domain, as well as in complex with three different inhibitors: the ATP analogue AMP-PNP; the pan Src kinase inhibitor PP2; and the BCR-Abl/Src-family inhibitor Dasatinib. The Lyn kinase domain was determined in its “active” conformation, but in the unphosphorylated state. All three inhibitors are bound at the ATP-binding site, with PP2 and Dasatinib extending into a hydrophobic pocket deep in the substrate cleft, thereby providing a basis for the Src-specific inhibition. Analysis of sequence and structural differences around the active site region of the Src-family PTKs were evident. Accordingly, our data provide valuable information for the further development of therapeutics targeting Lyn and the important Src-family of kinases.

The erythropoietin receptor.

The erythropoietin (epo) receptor is a member of the cytokine receptor family. It is expressed almost exclusively on erythroid precursor cells and controls the development of red blood cells. The epo receptor has no intrinsic kinase activity, but binds intracellular tyrosine kinases to elicit its signals. Alterations in the transmission of the signalling cascade lead to clinically abnormal red blood cell production.

Csk-binding protein mediates sequential enzymatic down-regulation and degradation of Lyn in erythropoietin-stimulated cells.

We have shown previously that the Src family kinase Lyn is involved in differentiation signals emanating from activated erythropoietin (Epo) receptors. The importance of Lyn to red cell maturation has been highlighted by Lyn-/- mice developing anemia. Here we show that Lyn interacts with C-terminal Src kinase-binding protein (Cbp), an adaptor protein that recruits negative regulators C-terminal Src kinase (Csk)/Csk-like protein-tyrosine kinase (Ctk). Lyn phosphorylated Cbp on several tyrosine residues, including Tyr314, which recruited Csk/Ctk to suppress Lyn kinase activity. Intriguingly, phosphorylated Tyr314 also bound suppressor of cytokine signaling 1 (SOCS1), another well characterized negative regulator of cell signaling, resulting in elevated ubiquitination, and degradation of Lyn. In Epo-responsive primary cells and cell lines, Lyn rapidly phosphorylated Cbp, suppressing Lyn kinase activity via Csk/Ctk within minutes of Epo stimulation; hours later, SOCS1 bound to Cbp and was involved in the ubiquitination and turnover of Lyn protein. Thus, a single phosphotyrosine residue on Cbp coordinates a two-phase process involving distinct negative regulatory pathways to inactivate, then degrade, Lyn.