Anemia due to chronic disease or chemotherapy often is ameliorated by erythropoietin (Epo). Present studies reveal that, unlike steady-state erythropoiesis, erythropoiesis during anemia depends sharply on an Epo receptor-phosphotyrosine-343-Stat5 signaling axis. In mice expressing a phosphotyrosine-null (PY-null) Epo receptor allele (EpoR-HM), severe and persistent anemia was induced by hemolysis or 5-fluorouracil. In shortterm transplantation experiments, donor EpoR-HM bone marrow cells also failed to efficiently repopulate the erythroid compartment. In each context, stress erythropoiesis was rescued to WT levels upon the selective restoration of an EpoR PY343 Stat5-binding site (EpoR-H allele). As studied using a unique primary culture system, EpoR-HM erythroblasts exhibited marked stage-specific losses in Epo-dependent growth and survival. EpoR-H PY343 signals restored efficient erythroblast expansion, and the selective Epo induction of the Stat5 target genes proviral integration site-1 (Pim-1) and oncostatin-M. Bcl2-like 1 (Bcl-x), in contrast, was not significantly induced via WT-EpoR, EpoR-HM, or EpoR-H alleles. In Kit + CD71 + erythroblasts, EpoR-PY343 signals furthermore enhanced SCF growth effects, and SCF modulation of Pim-1 kinase and oncostatin-M expression. In maturing Kit -CD71 + erythroblasts, oncostatin-M exerted antiapoptotic effects that likewise depended on EpoR PY343-mediated events. Stress erythropoiesis, therefore, requires stage-specific EpoR-PY343-Stat5 signals, some of which selectively bolster SCF and oncostatin-M action.

Lyn kinase is known to modulate the formation and function of B cells, monocytes, and mast cells. However, Lyn ؊/؊ mice also develop erythrosplenomegaly, and cases for both negative and positive erythropoietic actions of Lyn recently have been outlined. In phenylhydrazinetreated Lyn ؊/؊ mice, extramedullary splenic erythropoiesis was hyperactivated, but this did not lead to accelerated recovery from anemia. Furthermore, ex vivo analyses of the development of bone marrow-derived Lyn ؊/؊ erythroblasts in unique primary culture systems indicated positive roles for Lyn at 2 stages. Latestage Lyn ؊/؊ erythroblasts exhibited deficit Ter119 pos cell formation, and this was paralleled by increased apoptosis (and decreased Bcl-xL expression). During early development, Lyn ؊/؊ erythroblasts accumulated at a Kit pos CD71 high stage, possessed decreased proliferative capacity, and were attenuated in entering an apparent G1/S cell-cycle phase. In proposed compensatory responses, Lyn ؊/؊ erythroblasts expressed increased levels of activated Akt and p60-Src and decreased levels of death-associated pro- IntroductionSrc family kinases (SFKs) modulate a broad spectrum of bioresponse pathways including antigen and immune complex signaling, cytokine responses, integrin actions, G-protein-coupled pathways, cytoskeletal organization, and ion channels. [1][2][3][4][5][6] Within hematopoietic tissues, SFKs can function as key regulators as illustrated by original p60-Src gene disruption experiments that identified roles for this SFK during osteoclast development. 7 Studies have since extended to include investigations of SFK actions in B-cell, myeloid, and mast cell lineages with one major focus on Lyn as a hematopoietic SFK that is expressed in all blood cell lineages (with the exception of T cells). 8 In part, this focus on Lyn derives from negative roles exerted on monocyte production and plasma cell function as revealed in Lyn Ϫ/Ϫ mice by M-phi tumorigenesis 9 and IgM hyperglobulinemia. 10 In B cells, negative actions involve Lyn-mediated phosphorylation of immunoreceptor tyrosine-based inhibitory motifs (ITIM) within B-cell receptorassociated CD22 and Fc␥RII chains. 11,12 In monocytes (and B cells), the phosphorylation of related motifs within inhibitory paired Ig-like receptor B (PIR-B) and/or signal recognition peptide receptor-alpha subunit (SIRP-1␣) accessory receptors also is Lyn mediated. 13 In each system, these phosphorylated motifs can recruit Src homology region 2 domain-containing phosphatase-1 (SHP-1) as a negatively acting tyrosine phosphatase. 14,15 Lyn also can stimulate p62 downstream of tyrosine kinase (Dok), 16 an adaptor protein that can repress B-cell proliferation. 17 Lyn, nonetheless, also can exert important positive hematopoietic effects. During B-cell development, this is illustrated by a requirement for Lyn within marginal and follicular zones 10,18,19 ; within recirculating B cells; and for pre-B-cell development in the absence of Fyn and Blk. 20 These positive effects involve Lynmediated phosphorylati...

DYRKs are a new family of dual-specificity tyrosineregulated kinases with emerging roles in cell growth and development. Recently, we discovered that DYRK3 is expressed primarily in erythroid progenitor cells and modulates late erythropoiesis. We now describe 1) roles for the DYRK3 YTY signature motif in kinase activation, 2) the coupling of DYRK3 to cAMP response element (CRE) Mammalian DYRKs 1 (1, 2) and HIPKs (3) are recently described subfamilies of MAPK-related protein kinases that target Ser/Thr sites, yet also appear to be activated by tyrosine (auto)phosphorylation at a conserved YXY motif (or loop) between consensus kinase subdomains VII and VIII (hence, the nomenclature dual-specificity tyrosine-regulated kinases, DYRKs) (1, 4). At least seven DYRK isoforms have been described (5) that appear to derive from four unique genes (dyrk1-4).-2 Within this family, DYRK1 has been best studied to date, is expressed at high levels in brain (6), maps to the critical region of the Down's syndrome locus (7), and precipitates learning and memory defects when expressed in transgenic mice (8). Also, mutation of a dyrk1 gene homolog in Drosophila (MNB, for minibrain kinase) disrupts neuroblast formation in the outer proliferation center and limits optic and central lobe development (9). By comparison, HIPKs contain a DYRK-type kinase domain, but, as a separable subfamily, differ in possessing N-terminal domains that interact with NK homeoproteins (3). HIPK2 has been best studied and recently has been discovered to play an important role in p53 regulation during radiation-induced apoptosis (10).Other DYRKs have not been well studied. Recently, our laboratory (11) and Lord et al. (12) discovered that DYRK3 is selectively expressed at high levels in hematopoietic cells of erythroid lineage. Using an antisense oligonucleotide approach, it has also been demonstrated, in primary murine and human hematopoietic progenitor cells, that inhibition of DYRK3 expression significantly and specifically affects the production of colony-forming units-erythroid (the penultimate progenitors of erythroblasts). Based on apparently preferred arginine-and proline-directed substrate sequences (13) and on the in vitro ability of DYRK1 to phosphorylate eukaryotic synthesis initiation factor-2B⑀ and tau microtubule-associated protein at priming sites (14), DYRK1 has been suggested to act as a glycogen synthase kinase-priming kinase. Via in vitro kinase assays, DYRK1 has also been shown to be capable of phosphorylating forkhead transcription factor FKHR (forkhead in rhabdomyosarcoma) (15), CREB (16), and STAT3 (signal transducer and activator of transcription-3) (17). However, factors that regulate DYRK3 and the nature of DYRK targets in general remain otherwise unclear.Homologs of mammalian DYRKs interestingly also occur in Saccharomyces cerevisiae and Dictyostelium (Yak1p and YakA, respectively) (18, 19); and recently, each of these DYRK-like kinases has been linked to PKA signaling pathways (20,21). In Dictyostelium, YakA is required for cAMP ...