Role of Flk-1 in mouse hematopoietic stem cells.

It was reported that human hematopoietic stem cells in bone marrow were restricted to the CD34(+)KDR(+) cell fraction. We found that expression levels of Flk-1, a mouse homologue of KDR, were low or undetectable in mouse Lin(-)c-Kit(+)Sca-1(+)CD34(low/-) cells as well as Hoechst33342(-) cells (side population), which have long-term reconstitution capacity. Furthermore, neither Flk-1(+)CD34(low/-) cells nor Flk-1(+)CD34(+) cells had long-term reconstitution capacity in mouse. Taken together with other observations using Flk-1-deficient mice, these results indicate that Flk-1 is essential for the development of hematopoietic stem cells in embryo but not for the function of hematopoietic stem cells in adult mouse bone marrow.

CENP-H, a constitutive centromere component, is required for centromere targeting of CENP-C in vertebrate cells.

CENP-H has recently been discovered as a constitutive component of the centromere that co-localizes with CENP-A and CENP-C throughout the cell cycle. The precise function, however, remains poorly understood. We examined the role of CENP-H in centromere function and assembly by generating a conditional loss-of-function mutant in the chicken DT40 cell line. In the absence of CENP-H, cell cycle arrest at metaphase, consistent with loss of centromere function, was observed. Immunocytochemical analysis of the CENP-H-deficient cells demonstrated that CENP-H is necessary for CENP-C, but not CENP-A, localization to the centromere. These findings indicate that centromere assembly in vertebrate cells proceeds in a hierarchical manner in which localization of the centromere-specific histone CENP-A is an early event that occurs independently of CENP-C and CENP-H.

A novel regulator of G-protein signaling bearing GAP activity for Galphai and Galphaq in megakaryocytes.

The regulator of G-protein signaling (RGS) negatively regulates the alpha subunit of G proteins by accelerating their intrinsic guanosine triphosphatase (GTPase) activity. Here are reported the isolation and characterization of a novel mouse RGS, termed RGS18, which is a new member of RGS subfamily B. Northern blot analysis showed that RGS18 messenger RNA was detected predominantly in spleen and hematopoietic cells, and immunohistochemical studies demonstrated that RGS18 was expressed in megakaryocytes, platelets, granulocytes/monocytes, and, weakly, in hematopoietic stem cells, but not in lymphocytes or erythrocytes. Although various subcellular localizations of RGS have been reported, RGS18 was found to be localized in cytoplasm in megakaryocytes. In vitro binding assays of RGS18 with megakaryocyte cell lysates with or without AlF(4)(-) treatment demonstrated that RGS18 specifically binds to 2 alpha subunits of the G protein, Galphai and Galphaq. Furthermore, RGS18 clearly exhibited GTPase-activating protein (GAP) activity for Galphai and Galphaq but not for Galphas or Galpha12. In addition, chemokine stromal-derived factor 1 (SDF-1), which has been reported to stimulate megakaryocyte colony formation in the presence of thrombopoietin, affected the binding of RGS18 to Galphai but not to Galphaq. Therefore, the newly isolated RGS18 turned out to be a new member of the RGS family bearing GAP activity for Galphai, which might be stimulated by SDF-1 in megakaryocytes, as well as for Galphaq. Thus, RGS18 may play an important role in proliferation, differentiation, and/or migration of megakaryocytes.

Isolation of a novel interleukin-1-inducible nuclear protein bearing ankyrin-repeat motifs.

We isolated a novel gene termed interleukin (IL)-1-inducible nuclear ankyrin-repeat protein (INAP), of which expression was specifically induced by IL-1 in OP9 stromal cells. The INAP has ankyrin-repeat motifs and shares weak amino acid sequence homology with Bcl-3 and other IkappaB family members. The human genomic INAP gene found in the NCBI data base is located at chromosome 3q3.11. Northern blot analyses revealed that INAP was not expressed in any examined tissues without stimulation, but INAP expression was rapidly and transiently induced by IL-1 although not by tumor necrosis factor alpha nor by phorbol 12-myristate 13-acetate in OP9 cells. Immunoblots with anti-INAP-specific antibody demonstrated that INAP was rapidly and specifically produced by IL-1 stimulation and was predominantly localized in the nucleus. Immunofluorescence stainings showed that the INAP newly synthesized by IL-1 stimulation was promptly translocated into the nucleus, and FLAG-tagged INAP forcibly expressed in NIH/3T3 cells was also specifically localized in the nucleus. The possible interaction of INAP with RelA/p65, NF-kappaB1/p50, NF-kappaB2/p52, C/EBPbeta, and retinoid X receptor was examined, but we could detect none of these interactions in the nuclear extracts of IL-1-stimulated cells. Unlike Bcl-3 and other IkappaB family members, INAP may play a unique role in IL-1-induced specific gene expression and/or signal transduction in the nucleus.

Human CENP-H multimers colocalize with CENP-A and CENP-C at active centromere--kinetochore complexes.

Centromere and kinetochore proteins have a pivotal role in centromere structure, kinetochore formation and sister chromatid separation. However, the molecular architecture and the precise dynamic function of the centromere-kinetochore complex during mitosis remain poorly understood. Here we report the isolation and characterization of human CENP-H. Confocal microscopic analyses of HeLa cells with anti-human CENP-H-specific antibody demonstrated that CENP-H colocalizes with inner kinetochore plate proteins CENP-A and CENP-C in both interphase and metaphase. CENP-H was present outside centromeric heterochromatin, where CENP-B is localized, and inside the kinetochore corona, where CENP-E is localized during prometaphase. Furthermore, CENP-H was detected at neocentromeres, but not at inactive centromeres in stable dicentric chromosomes. In vitro binding assays of human CENP-H with centromere-kinetochore proteins suggest that the CENP-H binds to itself and MCAK, but not to CENP-A, CENP-B or CENP-C. CENP-H multimers were observed in cells in which both FLAG-tagged CENP-H and hemagglutinin-tagged CENP-H were expressed. These results suggest that CENP-H multimers localize constitutively to the inner kinetochore plate and play an important fundamental role in organization and function of the active human centromere-kinetochore complex.

Cell-growth control by monomeric antigen: the cell surface expression of lysozyme-specific Ig V-domains fused to truncated Epo receptor.

Previously we have shown that the V(H) and V(L) fragments of an anti-hen egg lysozyme (HEL) antibody HyHEL-10 are weakly associated but can be driven together by antigen. By joining these antibody variable domains to the cytoplasmic portion of the murine erythropoietin receptor, we created a chimeric growth factor receptor that could be activated by HEL. After co-transfection with two plasmids encoding the respective chimeric receptors in IL-3 dependent murine pro-B Ba/F3 cells, a portion of the cells survived under antigen dependent stimulation without IL-3. These surviving cells all showed coexpression of the two chimeric receptor chains and demonstrated HEL dose-dependent growth stimulation without IL-3. When another IL-3 dependent cell line 32D was transfected with a variant of such chimeric receptor with a linker peptide (Gly-Ser-Gly) inserted between V(H)/V(L) and EpoR domains, an improved growth response was attained. These observations suggest the utility of heterodimeric Fv chimeric receptors in creating cells that respond to monomeric antigen.

Ste20-like kinase (SLK), a regulatory kinase for polo-like kinase (Plk) during the G2/M transition in somatic cells.

BACKGROUND: Activation of the cyclin-dependent kinase cdc2-cyclin B1 at the G2/M transition of the cell cycle requires dephosphorylation of threonine-14 and tyrosine-15 in cdc2, which in higher eukaryotes is brought about by the Cdc25C phosphatase. In Xenopus, there is evidence that a kinase cascade comprised of xPlkk1 and Plx1, the Xenopus polo-like kinase 1, plays a key role in the activation of Cdc25C during oocyte maturation. In the mammalian somatic cell cycle, a polo-like kinase homologue (Plk1) also functions during mitosis, but a kinase upstream of Plk is still unknown. RESULTS: We show here that human Ste20-like kinase (SLK), which is a ubiquitously expressed mammalian protein related to xPlkk1, can phosphorylate and activate murine Plk1. During progression through the G2 phase of the mammalian cell cycle, the activity of endogenous SLK is increased. The amount of SLK protein is decreased in quiescent and differentiating cells. Treatment with okadaic acid induces a phosphorylation-dependent enhancement of SLK activity. CONCLUSIONS: We propose that SLK has a role in the regulation of Plk1 activity in actively dividing cells during the somatic cell cycle. SLK itself is suggested to be regulated by phosphorylation.

Identification of human APC10/Doc1 as a subunit of anaphase promoting complex.

Anaphase-promoting complex or cyclosome (APC) is a ubiquitin ligase which specifically targets mitotic regulatory factors such as Pds1/Cut2 and cyclin B. Identification of the subunits of multiprotein complex APC in several species revealed the highly conserved composition of APC from yeast to human. It has been reported, however, that vertebrate APC is composed of at least eight subunits, APC1 to APC8, while budding yeast APC is constituted of at least 12 components, Apc1 to Apc13. It has not yet been clearly understood whether additional components found in budding yeast, Apc9 to Apc13, are actually composed of mammalian APC. Here we isolated and characterized human APC10/Doc1, and found that APC10/Doc1 binds to APC core subunits throughout the cell cycle. Further, it was found that APC10/Doc1 is localized in centrosomes and mitotic spindles throughout mitosis, while it is also localized in kinetochores from prophase to anaphase and in midbody in telophase and cytokinesis. These results strongly support the notion that human APC10/Doc1 may be one of the APC core subunits rather than the transiently associated regulatory factor.

Characterization of a novel kinetochore protein, CENP-H.

Macromolecular centromere-kinetochore complex plays a critical role in sister chromatid separation, but its complete protein composition as well as its precise dynamic function during mitosis has not yet been clearly determined. Here we report the isolation of a novel mouse kinetochore protein, CENP-H. The CENP-H, with an apparent molecular mass of 33 kDa, was found to contain a coiled-coil structure and a nuclear localization signal. The CENP-H transcripts were relatively scarce but were detectable in most tissues and embryos at various stages of development. Immunofluorescence stainings of mouse fibroblast cells with anti-CENP-H-specific antibody demonstrated that the CENP-H is specifically and constitutively localized in kinetochores throughout the cell cycle; this was also confirmed by stainings with anti-centromere-specific antibody. Thus the newly isolated CENP-H may play a role in kinetochore organization and function throughout the cell cycle.

Regulation of APC activity by phosphorylation and regulatory factors.

Ubiquitin-dependent proteolysis of Cut2/Pds1 and Cyclin B is required for sister chromatid separation and exit from mitosis, respectively. Anaphase-promoting complex/cyclosome (APC) specifically ubiquitinates Cut2/Pds1 at metaphase-anaphase transition, and ubiquitinates Cyclin B in late mitosis and G1 phase. However, the exact regulatory mechanism of substrate-specific activation of mammalian APC with the right timing remains to be elucidated. We found that not only the binding of the activators Cdc20 and Cdh1 and the inhibitor Mad2 to APC, but also the phosphorylation of Cdc20 and Cdh1 by Cdc2-Cyclin B and that of APC by Polo-like kinase and cAMP-dependent protein kinase, regulate APC activity. The cooperation of the phosphorylation/dephosphorylation and the regulatory factors in regulation of APC activity may thus control the precise progression of mitosis.

Requirement of activation of JNK and p38 for environmental stress-induced erythroid differentiation and apoptosis and of inhibition of ERK for apoptosis.

C-Jun amino terminal kinase/stress-activated protein kinases (JNK/SAPK) and p38 subgroups of mitogen-activated protein kinases have been suggested to play a critical role in apoptosis, cell growth, and/or differentiation. We found that a short exposure of SKT6 cells, which respond to erythropoietin (Epo) and induce erythroid differentiation, to osmotic or heat shock induced transient activation of JNK/SAPK and p38 and inactivation of ERK and resulted in erythroid differentiation without Epo, whereas long exposure of the cells to these stresses induced prolonged activation/inactivation of the same kinases and caused apoptosis. Inhibition of JNK/SAPK and p38 resulted in inhibition of stress-induced erythroid differentiation and apoptosis. Inhibition of ERK had no effect on stress-induced erythroid differentiation, but stimulated apoptosis. Activation of p38 and/or JNK/SAPK for a short time caused erythroid differentiation without Epo, although its prolonged activation induced apoptosis. Activation of ERK suppressed stress-induced apoptosis. These results indicate that short cellular stresses, inducing transient activation of JNK/SAPK and p38, lead to cell differentiation rather than apoptosis. Furthermore, activation of JNK/SAPK and p38 is required for both cell differentiation and apoptosis, and the duration of their activation may determine the cell fate, cell differentiation, and apoptosis. In contrast, inactivation of ERK is required for stress-induced apoptosis but not cell differentiation.

Activation of hematopoietic progenitor kinase-1 by erythropoietin.

Hematopoietic progenitor kinase-1 (HPK1), which is expressed predominantly in hematopoietic cells, was identified as a mammalian Ste20 homologue that, upon transfection, leads to activation of JNK/SAPK in nonhematopoietic cells. The JNK/SAPK pathway is activated by various environmental stresses and proinflammatory and hematopoietic cytokines. Upstream activators of HPK1 currently remain elusive, and its precise role in hematopoiesis has yet to be defined. We therefore examined the possible involvement of HPK1 in erythropoietin (Epo) and environmental stress-induced JNK/SAPK activation in the Epo-dependent FD-EPO cells and Epo-responsive SKT6 cells. We found that Epo, but not environmental stresses, induced rapid and transient activation of HPK1, whereas both induced activation of JNK/SAPK. A screen for HPK1 binding proteins identified the hematopoietic cell-specific protein 1 (HS1) as a potential HPK1 interaction partner. We found HPK1 constitutively associated with HS1 and that HS1 was tyrosine-phosphorylated in response to cellular stresses as well as Epo stimulation. Furthermore, antisense oligonucleotides to HPK1 suppressed Epo-dependent cell growth and Epo-induced erythroid differentiation. We therefore conclude that Epo induces activation of both HPK1 and HS1, whereas cellular stresses activate only HS1, and that the HPK1-JNK/SAPK pathway is involved in Epo-induced growth and differentiation signals.

Immature megakaryocytes undergo apoptosis in the absence of thrombopoietin.

We examined withdrawal effects of recombinant mouse Tpo (rm-Tpo) on the apoptosis of mature and immature megakaryocytes in in vitro experiments. Apoptotic megakaryocytes were detected by double staining for acetylcholinesterase and by the TdT-mediated dUTP-biotin nick end labeling (TUNEL) method. When the purified mature megakaryocytes were cultured with or without rm-Tpo, the numbers of viable megakaryocytes, apoptotic megakaryocytes, and megakaryocytes with cytoplasmic processes were not significantly different between the two groups. In contrast, purified immature megakaryocytes underwent apoptosis when rm-Tpo was absent from the culture system. Murine bone marrow cells were cultured with rm-Tpo (50 U/mL) on days 1-7 to generate immature megakaryocytes and subsequently were cultured with different concentrations of rm-Tpo (0-50 U/mL) on days 8-14. The number of viable megakaryocytes was decreased and that of apoptotic megakaryocytes was increased by rm-Tpo in a dose-dependent manner. These results indicated a clear relation between the rm-Tpo level and the apoptosis of immature megakaryocytes.

Activation of p38 MAP kinase and JNK but not ERK is required for erythropoietin-induced erythroid differentiation.

p38 MAP kinase (p38) and JNK have been described as playing a critical role in the response to a variety of environmental stresses and proinflammatory cytokines. It was recently reported that hematopoietic cytokines activate not only classical MAP kinases (ERK), but also p38 and JNK. However, the physiological function of these kinases in hematopoiesis remains obscure. We found that all MAP kinases examined, ERK1, ERK2, p38, JNK1, and JNK2, were rapidly and transiently activated by erythropoietin (Epo) stimulation in SKT6 cells, which can be induced to differentiate into hemoglobinized cells in response to Epo. Furthermore, p38-specific inhibitor SB203580 but not MEK-specific inhibitor PD98059 significantly suppressed Epo-induced differentiation and antisense oligonucleotides of p38, JNK1, and JNK2, but neither ERK1 nor ERK2 clearly inhibited Epo-induced hemoglobinization. However, in Epo-dependent FD-EPO cells, inhibition of either ERKs, p38, or JNKs suppressed cell growth. Furthermore, forced expression of a gain-of-function MKK6 mutant, which specifically activated p38, induced hemoglobinization of SKT6 cells without Epo. These results indicate that activation of p38 and JNKs but not of ERKs is required for Epo-induced erythroid differentiation of SKT6 cells, whereas all of these kinases are involved in Epo-induced mitogenesis of FD-EPO cells.

Erythropoietin receptor and STAT5-specific pathways promote SKT6 cell hemoglobinization.

Erythrocyte production in mammals is known to depend on the exposure of committed progenitor cells to the glycoprotein hormone erythropoietin (Epo). In chimeric mice, gene disruption experiments have demonstrated a critical role for Epo signaling in development beyond the erythroid colony-forming unit (CFU-e) stage. However, whether this might include the possible Epo-specific induction of red blood cell differentiation events is largely unresolved. To address this issue, mechanisms of induced globin expression in Epo-responsive SKT6 cells have been investigated. Chimeric receptors containing an epidermal growth factor (EGF) receptor extracellular domain and varied Epo receptor cytoplasmic domains first were expressed stably at physiological levels in SKT6 cells, and their activities in mediating induced hemoglobinization were assayed. While activity was exerted by a full-length chimera (EE483), truncation to remove 7 of 8 carboxyl-terminal tyrosine sites (EE372) markedly enhanced differentiation signaling. Moreover, mutation of a STAT5 binding site in this construct (EE372-Y343F) inhibited induced globin expression and SKT6 cell hemoglobinization, as did the ectopic expression of dominant-negative forms of STAT5 in parental SKT6 cells. As in normal CFU-e, SKT6 cells also were shown to express functional receptors for stem cell factor (SCF). To further define possible specific requirements for differentiation signaling, effects of SCF on SKT6 cell hemoglobinization were tested. Interestingly, SCF not only failed to promote globin expression but inhibited this Epo-induced event in a dose-dependent, STAT5-independent fashion. Thus, effects of Epo on globin expression may depend specifically on STAT5-dependent events, and SCF normally may function to attenuate terminal differentiation while promoting CFU-e expansion.

PKA and MPF-activated polo-like kinase regulate anaphase-promoting complex activity and mitosis progression.

Ubiquitin-mediated proteolysis is the key to cell cycle control. Anaphase-promoting complex/cyclosome (APC) is a ubiquitin ligase that targets cyclin B and factors regulating sister chromatid separation for proteolysis by the proteasome and, consequently, regulates metaphase-anaphase transition and exit from mitosis. Here we report that Cdc2-cyclin B-activated Polo-like kinase (Plk) specifically phosphorylates at least three components of APC and activates APC to ubiquitinate cyclin B in the in vitro-reconstituted system. Conversely, protein kinase A (PKA) phosphorylates two subunits of APC but suppresses APC activity. PKA is superior to Plk in its regulation of APC, and Plk activity peaks whereas PKA activity is falling at metaphase. These results indicate that Plk and PKA regulate mitosis progression by controlling APC activity.

Tec is involved in G protein-coupled receptor- and integrin-mediated signalings in human blood platelets.

Tec is a non-receptor type tyrosine kinase which is tyrosine phosphorylated and activated upon stimulation of hematopoietic cells with various cytokines. The role of Tec in G protein-coupled receptor- and integrin-mediated signalings has not been elucidated. We therefore investigated the regulation of Tec in human blood platelets. Tec was rapidly tyrosine phosphorylated in response to platelet agonists which activate G protein-coupled receptors such as thromboxane A2 analog (U46619), thrombin, and thrombin receptor activating peptide (TRAP). TRAP-induced phosphorylation in Tec was significantly reduced under the conditions which abrogate fibrinogen binding to GP IIb-IIIa and subsequent platelet aggregation. However, TRAP induced significant levels of the phosphorylation even under these conditions and also in thrombasthenic platelets which lack functional GP IIb-IIIa molecules, suggesting that activation of G-protein-coupled receptor causes the phosphorylation. To clarify whether integrin engagement by itself causes the phosphorylation in Tec, we examined the state of the phosphorylation in platelets activated by integrin engagement. Platelet adhesion to immobilized fibrinogen or collagen induced significant levels of the phosphorylation. Furthermore, Tec was translocated to cytoskeleton in response to TRAP in a manner dependent on platelet aggregation, suggesting that Tec can be a component of integrin-mediated signalings. These results collectively indicate that Tec is involved in G protein-coupled receptor- and integrin-mediated signalings in human blood platelets.

Hematopoietic cell phosphatase negatively regulates erythropoietin-induced hemoglobinization in erythroleukemic SKT6 cells.

In an increasing number of hematopoietic cytokine receptor systems (T-cell receptor, B-cell receptor, and macrophage colony-stimulating factor, stem cell factor, interleukin-3, and erythropoietin [EPO] receptors), inhibitory roles for the protein tyrosine phosphatase hematopoietic cell phosphatase (HCP; SHPTP1, PTP1C, and SHP1) have been defined in proliferative signaling. However, evidence exists to suggest that HCP also may exert important effects on blood cell differentiation. To investigate possible roles for HCP during late erythroid differentiation, effects of manipulating HCP expression or recruitment on EPO-induced hemoglobinization in erythroleukemic SKT6 cells have been investigated. No effects of EPO on levels of HCP, Syp, Stat5, the EPO receptor, or GATA-1 expression were observed during induced differentiation. However, the tyrosine phosphorylation of JAK2, the EPO receptor, and Stat5 was efficiently activated, and HCP was observed to associate constitutively with the EPO receptor in this differentiation-specific system. In studies of HCP function, inhibition of HCP expression by antisense oligonucleotides enhanced hemoglobinization, whereas the enforced ectopic expression of wild-type (wt) HCP markedly inhibited EPO-induced globin expression and Stat5 activation. Based on these findings, epidermal growth factor (EGF) receptor/EPO receptor chimeras containing either the wt EPO receptor cytoplasmic domain (EECA) or a derived HCP binding site mutant (EECA-Y429,431F) were expressed in SKT6 cells, and their abilities to mediate differentiation were assayed. Each chimera supported EGF-induced hemoglobinization, but efficiencies for EECA-Y429,431F were enhanced 400% to 500%. Thus, these studies show a novel role for HCP as a negative regulator of EPO-induced erythroid differentiation. In normal erythroid progenitor cells, HCP may act to prevent premature commitment to terminal differentiation. In erythroleukemic SKT6 cells, this action also may enforce mitogenesis.

Thrombopoietin-induced polyploidization of bone marrow megakaryocytes is due to a unique regulatory mechanism in late mitosis.

Megakaryocytes undergo a unique differentiation program, becoming polyploid through repeated cycles of DNA synthesis without concomitant cell division. However, the mechanism underlying this polyploidization remains totally unknown. It has been postulated that polyploidization is due to a skipping of mitosis after each round of DNA replication. We carried out immunohistochemical studies on mouse bone marrow megakaryocytes during thrombopoietin- induced polyploidization and found that during this process megakaryocytes indeed enter mitosis and progress through normal prophase, prometaphase, metaphase, and up to anaphase A, but not to anaphase B, telophase, or cytokinesis. It was clearly observed that multiple spindle poles were formed as the polyploid megakaryocytes entered mitosis; the nuclear membrane broke down during prophase; the sister chromatids were aligned on a multifaced plate, and the centrosomes were symmetrically located on either side of each face of the plate at metaphase; and a set of sister chromatids moved into the multiple centrosomes during anaphase A. We further noted that the pair of spindle poles in anaphase were located in close proximity to each other, probably because of the lack of outward movement of spindle poles during anaphase B. Thus, the reassembling nuclear envelope may enclose all the sister chromatids in a single nucleus at anaphase and then skip telophase and cytokinesis. These observations clearly indicate that polyploidization of megakaryocytes is not simply due to a skipping of mitosis, and that the megakaryocytes must have a unique regulatory mechanism in anaphase, e.g., factors regulating anaphase such as microtubule motor proteins might be involved in this polyploidization process.

Activation of p38 MAP kinase pathway by erythropoietin and interleukin-3.

Activation of p38 MAP kinase (p38) as well as JNK/SAPK has been described as being induced by a variety of environmental stresses such as osmotic shock, ultraviolet radiation, and heat shock, or the proinflammatory cytokines tumor necrosis factor-alpha and interleukin-1 (IL-1). We found that the hematopoietic cytokines erythropoietin (Epo) and IL-3, which regulate growth and differentiation of erythroids and hematopoietic progenitors, respectively, also activate a p38 cascade. Immunoblot analyses and in vitro kinase assay clearly showed that Epo and IL-3 rapidly and transiently phosphorylated and activated p38 in Epo- or IL-3-dependent mouse hematopoietic progenitor cells. p38 can generally be activated by the upstream kinase MKK3 or MKK6. However, in vitro kinase assays in the immunoprecipitates with anti-MKK6 antibody and anti-phosphorylated MKK3/MKK6 antibody showed that activation of neither MKK3 nor MKK6 was detected after Epo or IL-3 stimulation, while osmotic shock clearly induced activation of both MKK3/MKK6 and p38. Together with previous observations, these results suggest that both p38 and JNK cascades play an important role not only in stress and proinflammatory cytokine responses but also in hematopoietic cytokine actions.