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Erythroid homoeostasis is primarily controlled by Epo (erythropoietin) receptor signalling; however, the Lyn tyrosine kinase plays an important subsidiary role in regulating the erythroid compartment. Nonetheless, specific erythroid pathways that require Lyn activity and their biological significance remain unclear. To address this, we asked what consequence loss of Lyn had on the ex vivo expansion and maturation of splenic erythroid progenitors and Epo receptor signalling. Pharmacological inhibition of Lyn with PP2 inhibited the survival of terminally differentiated erythroblasts. Less committed erythroid progenitors expanded well, whereas early splenic Lyn−/− erythroblasts had attenuated ex vivo expansion, and late stage Lyn−/− erythroblasts were retarded in completing morphological maturation ex vivo. Furthermore, immortalized Lyn−/− erythroblasts were slower growing, less viable and inhibited in their differentiation. Signalling studies showed that Lyn was required for both positive GAB2/Akt/FoxO3 (forkhead box O3) survival signals as well as negative feedback of JAK2 (Janus kinase 2)/STAT5 (signal transducer and activator of transcription 5) and ERK1/2 (extracellular-signal-regulated kinase 1/2) signals via SHP-1 (Src homology 2 domain-containing protein tyrosine phosphatase 1). During differentiation, Lyn controls survival and cell cycle exit as demonstrated by reduced STAT5 and FoxO3/GSKα/β (glycogen synthase kinase α/β) phosphorylation and diminished p27Kip1 induction in Lyn-deficient erythroblasts. Lyn deficiency alters the balance of pro- and anti-apoptotic molecules (BAD and BclXL), thereby reducing survival and preventing cell cycle exit. Consequently, Lyn facilitates normal erythrocyte production by influencing different stages of erythroid progenitor expansion, and mature cell development and survival signalling.

Lyn is involved in erythropoietin (Epo)-receptor signaling and erythroid homeostasis. Downstream pathways influenced following Lyn activation and their significance to erythropoiesis remain unclear. To address this, we assessed a gain-of-function Lyn mutation (Lyn(up/up)) on erythropoiesis and Epo receptor signaling. Adult Lyn(up/up) mice were anemic, with dysmorphic red cells (spherocyte-like, acanthocytes) in their circulation, indicative of hemolytic anemia and resembling the human disorder chorea acanthocytosis. Heterozygous Lyn(+/up) mice became increasingly anemic with age, indicating that the mutation was dominant. In an attempt to overcome this anemia, extramedullary erythropoiesis was activated. As the mice aged, the levels of different immature erythroid populations changed, indicating compensatory mechanisms to produce more erythrocytes were dynamic. Changes in Epo signaling were observed in Lyn(+/up) erythroid cell lines and primary CD71(+) Lyn(up/up) erythroblasts, including significant alterations to the phosphorylation of Lyn, the Epo receptor, Janus kinase 2, Signal Transducer and Action of Transcription-5, GRB2-associated-binding protein-2, Akt, and Forkhead box O3. As a consequence of altered Lyn signaling, Lyn(+/up) cells remained viable in the absence of Epo but displayed delayed Epo-induced differentiation. These data demonstrate that Lyn gene dosage and activity are critical for normal erythropoiesis; constitutively active Lyn alters Epo signaling, which in turn produces erythroid defects.

Erythropoiesis is controlled principally through erythropoietin (Epo) receptor signaling, which involves Janus kinase 2 (JAK2) and Lyn tyrosine kinase, both of which are important for regulating red blood cell (RBC) development. Negative regulation of Lyn involves C-Src kinase (Csk)-mediated phosphorylation of its C-terminal tyrosine, which is facilitated by the transmembrane adaptor Csk-binding protein (Cbp). Although Cbp has significant functions in controlling Lyn levels and activity in erythroid cells in vitro, its importance to primary erythroid cell development and signaling has remained unclear. To address this, we assessed the consequence of loss of Cbp on the erythroid compartment in vivo and whether Epo-responsive cells isolated from Cbp-knockout mice exhibited altered signaling. Our data show that male Cbp−/− mice display a modest but significant alteration to late erythroid development in bone marrow with evidence of increased erythrocytes in the spleen, whereas female Cbp−/− mice exhibit a moderate elevation in early erythroid progenitors (not seen in male mice) that does not influence the later steps in RBC development. In isolated primary erythroid cells and cell lines generated from Cbp−/− mice, survival signaling through Lyn/Akt/FoxO3 was elevated, resulting in sustained viability during differentiation. The high Akt activity disrupted GAB2/SHP-2 feedback inhibition of Lyn; however, the elevated Lyn activity also increased inhibitory signaling via SHP-1 to restrict the Erk1/2 pathway. Interestingly, whereas loss of Cbp led to mild changes to late RBC development in male mice, this was not apparent in female Cbp−/− mice, possibly due to their elevated estrogen, which is known to facilitate early progenitor self-renewal.

A yeast two-hybrid screen performed to identify binding partners of the CaR (calcium-sensing receptor) intracellular tail identified the adaptor protein 14-3-3θ as a novel binding partner that bound to the proximal membrane region important for CaR expression and signalling. The 14-3-3θ protein directly interacted with the CaR tail in pull-down studies and FLAG-tagged CaR co-immunoprecipitated with EGFP (enhanced green fluorescent protein)-tagged 14-3-3θ when co-expressed in HEK (human embryonic kidney)-293 or COS-1 cells. The interaction between the CaR and 14-3-3θ did not require a putative binding site in the membrane-proximal region of the CaR tail and was independent of PKC (protein kinase C) phosphorylation. Confocal microscopy demonstrated co-localization of the CaR and EGFP–14-3-3θ in the ER (endoplasmic reticulum) of HEK-293 cells that stably expressed the CaR (HEK-293/CaR cells), but 14-3-3θ overexpression had no effect on membrane expression of the CaR. Overexpression of 14-3-3θ in HEK-293/CaR cells attenuated CaR-mediated Rho signalling, but had no effect on ERK (extracellular-signal-regulated kinase) 1/2 signalling. Another isoform identified from the library, 14-3-3ζ, exhibited similar behaviour to that of 14-3-3θ with respect to CaR tail binding, cellular co-localization and impact on receptor-mediated signalling. However, unlike 14-3-3θ, this isoform, when overexpressed, significantly reduced CaR plasma membrane expression. Results indicate that 14-3-3 proteins mediate CaR-dependent Rho signalling and may modulate the plasma membrane expression of the CaR.

The calcium-sensing receptor (CaR) plays an integral role in calcium homeostasis and the regulation of other cellular functions including cell proliferation and cytoskeletal organisation. The multifunctional nature of the CaR is manifested through ligand-dependent stimulation of different signalling pathways that are also regulated by partner binding proteins. Following a yeast two-hybrid library screen using the intracellular tail of the CaR as bait, we identified several novel binding partners including the focal adhesion protein, testin. Testin has not previously been shown to interact with cell surface receptors. The sites of interaction between the CaR and testin were mapped to the membrane proximal region of the receptor tail and the second zinc-finger of LIM domain 1 of testin, the integrity of which was found to be critical for the CaR-testin interaction. The CaR-testin association was confirmed in HEK293 cells by coimmunoprecipitation and confocal microscopy studies. Ectopic expression of testin in HEK293 cells stably expressing the CaR enhanced CaR-stimulated Rho activity but had no effect on CaR-stimulated ERK signalling. These results suggest an interplay between the CaR and testin in the regulation of CaR-mediated Rho signalling with possible effects on the cytoskeleton.

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.

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.

Stress-activated protein kinase-3 (SAPK3) is unique amongst the mitogen-activated protein kinase (MAPK) family with its C-terminal 5 amino acids directing interaction with the PDZ domain-containing substrates alpha1-Syntrophin and SAP90/PSD95. Here, we identify three additional PDZ domain-containing binding partners, Lin-7C, Scribble, and outer membrane protein 25 (OMP25). This latter protein is localised together with SAPK3 at the mitochondria but it is not a SAPK3 substrate. Instead, OMP25 inhibits SAPK3 activity towards PDZ domain-containing substrates such as alpha1-Syntrophin and substrates without PDZ domains such as the mitochondrial protein Sab. This is a new mechanism for the regulation of SAPK3 and suggests that its intracellular activity should not be solely assessed by its phosphorylation status.

Myeloid leukemia factor 1 (MLF1) is a novel oncoprotein involved in translocations associated with acute myeloid leukemia (AML), especially erythroleukemias. In this study, we demonstrate that ectopic expression of Mlf1 prevented J2E erythroleukemic cells from undergoing biological and morphological maturation in response to erythropoietin (Epo). We show that Mlf1 inhibited Epo-induced cell cycle exit and suppressed a rise in the cell cycle inhibitor p27(Kip1). Unlike differentiating J2E cells, Mlf1-expressing cells did not downregulate Cul1 and Skp2, components of the ubiquitin E3 ligase complex SCF(Skp2) involved in the proteasomal degradation of p27(Kip1). In contrast, Mlf1 did not interfere with increases in p27(Kip1) and terminal differentiation initiated by thyroid hormone withdrawal from erythroid cells, or cytokine-stimulated maturation of myeloid cells. These data demonstrate that Mlf1 interferes with an Epo-responsive pathway involving p27(Kip1) accumulation, which inhibits cell cycle arrest essential for erythroid terminal differentiation.

The SOCS family of genes are negative regulators of cytokine signalling with SOCS-1 displaying tumor suppressor activity. SOCS-1, CIS and SOCS-3 have been implicated in the regulation of red blood cell production. In this study, a detailed examination was conducted on the expression patterns of these three SOCS family members in normal erythroid progenitors and a panel of erythroleukemic cell lines. Unexpectedly, differences in SOCS gene expression were observed during maturation of normal red cell progenitors, viz changes to CIS were inversely related to the alterations of SOCS-1 and SOCS-3. Similarly, these SOCS genes were differentially expressed in transformed erythoid cells - erythroleukemic cells immortalized at an immature stage of differentiation expressed SOCS-1 and SOCS-3 mRNA constitutively, whereas in more mature cell lines SOCS-1 and CIS were induced only after exposure to erythropoietin (Epo). Significantly, when ectopic expression of the tyrosine kinase Lyn was used to promote differentiation of immature cell lines, constitutive expression of SOCS-1 and SOCS-3 was completely suppressed. Modulation of intracellular signalling via mutated Epo receptors in mature erythroleukemic lines also highlighted different responses by the three SOCS family members. Close scrutiny of SOCS-1 revealed that, despite large increases in mRNA levels, the activity of the promoter did not alter after erythropoietin stimulation; in addition, erythroid cells from SOCS-1-/- mice displayed increased sensitivity to Epo. These observations indicate complex, stage-specific regulation of SOCS genes during normal erythroid maturation and in erythroleukemic cells.

Invasion and metastasis are controlled by the invadopodia, which delivers matrix-degrading enzymes to the invasion interface permitting cancer cell penetration and spread into healthy tissue. We have identified a novel pathway that directs Lyn/Src family tyrosine kinase signals to the invadopodia to regulate sarcoma cell invasion via the molecule AFAP-1-like-1 (AFAP1L1), a new member of the AFAP (actin filament-associated protein) family. We show that AFAP1L1 can transform cells, promote migration and co-expression with active Lyn profoundly influences cell morphology and movement. AFAP1L1 intersects several invadopodia pathway components through its multiple domains and motifs, including the following (i) pleckstrin homology domains that bind phospholipids generated at the plasma membrane by phosphoinositide 3-kinase, (ii) a direct filamentous-actin binding domain and (iii) phospho-tyrosine motifs (pY136 and pY566) that specifically bind Vav2 and Nck2 SH2 domains, respectively. These phosphotyrosine motifs are essential for AFAP1L1-mediated cytoskeleton regulation. Through its interaction with Vav2, AFAP1L1 regulates Rac activity and downstream control of PAK1/2/3 (p21-activated kinases) phosphorylation of myosin light chain (MLC) kinase and MLC2. AFAP1L1 interaction with Nck2 recruits actin-nucleating complexes. Significantly, in osteosarcoma cell lines, knockdown of AFAP1L1 inhibits phosphorylated MLC2 recruitment to filamentous-actin structures, disrupts invadopodia formation, cell attachment, migration and invasion. These data define a novel pathway that directs Lyn/Src family tyrosine kinase signals to sarcoma cell invadopodia through specific recruitment of Vav2 and Nck2 to phosphorylated AFAP1L1, to control cell migration and invasion.

Although expression quantitative trait locus, eQTL, serves as an explicit indicator of gene-gene associations, challenges remain to disentangle the mechanisms by which genetic variations alter gene expression. Here we combined eQTL and molecular analyses to identify an association between two seemingly non-associated genes in brain expression data from BXD inbred mice, namely Ptpn21 and Nrg3. Using biotinylated receptor tracking and immunoprecipitation analyses, we determined that PTPN21 de-phosphorylates the upstream receptor tyrosine kinase ErbB4 leading to the up-regulation of its downstream signaling. Conversely, kinase-dead ErbB4 (K751R) or phosphatase-dead PTPN21 (C1108S) mutants impede PTPN21-dependent signaling. Furthermore, PTPN21 also induced Elk-1 activation in embryonic cortical neurons and a novel Elk-1 binding motif was identified in a region located 1919bp upstream of the NRG3 initiation codon. This enables PTPN21 to promote NRG3 expression through Elk-1, which provides a biochemical mechanism for the PTPN21-NRG3 association identified by eQTL. Biologically, PTPN21 positively influences cortical neuronal survival and, similar to Elk-1, it also enhances neuritic length. Our combined approaches show for the first time, a link between NRG3 and PTPN21 within a signaling cascade. This may explain why these two seemingly unrelated genes have previously been identified as risk genes for schizophrenia.

J2E cells produce rapid, fatal erythroleukemias in vivo but still respond to erythropoietin (epo) in vitro by differentiating, proliferating and remaining viable in the absence of serum. Mutant epo receptors were introduced into these cells to determine whether they could influence the different biological responses to epo in vitro and the development of erythroleukemias. Three mutant receptors were used as cytoplasmic truncation mutants Delta257 and Delta321 (above box 1 and below box 2 respectively), and the cytoplasmic point mutant W282R (defective for JAK2 activation). Strikingly, the Delta321 mutation produced a hyper-sensitive response in vitro to epo-induced differentiation and viability, but not to proliferation. In contrast with the Delta321 receptor, the Delta257 and W282R mutants inhibited all biological responses to epo due to impaired JAK2 phosphorylation. Significantly, erythroleukemias took almost twice as long to develop with cells containing the W282R mutation, indicating that JAK2 plays an important role in the emergence of these leukemias. These data demonstrate that mutant epo receptors dominantly altered responses of J2E cells to epo in culture and the development of erythroleukemias. Oncogene (2000) 19, 953 - 960.

Structurally related tetratricopeptide repeat motifs in steroid receptor-associated immunophilins and the STI1 homolog, Hop, mediate the interaction with a common cellular target, hsp90. We have identified the binding domain in hsp90 for cyclophilin 40 (CyP40) using a two-hybrid system screen of a mouse cDNA library. All isolated clones encoded the intact carboxyl terminus of hsp90 and overlapped with a common region corresponding to amino acids 558-724 of murine hsp84. The interaction was confirmed in vitro with bacterially expressed CyP40 and deletion mutants of hsp90beta and was delineated further to a 124-residue COOH-terminal segment of hsp90. Deletion of the conserved MEEVD sequence at the extreme carboxyl terminus of hsp90 precludes interaction with CyP40, signifying an important role for this motif in hsp90 function. We show that CyP40 and Hop display similar interaction profiles with hsp90 truncation mutants and present evidence for the direct competition of Hop and FK506-binding protein 52 with CyP40 for binding to the hsp90 COOH-terminal region. Our results are consistent with a common tetratricopeptide repeat interaction site for Hop and steroid receptor-associated immunophilins within a discrete COOH-terminal domain of hsp90. This region of hsp90 mediates ATP-independent chaperone activity, overlaps the hsp90 dimerization domain, and includes structural elements important for steroid receptor interaction.

The Src family kinase Lyn is involved in differentiation signals emanating from activated erythropoietin (Epo) receptors, it interacts with COOH-terminal Src kinase-binding protein (Cbp), an adaptor protein that recruits negative regulators COOH-terminal Src kinase (Csk) and suppressor of cytokine signaling-1 (SOCS1). Lyn phosphorylates Cbp on several tyrosine residues, including Tyr314, which recruits Csk/SOCS1, as well as Tyr381 and Tyr409 that bind Lyns own SH2 domain. We show that Cbp alters not only the ability of erythroid cells to differentiate but also their colony morphology. Consequently, we detailed the ability of Cbp to interact with and influence Lyns ability to initiate changes in cellular architecture, which affect cell-cell and cell-substratum interactions. Over-expression of active Lyn promotes filopodia formation while inactive Lyn promotes lamellipodia formation. Conversely, Cbp over-expression, which inhibits Lyn activity, promotes lamellipodia formation, while Cbp mutants preventing its interaction/signaling consequently allow Lyn to promote filopodia formation. Thus, the Lyn-Cbp pathway and subsequent regulation of Lyn signaling and cell morphology involves a dynamic and complex series of interactions.

Thyroid hormone and its cognate receptor (TR) have been implicated in the production of red blood cells. Here, we show mice deficient for TRalpha have compromised fetal and adult erythropoiesis. Erythroid progenitor numbers were significantly reduced in TRalpha(-/-) fetal livers, and transit through the final stages of maturation was impeded. In addition, immortalized TRalpha(-/-) erythroblasts displayed increased apoptosis and reduced capacity for proliferation and differentiation. Adult TRalpha(-/-) mice had lower hematocrit levels, elevated glucocorticoid levels, and an altered stress erythropoiesis response to hemolytic anemia. Most TRalpha(-/-) animals contained markedly altered progenitor numbers in their spleens. Strikingly, 20% of TRalpha(-/-) mice failed to elicit a stress erythropoiesis response and recovered very poorly from hemolytic anemia. We conclude that an underlying erythroid defect exists in TRalpha(-/-) mice, demon-strating the importance of TRalpha to the erythroid compartment.

Hereditary Motor and Sensory Neuropathy Lom (HMSNL) is a severe autosomal recessive peripheral neuropathy, the most common form of demyelinating Charcot-Marie-Tooth (CMT) disease in the Roma (Gypsy) population. The mutated gene, N-myc downstream-regulated gene 1 (NDRG1), is widely expressed and has been implicated in a range of processes and pathways. To gain an insight into NDRG1 function we performed yeast two-hybrid screening and identified interacting proteins whose known functions suggest involvement in cellular trafficking. Further analyses, focusing on apolipoproteins A-I and A-II, confirmed their interaction with NDRG1 in mammalian cells and suggest a defect in Schwann cell lipid trafficking as a major pathogenetic mechanism in HMSNL. At the same time, the chromosomal location of NDRG1 coincides with a reported HDL-C QTL in humans and in mice. A putative role of NDRG1 in the general mechanisms of HDL-mediated cholesterol transport was supported by biochemical studies of blood lipids, which revealed an association between the Gypsy founder mutation, R148X, and decreased HDL-C levels.

In vitro studies have implicated the Lyn tyrosine kinase in erythropoietin signaling. In this study, we show that J2E erythroid cells lacking Lyn have impaired signaling and reduced levels of transcription factors STAT5a, EKLF and GATA-1. Since mice lacking STAT5, EKLF or GATA-1 have red cell abnormalities, this study also examined the erythroid compartment of Lyn(-/-) mice. Significantly, STAT5, EKLF and GATA-1 levels were appreciably lower in Lyn(-/-) erythroblasts, and the phenotype of Lyn(-/-) animals was remarkably similar to GATA-1(low) animals. Although young adult Lyn-deficient mice had normal hematocrits, older mice developed anemia. Grossly enlarged erythroblasts and florid erythrophagocytosis were detected in the bone marrow of mice lacking Lyn. Markedly elevated erythroid progenitors and precursor levels were observed in the spleens, but not bone marrow, of Lyn(-/-) animals indicating that extramedullary erythropoiesis was occurring. These data indicate that Lyn(-/-) mice display extramedullary stress erythropoiesis to compensate for intrinsic and extrinsic erythroid defects.

Hemopoietic cells, apparently committed to one lineage, can be reprogrammed to display the phenotype of another lineage. The J2E erythroleukemic cell line has on rare occasions developed the features of monocytic cells. Subtractive hybridization was used in an attempt to identify genes that were up-regulated during this erythroid to myeloid transition. We report here on the isolation of hemopoietic lineage switch 5 (Hls5), a gene expressed by the monocytoid variant cells, but not the parental J2E cells. Hls5 is a novel member of the RBCC (Ring finger, B box, coiled-coil) family of genes, which includes Pml, Herf1, Tif-1alpha, and Rfp. Hls5 was expressed in a wide range of adult tissues; however, at different stages during embryogenesis, Hls5 was detected in the branchial arches, spinal cord, dorsal root ganglia, limb buds, and brain. The protein was present in cytoplasmic granules and punctate nuclear bodies. Isolation of the human cDNA and genomic DNA revealed that the gene was located on chromosome 8p21, a region implicated in numerous leukemias and solid tumors. Enforced expression of Hls5 in HeLa cells inhibited cell growth, clonogenicity, and tumorigenicity. It is conceivable that HLS5 is one of the tumor suppressor genes thought to reside at the 8p21 locus.

The PKB (protein kinase B, also called Akt) family of protein kinases plays a key role in insulin signaling, cellular survival, and transformation. PKB is activated by phosphorylation on residues threonine 308, by the protein kinase PDK1, and Serine 473, by a putative serine 473 kinase. Several protein binding partners for PKB have been identified. Here, we describe a protein partner for PKBα termed CTMP, or carboxyl-terminal modulator protein, that binds specifically to the carboxyl-terminal regulatory domain of PKBα at the plasma membrane. Binding of CTMP reduces the activity of PKBα by inhibiting phosphorylation on serine 473 and threonine 308. Moreover, CTMP expression reverts the phenotype of v-Akt–transformed cells examined under a number of criteria including cell morphology, growth rate, and in vivo tumorigenesis. These findings identify CTMP as a negative regulatory component of the pathway controlling PKB activity.

Erythropoietin (Epo) and thyroid hormone (T(3)) are key molecules in the development of red blood cells. We have shown previously that the tyrosine kinase Lyn is involved in differentiation signals emanating from an activated erythropoietin receptor. Here we demonstrate that Lyn interacts with thyroid hormone receptor-interacting protein 1 (Trip-1), a transcriptional regulator associated with the T(3) receptor, providing a link between the Epo and T(3) signaling pathways. Trip-1 co-localized with Lyn and the T(3) receptor alpha in the cytoplasm/plasma membrane of erythroid cells but translocated to discrete nuclear foci shortly after Epo-induced differentiation. Our data reveal that T(3) stimulated the proliferation of immature erythroid cells, and inhibited maturation promoted by erythropoietin. Removal of T(3) reduced cell division and enhanced terminal differentiation. This was accompanied by large increases in the cell cycle inhibitor p27(Kip1) and by increasing expression of erythroid transcription factors GATA-1, EKLF, and NF-E2. Strikingly, a truncated Trip-1 inhibited both erythropoietin-induced maturation and T(3)-initiated cell division. This mutant Trip-1 acted in a dominant negative fashion by eliminating endogenous Lyn, elevating p27(Kip1), and blocking T(3) response elements. These data demonstrate that Trip-1 can simultaneously modulate responses involving both cytokine and nuclear receptors.

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.

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.

Erythropoiesis is primarily controlled by erythropoietin (Epo), which stimulates proliferation, differentiation and survival of erythroid precursors. We have previously shown that the tyrosine kinase Lyn is critical for transducing differentiation signals emanating from the activated Epo receptor. A yeast two-hybrid screen for downstream effectors of Lyn identified a novel protein, Liar (Lyn interacting ankyrin repeat), which forms a multi-protein complex with Lyn and HS1 in erythroid cells. Interestingly, the ankyrin repeats of Liar define a novel SH3 binding region for Lyn and HS1. Liar also contains functional nuclear localisation and nuclear export sequences, and shuttles rapidly between the nucleus and cytoplasm. Ectopic expression of Liar inhibited the differentiation of normal erythroid progenitors, as well as immortalised erythroid cells. Significantly, Liar affected Epo-activated signaling molecules including Erk2, STAT5 and Lyn. These results show that Liar is a novel Lyn-interacting molecule that plays an important role in regulating intracellular signaling events associated with erythroid terminal differentiation.

We report here the isolation of a new member of the ADP-ribosylation factor (ARF)-like family (ARL-6) present in the J2E erythroleukemic cell line, but not its myeloid variants. Consistent with this lineage-restricted expression, ARL-6 mRNA increased with erythropoietin-induced maturation of J2E cells, and decreased with interleukin 6-induced differentiation of M1 monoblastoid cells. In tissues, ARL-6 mRNA was most abundant in brain and kidney. While ARL-6 protein was predominantly cytosolic, its membrane association increased following exposure to GTP-gammaS, like many members of the ARF/ARL family. Using the yeast two-hybrid system, six molecules which interact with ARL-6 were identified including SEC61beta, a subunit of the heterotrimeric protein conducting channel SEC61p. Co-immunoprecipitation of ARL-6 confirmed a stable association between ARL-6 and SEC61beta in COS cells. These results demonstrate that ARL-6, a novel member of the ADP-ribosylation factor-like family, interacts with the SEC61beta subunit.