Erythroid homeostasis depends critically upon erythropoietin (Epo) and stem cell factor cosignaling in late progenitor cells. Epo bioresponses are relayed efficiently by minimal receptor forms that retain a single Tyr-343 site for STAT5 binding, while forms that lack all cytoplasmic Tyr(P) sites activate JAK2 and the transcription of c-Myc plus presumed additional target genes. In FDCER cell lines, which express endogenous c-Kit, the signaling capacities of such minimal Epo receptor forms (ER-HY343 and ER-HY343F) have been dissected to reveal: 1) that Epo-dependent mitogenesis, survival, and bcl-x gene expression via ER-HY343 depend upon the intactness of the Tyr-343 STAT5 binding site; 2) that ER-HY343-dependent bcl-x L gene transcription is enhanced markedly via c-Kit; 3) that socs-3, plfap, dpp-1, and cacy-bp gene transcription is induced via ER-HY343, whereas dpp-1 and cacy-bp gene expression is also supported by ER-HY343F; 4) that ectopically expressed SOCS-3 suppresses proliferative signaling by not only ER-HY343 but also c-Kit; and 5) that in FDCER and primary erythroid cells, c-Kit appears to provide the primary route to MAPK activation. Thus, integration circuits exist in only select downstream pathways within Epo and stem call factor receptor signaling. Epo,1 the prime hormonal regulator of red cell development, initiates its effects by binding to receptor dimers on the surface of erythroid burst-and colony-forming units and activating the tethered Janus family kinase (JAK) 2 (1, 2). JAK2 then mediates the phosphorylation of eight cytoplasmic tyrosine sites within the Epo receptor, and via these sites, a complex set of Src homology 2 domain-encoding effectors (and associated cofactors) are engaged. These include STAT 5A and B; Grb2/ mSOS/Raf/Ras; phosphatidylinositol 3-kinase, phospholipase-␥1; and SHIP; Lyn, Syk, and Tec; SHPTP-1 and 2; the nucleotide exchange factors Vav and C3G (via Cbl); Cis and SOCS-3; and the adaptors Shc, Gab1, Gab2, CrkL, APS, and IRS-2 (reviewed by Wojchowski et al. (Ref. 3)). Although many of these are proto-oncogenic growth regulators, others are negative effectors whose action in terminating Epo-stimulated events is likewise crucial to regulated erythropoiesis. These include Cis, which appears to compete with STAT5 for binding at Tyr-343 (4, 5); SHPTP-1, which acts to dephosphorylate JAK2 (6), and the suppressor of cytokine signaling, SOCS-3, which also binds and inhibits JAK2 (7). In SOCS-3 Ϫ/Ϫ mice, in fact, a fatal erythrocytosis is precipitated (8).Despite the complexity of this signaling network, studies of tyrosine-mutated and -truncated Epo receptors in cell lines (9, 10), murine fetal liver (11, 12), and adult murine marrow and spleen (13) 2)), definitive erythropoiesis fails and lethal embryonic anemias are engendered. In at least certain systems, however, Epo receptor forms that lack all cytoplasmic Tyr(P) sites (yet activate JAK2) also have been reported to retain significant bioactivity (4), and among STAT5 a Ϫ/Ϫ and b Ϫ/Ϫ mice those which survive embryonic stre...

The hematopoietic cell S/T kinase Pim-1 was originally discovered as a target of murine leukemia provirus integration, and when expressed at increased levels is predisposing to lymphomagenesis. Recently, Pim-1 has been shown to enhance the activities of p100, c-Myb and cdc25a, and in part this might explain reported e ects on mitogenesis. In the context of cytokine withdrawal, Pim-1 also can attenuate programmed cell death (PCD). Cytokine withdrawal, however, alters signaling pathways and can complicate the dissection of mitogenic vs apoptotic responses. To better study possible e ects of Pim-1 on PCD, a hematopoietic cell model was developed in which proliferation was supported e ciently by SCF plus EPO in the absence of endogenous Pim-1 gene expression. This was provided by factor-dependent FDCW2 cells that express endogenous and functional cKit, and were transfected stably with truncated Epo receptor form mutated at a Y343 STAT5 binding site. In proliferating cells, exogenously expressed Pim-1 was observed to e ciently inhibit PCD as induced by either Co 60 or adriamycin, and the dose-dependent nature of this e ect was established in several independent clones. By comparison, e ects of exogenous Pim-1 on mitogenesis were nominal. In addition, in cell fractionation studies an estimated 25% of M r 34 000 Pim-1 (but not M r 44 000 Pim-1) was present in nuclear extracts. Thus, Pim-1 e ciently bu ers hematopoietic progenitor cells against death as induced by several clinically important apoptotic agents, and may directly target nuclear e ectors. Oncogene (2000) 19, 3684 ± 3692.

FOG is a multitype zinc finger protein that is essential for megakaryopoiesis, binds to the amino-terminal finger of GATA-1, and modulates the transcription of GATA-1 target genes. Presently investigated are effects of FOG and GATA-1 on the transcription of the megakaryocytic integrin gene, ␣IIb. In GATA-1-deficient FDCER cells (in the presence of endogenous FOG), ectopically expressed GATA-1 activated transcription 3-10-fold both from ␣IIb templates and the endogenous ␣IIb gene. The increased expression of FOG increased reporter construct transcription 30-fold overall. Unexpectedly, ␣IIb gene transcription also was stimulated efficiently upon the ectopic expression in of FOG per se. This occurred in the absence of any detectable expression of GATA-1 and was observed in multiple independent sublines for both the endogenous ␣IIb gene and transfected constructs yet proved to depend largely upon conserved GATA elements 457 and 55 base pairs upstream from the transcriptional start site. In 293 cells, FOG plus GATA-1 but not FOG alone only moderately stimulated ␣IIb transcription, and no direct interactions of FOG with the ␣IIb promoter were detectable. Thus, FOG acts in concert with GATA-1 to stimulate ␣IIb expression but also can act via a GATA-1-independent route, which is proposed to involve additional hematopoietic-restricted cofactors (possibly GATA-2).

DYRKs are a new subfamily of dual-specificity kinases that was originally discovered on the basis of homology to Yak1, an inhibitor of cell cycle progression in yeast. At present, mDYRK-3 and mDYRK-2 have been cloned, and mDYRK-3 has been characterized with respect to kinase activity, expression among tissues and hematopoietic cells, and possible function during erythropoiesis. In sequence, mDYRK-3 diverges markedly in noncatalytic domains from mDYRK-2 and mDYRK-1a, but is 91.3% identical overall to hDYRK-3. Catalytically, mDYRK-3 readily phosphorylated myelin basic protein (but not histone 2B) and also appeared to autophosphorylate in vitro. Expression of mDYRK-1a, mDYRK-2, and mDYRK-3 was high in testes, but unlike mDYRK1a and mDYRK 2, mDYRK-3 was not expressed at appreciable levels in other tissues examined. Among hematopoietic cells, however, mDYRK-3 expression was selectively elevated in erythroid cell lines and primary pro-erythroid cells. In developmentally synchronized erythroid progenitor cells, expression peaked sharply following exposure to erythropoietin plus stem cell factor (SCF) (but not SCF alone), and in situ hybridizations of sectioned embryos revealed selective expression of mDYRK-3 in fetal liver. Interestingly, antisense oligonucleotides to mDYRK-3 were shown to significantly and specifically enhance colony-forming unit-erythroid colony formation. Thus, it is proposed that mDYRK-3 kinase functions as a lineage-restricted, stage-specific suppressor of red cell development. (Blood. 2001;97:901-910)