Regulation of JAK3 expression in human monocytes: phosphorylation in response to interleukins 2, 4, and 7.

The Janus family of kinases (JAKs) has been shown to be involved in the signal transduction of a number of cytokine receptors. Recently, we have cloned a novel JAK family member, JAK3, that is expressed in natural killer and activated T cells and is coupled functionally and physically to the interleukin 2 (IL-2) receptor in these cells. Here we report that JAK3 was expressed at low but detectable levels in human monocytes. In contrast, JAK3 expression was strongly induced during activation by interferon gamma (IFN-gamma) or lipopolysaccharide. Moreover, JAK3 became tyrosine phosphorylated in response to IL-2, IL-4, and IL-7 but not response to IFN-gamma or granulocyte/macrophage colony-stimulating factor. Together, these findings suggest that JAK3 is functionally important in activated monocytes and cells of the myeloid lineage and is involved in signaling responses of cytokines that use the common gamma-chain of the IL-2 receptor.

Lck associates with and is activated by Kit in a small cell lung cancer cell line: inhibition of SCF-mediated growth by the Src family kinase inhibitor PP1.

At least 70% of small cell lung cancers (SCLCs) express the Kit receptor tyrosine kinase and its ligand, stem cell factor (SCF). In an effort to define the signal transduction pathways activated by Kit in SCLC, we focused on Src family kinases and, in particular, Lck, a Src-related tyrosine kinase that is expressed in hemopoietic cells and certain tumors, including SCLC. SCF treatment of the H526 cell line induced a physical association between Kit and Lck that, in vitro, was dependent on phosphorylation of the juxtamembrane domain of Kit. Stimulation of Kit with recombinant SCF resulted in a rapid 3-6-fold increase in the specific activity of Lck, which was similar in magnitude to the activation of Lck resulting from the cross-linking of the T-cell receptor complex of Jurkat cells. Lck activity peaked by 5 min after SCF addition, and the elevated activity persisted for at least 30 min in the presence of SCF, with kinetics similar to the activation of mitogen-activated protein kinase. PP1, an inhibitor of Src family kinases with selectivity for Lck, completely inhibited SCF-mediated growth but had little effect on insulin-like growth factor-I-mediated growth. PP1 antagonized both SCF-mediated proliferation and inhibition of apoptosis. PP1 had no effect on Kit kinase activity but was shown to block total Lck activity by at least 90% by immune complex kinase assay. Low levels of Src, Hck, and Yes were also expressed in the H526 cell line; only Yes showed a consistent increase in specific activity, which was also inhibited by PP1 following SCF treatment. These data demonstrate that, in the H526 SCLC cell line, Lck and, possibly, Yes are downstream of Kit in a signal transduction pathway; the inhibition by PP1 of SCF-mediated proliferation and inhibition of apoptosis suggests that Src family kinases are intermediates in the signaling pathways that regulate these processes.

Maturation of erythroid cells and erythroleukemia development are affected by the kinase activity of Lyn.

This study examined the impact of the tyrosine kinase Lyn on erythropoietin-induced intracellular signaling in erythroid cells. In J2E erythroleukemic cells, Lyn coimmunoprecipitated with numerous proteins, including SHP-1, SHP-2, ras-GTPase-activating protein, signal transducers and activators of transcription (STAT) 5a, STAT5b, and mitogen-activated protein kinase; however, introduction of a dominant-negative Lyn (Y397F Lyn) inhibited the interaction of Lyn with all of these molecules except SHP-1. Cells containing the dominant-negative Lyn displayed altered intracellular phosphorylation patterns, including mitogen-actiated protein kinase, but not erythropoietin receptor, Janus-activated kinase (JAK) 2, or STAT5. As a consequence, erythropoietin-initiated differentiation and basal proliferation were severely impaired. Y397F Lyn reduced the protein levels of erythroid transcription factors erythroid Kruppel-like factor and GATA-1 up to 90%, which accounts for the inability of J2E cells expressing Y397F Lyn to synthesize hemoglobin. Although Lyn was shown to bind several sites on the cytoplasmic domain of the erythropoietin receptor, it was not activated when a receptor mutated at the JAK2 binding site was ectopically expressed in J2E cells indicating that JAK2 is the primary kinase in erythropoietin signaling and that Lyn is a secondary kinase. In normal erythroid progenitors, erythropoietin enhanced phosphorylation of Lyn; moreover, exogenous Lyn increased colony forming unit-erythroid, but not burst forming uniterythroid, colonies from normal progenitors, demonstrating a stage-specific effect of the kinase. Significantly, altering Lyn activity in J2E cells had a profound effect on the development of erythroleukemias in vivo: the mortality rate was markedly reduced and latent period extended when either wild-type Lyn or Y397F Lyn was introduced into these cells. Taken together, these data show that Lyn plays an important role in intracellular signaling in nontransformed and leukemic erythroid cells.

The effect of c-raf antisense oligonucleotides on growth factor-induced proliferation of hematopoietic cells.

While it is well established that Raf-1 kinase is activated by phosphorylation in growth factor-dependent hematopoietic cell lines stimulated with a variety of hematopoietic growth factors, little is known about the biological effects of Raf-1 activation on normal hematopoietic cells. Therefore, we examined the requirement for Raf-1 in growth factor-regulated proliferation and differentiation of hematopoietic cells using c-faf antisense oligonucleotide. Raf-1 required for the proliferation of growth factor dependent cell lines stimulated by IL-2, IL-3, G-CSF, GM-CSF and EPO that bind to the hematopoietin class of receptors. Raf-1 is also required for the proliferation of cell lines stimulated by growth factors that use the tyrosine kinase containing receptor class, including SLF and CSF-1. In addition, Raf-1 is also required for IL-6, LIF- and OSM-induced proliferation whose receptors share the gp 130 subunit. In contrast to previous results which demonstrated that IL-4 could not activate Raf-1 kinase, c-raf antisense oligonucleotides also inhibited IL-4-induced proliferation of T cell and myeloid cell lines. Using normal hematopoietic cells, c-raf antisense oligonucleotides completely suppressed the colony formation of murine hematopoietic progenitors in response to single growth factors, such as IL-3, CSF-1 or GM-CSF. Further, c-raf antisense oligonucleotides inhibited the growth of murine progenitors stimulated with synergistic combinations of growth factors (required for primitive progenitor growth) including two, three and four factor combinations. In comparison to murine hematopoietic cells, c-raf antisense oligonucleotides also inhibited both IL-3 and GM-CSF-induced colony formation of CD 34+ purified human progenitors. In addition, Raf-1 is required for the synergistic response of CD 34+ human bone marrow progenitors to multiple cytokines; however, this effect was only observed when additional antisense oligonucleotides were added to the cultures at day 7 of a 14 day assay. Finally, Raf-1 is required for the synergistic response of human Mo-7e cells and of normal human fetal liver cells to five factor combinations. Thus, Raf-1 is required to transduce growth factor-induced proliferative signals in factor-dependent progenitor cells lines for all known classes of hematopoietic growth factor receptors, and is required for the growth of normal murine and human bone marrow-derived progenitors.

Early signaling pathways activated by c-Kit in hematopoietic cells.

c-Kit is a receptor tyrosine kinase that binds stem cell factor (SCF). Structurally, c-Kit contains five immunoglobulin-like domains extracellularly and a catalytic domain divided into two regions by a 77 amino acid insert intracellularly. Studies in white spotting and steel mice have shown that functional SCF and c-Kit are critical in the survival and development of stem cells involved in hematopoiesis, pigmentation and reproduction. Mutations in c-Kit are associated with a variety of human diseases. Interaction of SCF with c-Kit rapidly induces receptor dimerization and increases in autophosphorylation activity. Downstream of c-Kit, multiple signal transduction components are activated, including phosphatidylinositol-3-kinase, Src family members, the JAK/STAT pathway and the Ras-Raf-MAP kinase cascade. Structure-function studies have begun to address the role of these signaling components in SCF-mediated responses. This review will focus on the biochemical mechanism of action of SCF in hematopoietic cells.

Stem cell factor, the JAK-STAT pathway and signal transduction.

Recent work has demonstrated the importance of Janus family kinases (JAKs) and signal transducers and activators of transcription (STATs) in the stimulus-response coupling of receptors lacking intrinsic tyrosine kinase activity. In particular, the JAK-STAT pathway appears critical in signal transduction by interferon as well as numerous hematopoietic growth factors interacting with members of the hemapoietin receptor superfamily. Although ligands that interact with receptor tyrosine kinases (RTK), such as epidermal growth factor (EGF), platelet-derived growth factor (PDGF) and colony stimulating factor-1 (CSF-1), have been shown to induce increases in phosphorylation of both JAKs and STATs, little is known about activation of this pathway by stem cell factor (SCF). This review will summarize what is known about the JAK/STAT pathway in relation to SCF signal transduction.

Interleukin-2 regulation of diadenosine 5',5'''-p1 p4-tetraphosphate (Ap4A) levels and DNA synthesis in cloned murine T lymphocytes.

The levels or diadenosine 5', 5'''-p1, p4, tetraphosphate (Ap4A), a putative signal molecule associated with DNA synthesis, has been measured in murine T lymphocytes. The level or Ap4A detected correlated with the stimulation of DNA synthesis in murine T lymphocytes. In interleukin-2 (IL-2) dependent cells previously deprived of IL-2, new DNA synthesis can be induced by adding IL-2; the synthesis of DNA is preceded by an increase in Ap4A levels. A significant increase in DNA synthesis was observed after the Ap4A concentration exceeded the Kd of DNA polymerase alpha for Ap4A. Similarly, in cells blocked from synthesizing DNA by hydroxyurea, the levels or Ap4A are maintained only in the presence of IL-2. Once IL-2 is removed, the potential to synthesize DNA decreases and is preceded by decreases in the level or Ap4A. The DNA synthesis potential decreases rapidly after the Ap4A concentration fell below the Kd of DNA polymerase alpha for Ap4A. It is possible that Ap4A is a second messenger molecule required for the proliferation of lymphocytes and that the production of Ap4A in IL-2 dependent murine T lymphocytes is regulated by the homologous growth factor.

Migration of dog polymorphonuclear neutrophilic leukocytes to formylated peptides.

Formylated peptides are potent stimulants of polymorphonuclear neutrophilic leukocyte (PMN) migration from species such as humans and rabbits. Interestingly, PMNs from dogs, cats, pigs and cows have been reported as refractory to N-formyl-l-methionyl-l-leucyl-l-phenylalanine (FMLP) and generally are believed not to express formylpeptide receptors. Formylpeptides are a major component of conditioned media from E. coli cultures and believed to be a significant element in inflammatory responses elicited by E. coli. Our studies have found that E. coli filtrate was a potent stimulant of dog PMN migration. Inhibition of migration to E. coli filtrates by the antagonist t-botyloxycarbonyl-l-methionyl-l-leucyl-l-phenylalanine (t-boc-MLP) demonstrated that the migration was mediated through the formylated peptide receptor. Migration in response to peptides with higher affinity for the formylpeptide receptor than FMLP was further evidence for these receptors on the dog PMN. PMNs from dogs migrated in response to FMLP at high concentrations (100 microM); however, pretreatment with phorbol myristate acetate resulted in increased migration of dog PMNs in response to concentrations of FMLP as low as 1 pM. These results demonstrate that dog PMNs are responsive to formylpeptides and that these responses can be up-regulated by PMA. Thus PMNs from a species previously thought incapable of responding to formylpeptides can respond to formylpeptide analogs with high affinity for the receptor as well as be primed for enhanced migration to FMLP by PMA.

Lyn is activated during late G1 of stem-cell-factor-induced cell cycle progression in haemopoietic cells.

Stem cell factor (SCF) binds the receptor tyrosine kinase c-Kit and is critical in haemopoiesis. Recently we found that the Src family member Lyn is highly expressed in SCF-responsive cells, associates with c-Kit and is activated within minutes of the addition of SCF. Here we show that SCF activates Lyn a second time, hours later, during SCF-induced cell cycle progression. In cells arrested at specific phases of the cell cycle with the drugs mimosine, aphidicolin and nocodazole, maximal Lyn kinase activity occurred in late G(1) and through the G(1)/S transition. Similarly, kinetic studies of SCF-induced cell cycle progression found that activation of Lyn preceded the G(1)/S transition and was maintained into early S-phase. Activation of Lyn was paralleled by two events critical for the G(1)/S transition, increases in cyclin-dependent kinase 2 (Cdk2) activity and phosphorylation of the retinoblastoma gene product (Rb). Lyn was associated with Cdk2; Cdk2-associated Lyn was heavily phosphorylated on serine and threonine residues both in vitro and in situ during S-phase. Inhibition of Lyn activity with PP1 disrupted association with Cdk2 and decreased the numbers of cells entering S-phase. The degree of phosphorylation of Rb in PP1-treated cells suggested an increased number of cells arrested in the middle of G(1). These findings demonstrate that SCF activates the Src family member Lyn before the G(1)/S transition of the cell cycle and suggest that Lyn is involved in SCF-induced cell cycle progression.

Signal transduction of human interleukin 3 and granulocyte-macrophage colony-stimulating factor through serine and tyrosine phosphorylation.

To elucidate the rapid events in signal transduction of human granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 3 (IL 3), we examined phosphorylation of proteins on both serine and tyrosine residues in a cytokine-stimulated human myeloid cell line. We found increases in tyrosine phosphorylation within 30 s of stimulation with GM-CSF or IL 3, with peak responses occurring within 2 min. IL 3 and GM-CSF also induced serine phosphorylation, though 10 min of stimulation was required for maximum phosphate incorporation. Interestingly, both IL 3 and GM-CSF stimulated phosphate incorporation in identical substrates, a 68 kDa seryl-phosphoprotein (p68) and a 140 kDa tyrosyl-phosphoprotein (p140). Treatment of AML 193 cells with phorbol myristate acetate resulted in serine phosphorylation of p68; however, p140 was not phosphorylated on tyrosine. Depletion of protein kinase C isoenzymes with high concentrations of phorbol myristate acetate resulted in p68 phosphorylation, which was not further increased by IL 3 or GM-CSF. In contrast, cytokine-induced phosphorylation on tyrosine of p140 was observed after protein kinase C depletion. These data demonstrate the co-ordinate yet independent serine and tyrosine phosphorylation in IL 3- and GM-CSF-treated human myeloid cells, and thus suggest a common set of protein kinases stimulated by each separate ligand.

Stat1 associates with c-kit and is activated in response to stem cell factor.

Interaction of stem cell factor (SCF), a haematopoietic growth factor, with the receptor tyrosine kinase c-kit leads to autophosphorylation of c-kit as well as tyrosine phosphorylation of various substrates. Little is known about the role of the JAK/STAT pathway in signal transduction via receptor tyrosine kinases, although this pathway has been well characterized in cytokine receptor signal transduction. We recently found that the Janus kinase Jak2 associates with c-kit and that SCF induces rapid and transient phosphorylation of Jak2. Here we present evidence that SCF activates the transcription factor Stat1. Phosphorylated c-kit co-immunoprecipitates with Stat1 within 1 min of SCF stimulation of the human cell line MO7e. Co-precipitation experiments using glutathione S-transferase fusion proteins indicate that association with c-kit is mediated by the Stat1 SH2 domain. Stat1 is rapidly tyrosine-phosphorylated in response to SCF in MO7e cells, the murine cell line FDCP-1 and normal progenitor cells. SCF-induced phosphorylation of Jak2 and Stat1 was also observed in murine 3T3 fibroblasts stably transfected with full-length human c-kit receptor. Furthermore c-kit directly phosphorylates Stat1 fusion proteins in in vitro kinase assays. Electrophoretic mobility-shift assays with nuclear extracts from SCF-stimulated cell lines and normal progenitor cells indicate that activated Stat1 binds the m67 oligonucleotide, a high-affinity SIE promoter sequence. These results demonstrate that Stat1 is activated in response to SCF, and suggest that Stat1 is a component of the SCF signal-transduction pathway.

Regulation of c-myc and ornithine decarboxylase expression by distinct protein kinase systems in IL-3-dependent myeloid cells.

We have investigated whether PK-C-regulated events are independent of those biochemical events related to IL-3-induced tyrosine kinase activation by 32Dcl cells. The depletion of functional PK-C isoform activity by prolonged PMA treatment reduced the proliferative response to IL-3 by half that of untreated control cells. PK-C-deficient 32Dcl cells were unable to respond to PMA for the induction of c-myc and ODC mRNA accumulation. PK-C down-regulation did not affect IL-3-induced tyrosine phosphorylation and inhibited IL-3-regulated c-myc and ODC mRNA expression by only 30%. However, PK-C down-regulation had a pronounced inhibitory effect on IL-3 regulation of ODC enzymatic activity. While a PK-C-dependent and -independent pathway for the regulation of c-myc and ODC mRNA expression could be demonstrated, the regulation of ODC enzymatic activity appeared to require an intact PK-C system. The data suggest that the optimum biological and biochemical responses to IL-3 requires both pathways intact, however, tyrosine kinase activation and significant increases in gene products associated with proliferation can be achieved in the absence of a functional PK-C system.

PDGF-independent activation of PDGF-beta receptors in NIH-3T3 cells transformed by c-met protooncogene.

We have previously reported that c-met protooncogene, a member of a new class of receptor tyrosine-kinase gene family, is transforming when overexpressed in NIH-3T3 cells. In this paper, we report that the c-met protooncogene-transformed cells proliferate in a serum- and growth factor-free medium and exhibit constitutive tyrosine phosphorylation of several cellular proteins including the met protooncogene-encoded p145 and p185. Further investigations revealed platelet-derived growth factor (PDGF)-independent phosphorylation of PDGF-beta receptors in the transformed cells. Phosphoamino acid analysis revealed phosphorylation of PDGF receptors at tyrosine and serine residues. The PDGF receptor phosphorylation is unlikely to occur via autocrine production of PDGF since we could not detect PDGF activity both at the RNA level and at a functional protein level. Additionally, phospholipase C-gamma (PLC-gamma) a substrate of activated PDGF receptors, was found to be physically associated with PDGF receptors in the absence of PDGF stimulation in transformed cells. Furthermore, PDGF receptors coimmunoprecipitated along with PLC-gamma. Taken together, our results demonstrate a PDGF-independent phosphorylation and activation of PDGF-beta receptor in NIH-3T3 cells transformed by c-met protooncogene.

Lyn associates with the juxtamembrane region of c-Kit and is activated by stem cell factor in hematopoietic cell lines and normal progenitor cells.

Stem cell factor (SCF) is a cytokine critical for normal hematopoiesis. The receptor for SCF is c-Kit, a receptor tyrosine kinase. Our laboratory is interested in delineating critical components of the SCF signal transduction pathway in hematopoietic tissue. The present study examines activation of Src family members in response to SCF. Stimulation of cell lines as well as normal progenitor cells with SCF rapidly increased tyrosine phosphorylation of the Src family member Lyn. Peak responses were noted 10-20 min after SCF treatment, and phosphorylation of Lyn returned to basal levels 60-90 min after stimulation. SCF also induced increases in Lyn kinase activity in vitro. Lyn coimmunoprecipitated with c-Kit, and studies with GST fusion proteins demonstrated that Lyn readily associated with the juxtamembrane region of c-Kit. Treatment of cells with either Lyn antisense oligonucleotides or PP1, a Src family inhibitor, resulted in dramatic inhibition of SCF-induced proliferation. These data demonstrate that SCF rapidly activates Lyn and suggest that Lyn is critical in SCF-induced proliferation in hematopoietic cells.

Haemopoietic growth factor regulation of protein kinases and genes associated with cell proliferation.

Haemopoietic growth factors stimulate a number of common biochemical and molecular events despite the high specificity of individual ligand-receptor interactions. Analysis of three distinct colony-stimulating factors (CSFs), interleukin 3 (IL-3), granulocyte-CSF and granulocyte macrophage-CSF, and the lymphocytotropic growth factor IL-2 revealed remarkably similar distal subcellular biochemical signals, although the mode of initial membrane signal transduction may differ significantly. Both early progenitor cell growth factors, such as IL-3, and late-acting factors, such as CSF-1, stimulate tyrosine and serine/threonine substrate phosphorylations. One substrate (p68) is phosphorylated in response to many CSFs and to IL-2, suggesting that it plays a highly conserved role in the signal transduction processes of many different receptor(s). The proliferative CSFs and IL-2 also stimulate the expression of many of the same genes, including protooncogenes, the ornithine decarboxylase gene, and members of the phylogenetically ancient family of stress response genes. Thus although initial membrane events may differ among the proliferative stimulants, the biochemical and molecular convergence of signalling pathways on highly conserved cellular substrates and on the programme of gene expression is seen.

Dissociation of human cytokine receptor expression and signal transduction.

We have examined the relationship between granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3) receptor expression and signal transduction in populations of HL-60 cells differing in proliferative capacity to these cytokines. GM-CSF or IL-3 stimulation of HL-60 cells pretreated with either dimethyl sulfoxide (DMSO) or retinoic acid results in increases in proliferative response as well as both tyrosine and serine phosphorylation. In contrast, neither GM-CSF or IL-3 stimulation of parental HL-60 cells (those not treated with DMSO or retinoic acid) produced any changes in either proliferation or protein phosphorylation. Thus, although parental HL-60 cells expressed both GM-CSF and IL-3 receptors, treatment with either DMSO or retinoic acid was necessary to confer the capacity for signal transduction as assessed by both a biologic and biochemical response. Pretreatment of cells with genistein, a protein tyrosine kinase inhibitor, resulted in inhibition of GM-CSF-induced protein tyrosine phosphorylation as well as proliferation. These data show a strong correlation between cytokine-induced increases in protein phosphorylation and subsequent biologic responses. Further, this work demonstrates that cytokine receptor expression and signal transduction can be disassociated and suggests the potential for independent regulation of these two components of signal transduction.

Characterization of a 97-kDa phosphotyrosylprotein regulated by multiple cytokines.

We have examined the signal transduction pathways of a number of cytokines that interact with receptors that are members of the hematopoietin receptor superfamily. A 97-kDa protein was phosphorylated on tyrosine in response to stimulation of appropriate target cells with interleukin (IL)-2, IL-3, granulocyte-macrophage colony-stimulating factor (CSF), granulocyte-CSF, or erythropoietin. These data suggest that a 97-kDa phosphotyrosylprotein represents a point of convergence for signal transduction by a number of growth factor receptors that do not have homology with any known protein tyrosine kinase. To address the possibility that p97 may represent a tyrosine kinase involved in multiple signal transduction pathways, we tested the capacity of this protein to bind a tyrosine kinase substrate or ATP. Indeed, a 97-kDa phosphotyrosylprotein purified from IL-2-stimulated lymphoid cells as well as granulocyte-macrophage-CSF-stimulated myeloid cells bound to a polymer of glutamic acid and tyrosine which is a tyrosine kinase substrate. Further, a 97-kDa phosphotyrosylprotein present in both lineages also bound 8-azido-ATP. These data indicate that a 97-kDa phosphotyrosylprotein with properties consistent with those of a protein tyrosine kinase is involved in the signal transduction pathways of certain members of the newly identified hematopoietin receptor superfamily and may represent an early point of convergence in the stimulus-response coupling of multiple cytokine receptors.

Phosphorylation of a Fes-related protein in response to granulocyte-macrophage colony stimulating factor.

Previous work has suggested that a 97-kDa protein (p97) is involved in the signal transduction pathway of granulocyte-macrophage colony stimulating factor (GM-CSF) as well as interleukin 3, erythropoietin, and interleukin 2. We have examined the relationship of p97 to the protein tyrosine kinase Fes in the GM-CSF signal transduction pathway in erythroid and myeloid cell lines. GM-CSF stimulation of three different cell lines induced tyrosine phosphorylation of p97 as well as a number of other phosphotyrosylproteins. Although each cell line expressed the proto-oncogene product Fes, antisera specific for Fes did not recognize p97 in immunoblotting experiments. Furthermore, immunodepletion of Fes did not reduce the amount of p97 in GM-CSF-treated cells. Two-dimensional gel electrophoresis demonstrated that p97 and Fes have similar charge to mass ratios, and limited proteolytic mapping of p97 and Fes suggested that these proteins may be related but are not identical. Our studies demonstrate that p97 is not Fes but is probably a Fes-related protein.

Hck expression correlates with granulocyte-macrophage colony-stimulating factor-induced proliferation in HL-60 cells.

The human myeloid cell line HL-60 expresses approximately 300 high-affinity granulocyte-macrophage colony-stimulating factor receptors (GM-CSFRs), yet treatment of these cells with GM-CSF does not result in enhanced cellular proliferation or increases in protein tyrosine phosphorylation. In contrast, GM-CSF induces rapid increases in protein tyrosine phosphorylation and proliferative responses in HL-60 cells pretreated for 3 days in dimethyl sulfoxide (DMSO). Similarly, HL-60 cells pretreated with retinoic acid or 1,25 dihydroxyvitamin D3 were also capable of responding to GM-CSF. Interestingly, each of these treatments resulted in increased expression of the src-like tyrosine kinase hck. Stimulation with GM-CSF increased hck autophosphorylation in DMSO-treated HL-60 cells, suggesting that hck is a component of the GM-CSF signal transduction pathway. To determine if hck has a role in the DMSO-induced recoupling of the GM-CSFR, we overexpressed hck in HL-60 cells. The resulting cell line (HL-60/hck) expresses hck mRNA and protein at levels comparable with DMSO-treated HL-60 cells. Stimulation of HL-60/hck cells with GM-CSF results in activation of hck, increases in protein tyrosine phosphorylation, and increased proliferation. These results show that cytokine receptors can exist in an uncoupled form and suggest that in HL-60 cells, appropriate levels of the src-like tyrosine kinase hck are critical for functional coupling of the GM-CSFR to biologic responses.