Spred-1 Negatively Regulates Interleukin-3-mediated ERK/Mitogen-activated Protein (MAP) Kinase Activation in Hematopoietic Cells

Abstract

Sprouty/Spred family proteins have been identified as negative regulators of growth factor-induced ERK/mitogen-activated protein (MAP) kinase activation. However, it has not been clarified whether these proteins regulate cytokine-induced ERK activity. We found that Spred-1 is highly expressed in interleukin-3 (IL-3)-dependent hematopoietic cell lines and bone marrow-derived mast cells. To investigate the roles of Spred-1 in hematopoiesis, we expressed wild-type Spred-1 and a dominant negative form of Spred-1, ⌬C-Spred, in IL-3-and stem cell factor (SCF)-dependent cell lines as well as hematopoietic progenitor cells from mouse bone marrow by retrovirus gene transfer. In IL-3-dependent Ba/F3 cells expressing c-kit, forced expression of Spred-1 resulted in a reduced proliferation rate and ERK activation in response to not only SCF but also IL-3. In contrast, ⌬C-Spred augmented IL-3-induced cell proliferation and ERK activation. Wild-type Spred-1 inhibited colony formation of bone marrow cells in the presence of cytokines, whereas ⌬C-Spred-1 expression enhanced colony formation. Augmentation of ERK activation and proliferation in response to IL-3 was also observed in Spred-1-deficient bone marrow-derived mast cells. These data suggest that Spred-1 negatively regulates hematopoiesis by suppressing not only SCFinduced but also IL-3-induced ERK activation.Receptor tyrosine kinases, such as stem cell factor (SCF) 1 receptor (c-kit), as well as cytokine receptors including interleukin (IL)-3 or erythropoietin (EPO) receptor activate the extracellular signal-regulated kinase (ERK) cascade. ERK activation is initiated by binding of Grb2 to the phosphorylated tyrosine residues of the receptor or phosphorylated adaptor molecules such as Shc, FRS-2, IRS-1/2, SHP-2, and Gab-1. The complex of Grb2 and SOS (son of sevenless) activates Ras by GTP loading. Ras-GTP recruits Raf1 to the plasma membrane (1, 2), which is then phosphorylated and activated by several, not well defined, kinases with complex regulatory mechanisms (3-5). Activated Raf then phosphorylates and activates the dual-specific kinase MEK, which phosphorylates and activates ERKs. In addition, the Ras-independent Raf1-ERK activation mechanism has been recently demonstrated, and members of the protein kinase C family of serine/threonine kinases have been implicated as potential activators of Raf (6).Mitogen-activated protein (MAP) kinases including ERKs play important roles in hematopoiesis. Most hematopoietic cytokines (hematopoietins) activate the JAK-STAT and Ras-ERK pathways, both being required for a satisfactory level of proliferation and differentiation of hematopoietic cells. For example, STAT5 activation is not sufficient for EPO-dependent growth of CTLL2 cells expressing EPO receptor, but additional activation of MAP kinases can support their cellular proliferation in response to EPO (7). MAP kinases have also been shown to play a critical role in megakaryopoiesis by c-mpl (8). However, little is known about how MAP kinase is regulated in hematopoietic cell...

Spreds, inhibitors of the Ras/ERK signal transduction, are dysregulated in human hepatocellular carcinoma and linked to the malignant phenotype of tumors

“…The kinase activity of ERK is fully activated through the phosphorylation of threonine (Thr 202) and tyrosine (Tyr 204) of ERK by MAP kinase kinase (MEK), which is activated by Raf. Recently, we reported that Spred-deficient cells showed increased cell proliferation with enhanced phosphorylation of ERK (Nonami et al, 2004). These findings suggest that the expression of Spred may have a profound effect on cell proliferation by regulating ERK activity.…”

Identification of SPRED2 (Sprouty-related Protein with EVH1 Domain 2) as a Negative Regulator of the Hypothalamic-Pituitary-Adrenal Axis

“…In line with these in vitro data, we detected higher CRH mRNA contents in hypothalami of KO mice, paralleled by an increased CRH secretion. Because the suppressive effect of SPRED2 is restricted to the MAPK pathway (23)(24)(25), it seems reasonable that the up-regulated MAPK signaling in our KO mice probably leads to an augmented ERK/ Ets-dependent CRH transcription and release, resulting in an overproduction of hormones at all levels of the HPA axis.…”

“…There is evidence that EVH1 domains of SPREDs act dominant negative (25), but for an efficient suppression of MAPK signaling, both the EVH1 and the SPR domains seem to be required (24). This led us to the assumption that the aberrant expression of EVH1-␤-geo does not affect the MAPK-dependent regulation of the HPA axis.…”

“…Both EVH1 and SPR domains are required for efficient suppression of MAPK signaling because deletion mutants lacking either the EVH1 or the SPR domain were unable to block growth factor-induced ERK activation (24). In summary, SPREDs are regarded as potent inhibitors of a wide range of mitogenic stimuli like growth factors, cytokines, and chemokines, but the suppressing effect seems to be restricted to the Ras/ERK/MAPK pathway (23)(24)(25). To date, promoter, RNA, and protein expression studies have revealed profound SPRED2 expression in neural and various glandular tissues and in kidney (26,27).…”

“…To date, promoter, RNA, and protein expression studies have revealed profound SPRED2 expression in neural and various glandular tissues and in kidney (26,27). The first studies using SPRED1 Ϫ/Ϫ and SPRED2 Ϫ/Ϫ mice revealed that SPREDs are not essential for survival (28,29) but play a role in hematopoiesis (25,29,30), and SPRED2 seems to be an important modulator of bone morphogenesis by inhibiting the fibroblast growth factor-induced MAPK pathway (28). Interestingly, a similar phenotype was also mentioned for SPRED1 Ϫ/Ϫ mice (29,31), underlining the role of SPRED proteins in growth regulation and supporting the idea of overlapping physiological functions.…”