Integrative Signaling by Minimal Erythropoietin Receptor Forms and c-Kit

Abstract

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...

Differential regulation of SOCS genes in normal and transformed erythroid cells

“…Similarly, erythroleukemic cell lines arrested at different stages of red cell maturation displayed unique patterns of SOCS gene expression -SOCS-1 and SOCS-3 were constitutively expressed in transformed cells bearing an immature phenotype, while SOCS-1 and CIS were inducible in cell lines immortalized at a more mature stage (Figure 2). These data suggest that SOCS genes, not only perform specific tasks within erythroid cells (Starr et al, 1997;Verdier et al, 1998a, b;Sasaki et al, 2000;Cacalano et al, 2001;Pircher et al, 2001), but are regulated independently in a stage-specific manner. It is conceivable, therefore, that alterations to the expression of SOCS genes must occur for maturation of erythroid cells to proceed.…”

“…They are either induced by Epo (Yoshimura et al, 1995;Starr et al, 1997;Pircher et al, 2001) or expressed in fetal liver (Starr et al, 1997;Marine et al, 1999). While CIS À/À mice have no detectable phenotype (Marine et al, 1999), SOCS-1À/À mice suffer from mild anemia and an excess of nucleated erythroid cells in the spleen Metcalf et al, 1999).…”

Functional and Biochemical Consequences of Abrogating the Activation of Multiple Diverse Early Signaling Pathways in Kit

“…Thus far, two mechanisms of cooperation between Kit and Epo-R have been described in cell line models. Studies by Pircher et al (24) and Sui et al (23) have provided evidence that mitogen-activated protein kinase extracellular signal-regulated kinase 1 and extracellular signal-regulated kinase 2 at least in part may function as downstream integrators of Epo-R and Kit signals (23,24). Wu et al (19) have shown that activation of Kit induces tyrosine phosphorylation of the Epo-R, and that Kit interacts with the Epo-R by physically associating with its cytoplasmic domain.…”

Translocation and Phosphorylation of Calcyclin Binding Protein during Retinoic Acid-induced Neuronal Differentiation of Neuroblastoma SH-SY5Y Cells

“…It showed a 93% sequence identity with mouse CacyBP. Two independent reports showed that the level of CacyBP increased upon erythropoietin receptor activation (Xia et al, 2000;Pircher et al, 2001). Recently, the subcellular localization of CacyBP was examined in neurons and neuroblastoma NB-2a cells at different [Ca 2+ ] i (Filipek et al, 2002).…”