Hematopoietic cell phosphatase associates with the interleukin-3 (IL-3) receptor beta chain and down-regulates IL-3-induced tyrosine phosphorylation and mitogenesis.

Hematopoietic cell phosphatase (HCP) is a tyrosine phosphatase with two Src homology 2 (SH2) domains that is predominantly expressed in hematopoietic cells, including cells whose growth is regulated by interleukin-3 (IL-3). The potential effects of HCP on IL-3-induced protein tyrosine phosphorylation and growth regulation were examined to assess the role of HCP in hematopoiesis. Our studies demonstrate that, following ligand binding, HCP specifically associates with the beta chain of the IL-3 receptor through the amino-terminal SH2 domain of HCP, both in vivo and in vitro, and can dephosphorylate the receptor chain in vitro. The effects of increasing or decreasing HCP levels in IL-3-dependent cells were assessed with dexamethasone-inducible constructs containing an HCP cDNA in sense and antisense orientations. Increased HCP levels were found to reduce the levels of IL-3-induced tyrosine phosphorylation of the receptor and to dramatically suppress cell growth. Conversely, decreasing the levels of HCP increased IL-3-induced tyrosine phosphorylation of the receptor and marginally increased growth rate. These results support a role for HCP in the regulation of hematopoietic cell growth and begin to provide a mechanistic explanation for the dramatic effects that the genetic loss of HCP, which occurs in motheaten (me) and viable motheaten (mev) mice, has on hematopoiesis.     

Protein tyrosine phosphatase containing SH2 domains: characterization, preferential expression in hematopoietic cells, and localization to human chromosome 12p12-p13.

Protein tyrosine phosphorylation has been implicated in the growth and functional responses of hematopoietic cells. Recently, approaches have been developed to characterize the protein tyrosine phosphatases that may contribute to regulation of protein tyrosine phosphorylation. One novel protein tyrosine phosphatase was expressed predominantly in hematopoietic cells. Hematopoietic cell phosphatase encodes a 68-kDa protein that contains a single phosphatase conserved domain. Unlike other known protein tyrosine phosphatases, hematopoietic cell phosphatase contains two src homology 2 domains. We also cloned the human homolog, which has 95% amino acid sequence identity. Both the murine and human gene products have tyrosine-specific phosphatase activity, and both are expressed predominantly in hematopoietic cells. Importantly, the human gene maps to chromosome 12 region p12-p13. This region is associated with rearrangements in approximately 10% of cases of acute lymphocytic leukemia in children.     

Induction of tyrosine phosphorylation by the erythropoietin receptor correlates with mitogenesis.

A role for tyrosine phosphorylation in the signal-transducing mechanisms of several hematopoietic growth factors has been hypothesized. To extend these observations, we have examined the effects of erythropoietin (Epo) on tyrosine phosphorylation in an Epo-responsive cell that was obtained by transfecting the murine erythropoietin receptor (EpoR) into an interleukin-3 (IL-3)-dependent cell line. By two-dimensional analysis of phosphotyrosine-containing proteins isolated with a monoclonal antibody (1G2) against phosphotyrosine, Epo and IL-3 were found to rapidly induce tyrosine phosphorylation of comparable substrates of 92, 70, and 56 kDa. In addition, Epo uniquely induced phosphorylation of a 72-kDa substrate while IL-3 uniquely induced phosphorylation of a 140-kDa substrate. Immunoprecipitation and mixing experiments indicated that the 72-kDa substrate may represent a small fraction of the EpoR. To explore the significance of tyrosine phosphorylation, we generated two mutants of the EpoR that lacked 108 or 146 amino acids at their carboxyl termini. In addition we constructed an internally deleted mutant that lacked 20 amino acids in a region of sequence homology with the IL-2 receptor beta chain. Although all mutants were expressed at comparable levels and had comparable binding affinities for Epo, only the mutant lacking 108 amino acids at the carboxyl terminus retained significant mitogenic activity or the ability to induce tyrosine phosphorylation.     

Association of hematopoietic cell phosphatase with c-Kit after stimulation with c-Kit ligand.

Protein tyrosine phosphorylation and dephosphorylation have been implicated in the growth and functional responses of hematopoietic cells. Recent studies have identified a novel protein tyrosine phosphatase, termed hematopoietic cell phosphatase (HCP) or PTP1C, that is predominantly expressed in hematopoietic cells. HCP encodes a cytoplasmic phosphatase that contains two src homology 2 (SH2) domains. Since SH2 domains have been shown to target the association of signal-transducing molecules with activated growth factor receptors containing intrinsic protein kinase activity, we assessed the association of HCP with two hematopoietic growth factor receptors, c-Kit and c-Fms. The results demonstrate that HCP transiently associates with ligand-activated c-Kit but not c-Fms and that this association occurs through the SH2 domains. In both colony-stimulating factor 1- and stem cell factor-stimulated cells, there is a marginal increase in tyrosine phosphorylation of HCP. Lastly, HCP can dephosphorylate autophosphorylated c-Kit and c-Fms in in vitro reactions. The potential role of HCP in stem cell factor signal transduction is discussed.     

The survival function of the Bcr-Abl oncogene is mediated by Bad-dependent and -independent pathways: roles for phosphatidylinositol 3-kinase and Raf

The Bcr-Abl tyrosine kinase constitutively activates cytokine signal transduction pathways that stimulate growth and prevent apoptosis in hematopoietic cells. The antiapoptotic action of interleukin-3 (IL-3) has been linked to a signaling pathway which inactivates the proapoptotic protein Bad by phosphorylation through kinases such as Akt and Raf. Here we report also that expression of Bcr-Abl leads to phosphorylation of Bad in hematopoietic cells. Bad phosphorylation induced by Bcr-Abl is kinase dependent, requires phosphatidylinositol 3-kinase (PI3-kinase), and mitochondrial targeting of Raf, and occurs independently of Erk. The ability of Bcr-Abl to confer cytokine-independent survival to hematopoietic cells was compromised by inhibitors of PI3-kinase, as well as by a dominant negative form of Raf targeted to the mitochondria. Furthermore, when the capacity of Bcr-Abl to phosphorylate Bad was completely blocked by dominant negative Raf, a subpopulation of cells remained viable, providing evidence for Bad-independent survival pathways. This alternative survival pathway remained PI3-kinase dependent. Finally, Bcr-Abl, but not IL-3, inhibited the proapoptotic activity of overexpressed Bad. We conclude that the antiapoptotic function of Bcr-Abl is mediated through pathways involving PI3-kinase and Raf and that survival can occur in the absence of Bad phosphorylation.     

Critical role for hematopoietic cell kinase (Hck)-mediated phosphorylation of Gab1 and Gab2 docking proteins in interleukin 6-induced proliferation and survival of multiple myeloma cells

Interleukin-6 (LI-6) is a known growth and survival factor in multiple myeloma via activation of extracellular signal-regulated kinase and phosphatidylinositol 3-kinase signaling cascade. In this report we show that Grb2-associated binder (Gab) family adapter proteins Gab1 and Gab2 are expressed by multiple myeloma cells; and that interleukin-6 induces their tyrosine phosphorylation and association with downstream signaling molecules. We further demonstrate that these events are Src family tyrosine kinase-dependent and specifically identify the role of hematopoietic cell kinase (Hck) as a new Gab family adapter protein kinase. Conversely, inhibition of Src family tyrosine kinases by the pyrazolopyrimidine PP2, as in kinase-inactive Hck mutants, significantly reduces IL-6-triggered activation of extracellular signal-regulated kinase and AKT-1, leading to significant reduction of multiple myeloma cell proliferation and survival. Taken together, these results delineate a key role for Hck-mediated phosphorylation of Gab1 and Gab2 docking proteins in IL-6-induced proliferation and survival of multiple myeloma cells and identify tyrosine kinases and downstream adapter proteins as potential new therapeutic targets in multiple myeloma.     

c-fps/fes protein-tyrosine kinase is implicated in a signaling pathway triggered by granulocyte-macrophage colony-stimulating factor and interleukin-3.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3) are hematopoietic growth factors which stimulate the proliferation and differentiation of myeloid progenitor cells. There is a considerable degree of overlap in target cell specificity and the functional effects of GM-CSF and IL-3. GM-CSF and IL-3 induce a nearly identical pattern of protein-tyrosine phosphorylation in certain cell lines, although their receptors have no kinase domains. Furthermore, their receptor complexes share one subunit (designated as beta). These observations raise the possibility that GM-CSF and IL-3 have a common signaling pathway. Here we show that both GM-CSF and IL-3 induce tyrosine phosphorylation and kinase activity of the c-fps/fes proto-oncogene product (p92c-fes), a non-receptor protein-tyrosine kinase, in a human erythro-leukemia cell line, TF-1, which requires GM-CSF or IL-3 for growth. In addition, GM-CSF induces physical association between p92c-fes and the beta chain of the GM-CSF receptor. p92c-fes is therefore a possible signal transducer of several hematopoietic growth factors including GM-CSF and IL-3 through the common beta chain.     

Interleukin 3-specific tyrosine phosphorylation of a membrane glycoprotein of Mr 150,000 in multi-factor-dependent myeloid cell lines.

Tyrosine phosphorylation of cellular proteins induced by various hematopoietic growth factors such as interleukin 3 (IL3), granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 4 (IL4) was studied in several multi-factor-dependent myeloid cell lines. Among the growth factors, IL3 specifically induced rapid tyrosine phosphorylation of a membrane glycoprotein of mol. wt 150 kd (gpp150) in the IL3-dependent cell lines, IC2 and DA-1. The IL3-induced tyrosine phosphorylation of gpp150 was detected within 30 s, reached a maximum at 3 min and decreased thereafter. The concentration of IL3 required for half-maximum stimulation of gpp150 tyrosine phosphorylation with 2.5 x 10(6)/ml cells was approximately 200 pM, which is the same as the dissociation constant for 125I-labeled IL3 binding. gpp150 was constitutively phosphorylated on tyrosine residue(s) in growth factor independent variants, IC2Tr and DA-1Tr, derived from IC2 and DA-1 respectively. Neither variant synthesized IL3. The present findings suggest that tyrosine phosphorylation of gpp150 is a critical event involved in both IL3-dependent and -independent growth.     

Ligand-independent activation of the platelet-derived growth factor beta receptor: requirements for bovine papillomavirus E5-induced mitogenic signaling.

The E5 protein of bovine papillomavirus type 1 binds to and activates the endogenous platelet-derived growth factor (PDGF) beta receptor in fibroblasts, resulting in cell transformation. We have developed a functional assay to test the ability of PDGF beta receptor mutants to mediate a mitogenic signal initiated by the E5 protein. Lymphoid Ba/F3 cells are strictly dependent on interleukin-3 for growth, but coexpression of the wild-type PDGF beta receptor and the E5 or v-sis-encoded protein generated a mitogenic signal which allowed Ba/F3-derived cells to proliferate in the absence of interleukin-3. In these cells, the E5 protein bound to and caused increased tyrosine phosphorylation of both the mature and the precursor forms of the wild-type PDGF beta receptor. The tyrosine kinase activity of the receptor was necessary for E5-induced receptor tyrosine phosphorylation and mitogenic activity but not for complex formation with the E5 protein. In contrast, the PDGF-binding domain of the receptor was not required for complex formation with the E5 protein, E5-induced tyrosine phosphorylation or mitogenic activity, demonstrating that E5-mediated receptor activation is ligand independent. Analysis of receptor mutants lacking various combinations of tyrosine phosphorylation sites revealed that the E5 and v-sis-encoded proteins display similar requirements for signaling and suggested that the wild-type PDGF beta receptor can generate multiple independent mitogenic signals. Importantly, these mutants dissociated two activities of the PDGF beta receptor tyrosine kinase, both of which are required for sustained mitogenic signaling: (i) receptor autophosphorylation and creation of binding sites for SH2 domain-containing proteins and (ii) phosphorylation of substrates other than the receptor itself.     

Evidence for the involvement of JAK/STAT pathway in the signaling mechanism of interleukin-17.

Interleukin-17 is a T-cell-derived pro-inflammatory cytokine, exhibiting multiple biological activities in a variety of cells and believed to fine tune all general phases of hematopoietic response. However, the signaling mechanism of this novel cytokine remains unknown. Here, we report for the first time that the early signaling events triggered by interleukin-17 involve tyrosine phosphorylation of several members of the JAK and STAT proteins in human U937 monocytic leukemia cells. Immunoprecipitation with specific antibodies followed by Western blot analysis with antiphosphotyrosine antibody has shown that in U937 cells, interleukin-17 induces time-dependent stimulation of tyrosine phosphorylation of JAK 1, 2 and 3, Tyk 2 and STAT 1, 2, 3 and 4 within 0.5 to 30 min. Interleukin-17-mediated tyrosine phosphorylation of these proteins strongly suggests that the JAK/STAT signaling pathway may play a major role in transducing signals from interleukin-17 receptors to the nucleus.     

Erythropoietin induces the tyrosine phosphorylation of its own receptor in human erythropoietin-responsive cells.

Using the human erythropoietin-responsive hematopoietic cell line UT-7, we showed that erythropoietin (Epo) rapidly and specifically induced the tyrosine phosphorylation of its own receptor (M(r) 75,000) and increased the tyrosine phosphorylation of other proteins of M(r) 140,000, 120,000, 95,000, 60,000, 57,000, and 42,000. Neither granulocyte-macrophage colony-stimulating factor, interleukin 3, interleukin 6, nor the kit ligand induced the phosphorylation of the M(r) 75,000 receptor protein, although these growth factors induced the phosphorylation of other proteins. Cross-linking experiments using 125I-Epo indicated that the UT-7 cells expressed three Epo receptor subunits, of M(r) 100,000, 85,000, and 75,000, among which only the M(r) 75,000 subunit was tyrosine-phosphorylated following activation with Epo.     

Tyrosine phosphorylation of common and specific sets of cellular proteins rapidly induced by insulin, insulin-like growth factor I, and epidermal growth factor in an intact cell

KB cells respond to insulin and insulin-like growth factor I (IGF-I) in a closely similar way (induction of membrane ruffling, stimulation of pinocytosis, and amino acid transport) but respond to epidermal growth factors (EGF) in a similar but distinct way. In the KB cells, using phosphotyrosine-specific antibody we have found that: the receptors for insulin (beta subunit), IGF-I (beta subunit), and EGF undergo tyrosine phosphorylation as early as 10 s after addition of their respective ligands; a 185-kDa protein is rapidly (less than 10 s) tyrosine phosphorylated by insulin and IGF-I through their respective receptor kinases but not EGF; tyrosine phosphorylation of a 190-kDa glycoprotein is rapidly (less than 10 s) induced by EGF through EGF receptor kinase; and tyrosine phosphorylation of a 240-kDa protein is stimulated within 30 s by all three growth factors. These patterns of tyrosine phosphorylation could be causally related to biological responses induced by the three growth factors.     

Tyrosine phosphorylation of a common 57-kDa protein in growth factor-stimulated and -transformed cells.

Protein tyrosine phosphorylation was studied in macrophages and fibroblasts to identify putative components of post-receptor mitogenic pathways that might be functionally conserved in different cell types. Nondenaturing conditions were established for the approximately quantitative recovery of anti-phosphotyrosine antibody (alpha PY)-reactive proteins from cells. A common, 57-kDa alpha PY-reactive protein was identified by V8 protease peptide mapping in colony-stimulating factor-1 (CSF-1)- or granulocyte-macrophage colony-stimulating factor (GM-CSF)-stimulated BAC1.2F5 macrophages, in platelet-derived growth factor-stimulated NIH-3T3 cells, and in CSF-1-stimulated NIH-3T3 cells expressing the c-fms/CSF-1 receptor. The 57-kDa protein was phosphorylated on serine and tyrosine and was the only alpha PY-reactive protein band whose phosphorylation was reproducibly increased in GM-CSF-stimulated cells. The effect of the growth factors on the tyrosine phosphorylation of the 57-kDa protein could be mimicked by treatment of the cells with orthovanadate, a phosphotyrosine protein phosphatase inhibitor. In the absence of growth factors, tyrosine phosphorylation of the 57-kDa protein was higher in v-fms or c-fms (F969, S301)-transformed NIH-3T3 cells than in untransformed NIH-3T3 (c-fms) and NIH-3T3 (c-fms, F969) cells. These data indicate that the 57-kDa protein is a common target for growth factor-stimulated tyrosine phosphorylation and potentially important for growth factor mitogenic signaling.     

PC12-E2 cells: A stable variant with altered responses to growth factor stimulation

A variant cell line, designated E2, characterized by more rapid responses to nerve growth factor (NGF) and basic fibroblast growth factor (bFGF) and markedly more robust responses to interleukin-6 and 8-Br-cAMP, has been subcloned from the rat PC12 cell line. The enhanced responsiveness to NGF in E2 cells is not due to receptor overexpression as judged by TrkA protein levels and tyrosine kinase activity, but may be associated with the increased and prolonged tyrosine phosphorylation of ERK1 (extracellular signal regulated kinase 1) and ERK2. The rapid morphological differentiation induced by different growth factors in E2 cells is constitutively express some differentiation-associated molecules that allow direct entry into the neuronal program. © 1995 Wiley-Liss, Inc.     

Differential effects of interleukin-2 and interleukin-4 on protein tyrosine phosphorylation in factor-dependent murine T cells

Interleukin-2 (IL-2) is a requisite factor for growth and proliferation of IL-2-dependent T cells. At present, the mechanism by which the high-affinity IL-2-IL-2 receptor interaction transmits a mitogenic signal to the cellular interior remains unclear. In this report we have used three murine T cell clones to demonstrate that IL-2 stimulates rapid tyrosine phosphorylation of several proteins. Two of these clones, CTLL-2 and CT6, exhibit a cytotoxic T cell phenotype, while the third, HT-2, was derived from a helper T cell line. All three T cell clones proliferated in response to IL-2 stimulation, but HT-2 cells also proliferated in response to interleukin-4 (IL-4). We comparatively examined the effects of IL-2 and IL-4 on protein tyrosine phosphorylation in these cells by immunoaffinity purification of phosphotyrosyl substrates with an anti-phosphotyrosine monoclonal antibody. Stimulation with concentrations of IL-2 resulting in maximal (10-30 U/ml) or sub-maximal (1-5 U/ml) proliferation caused the rapid tyrosine phosphorylation of 97 and 57 kDa proteins in all three cell lines. The 97 kDa protein was localized in the cytosol, while the 57 kDa protein was detected in both cytosolic and crude membrane fractions. IL-2-dependent tyrosine phosphorylation of an 86 kDa cytosolic protein was observed only in CT6 cells. Tyrosine phosphorylation of 22, 23 and 200 kDa proteins was also observed, but only in the cytotoxic T cell clones. Phosphoamino acid analyses revealed that the 97, 86 and 57 kDa proteins contained phosphotyrosine and phosphoserine residues. Concentrations of IL-2 below the threshold concentration for induction of a proliferative response correspondingly failed to stimulate protein tyrosine phosphorylation. In contrast, growth stimulation of HT-2 cells by IL-4 was not preceded by early changes in protein tyrosine phosphorylation, suggesting that protein tyrosine phosphorylation may not be essential for the induction of IL-4-dependent cell-cycle progression. These results demonstrate that high-affinity IL-2 receptors are coupled to tyrosine kinase activity(s) in T cells. However, the failure of IL-4 to stimulate protein tyrosine phosphorylation in the same cells indicates that enhanced protein tyrosine phosphorylation may not be requisite for growth factor-dependent T cell proliferation.     

Inhibition of interleukin 3 and granulocyte-macrophage colony-stimulating factor stimulated increase of active ras.GTP by herbimycin A, a specific inhibitor of tyrosine kinases.

Interleukin 3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulate the proliferation of several kinds of cultured hematopoietic cell lines. Growth signals from IL-3 and GM-CSF cause accumulation of active Ras.GTP complexes in PT18 mouse mast cell line (Satoh, T., Nakafuku, M., Miyajima, A., and Kaziro, Y. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 3314-3318). In this paper we describe the effect of herbimycin A, a tyrosine kinase-specific inhibitor, on the activation of Ras. The increase of Ras.GTP induced by IL-3 and GM-CSF diminished in cells treated with 0.5 approximately 1 micrograms/ml of herbimycin A for 24 h prior to the addition of the growth factors. Under this condition, the extent of phosphorylation on tyrosine residues of proteins decreased. However, the activity of cAMP-dependent protein kinase and protein kinase C did not change. Growth of cells in the presence of IL-3 or GM-CSF was also completely inhibited. These observations suggest that tyrosine kinases are involved in the pathways between IL-3 and GM-CSF receptors and Ras and that they are essential for the growth stimulated by these growth factors.     

Vav2 activates Rac1, Cdc42, and RhoA downstream from growth factor receptors but not beta1 integrins

The Rho family of GTPases plays a major role in the organization of the actin cytoskeleton. These G proteins are activated by guanine nucleotide exchange factors that stimulate the exchange of bound GDP for GTP. In their GTP-bound state, these G proteins interact with downstream effectors. Vav2 is an exchange factor for Rho family GTPases. It is a ubiquitously expressed homologue of Vav1, and like Vav1, it has previously been shown to be activated by tyrosine phosphorylation. Because Vav1 becomes tyrosine phosphorylated and activated following integrin engagement in hematopoietic cells, we investigated the tyrosine phosphorylation of Vav2 in response to integrin-mediated adhesion in fibroblasts and epithelial cells. However, no tyrosine phosphorylation of Vav2 was detected in response to integrin engagement. In contrast, treating cells with either epidermal growth factor or platelet-derived growth factor stimulated tyrosine phosphorylation of Vav2. We have examined the effects of overexpressing either wild-type or amino-terminally truncated (constitutively active) forms of Vav2 as fusion proteins with green fluorescent protein. Overexpression of either wild-type or constitutively active Vav2 resulted in prominent membrane ruffles and enhanced stress fibers. These cells revealed elevated rates of cell migration that were inhibited by expression of dominant negative forms of Rac1 and Cdc42. Using a binding assay to measure the activity of Rac1, Cdc42, and RhoA, we found that overexpression of Vav2 resulted in increased activity of each of these G proteins. Expression of a carboxy-terminal fragment of Vav2 decreased the elevation of Rac1 activity induced by epidermal growth factor, consistent with Vav2 mediating activation of Rac1 downstream from growth factor receptors.     

Phorbol 12-myristate 13-acetate inhibits granulocyte-macrophage colony stimulating factor-induced protein tyrosine phosphorylation in a human factor-dependent hematopoietic cell line.

The human myeloid cell line MO7 requires either granulocyte-macrophage colony stimulating factor (GM-CSF) or interleukin 3 (IL-3) for proliferation. We have previously shown that both GM-CSF and IL-3 transiently induce tyrosine phosphorylation of a number of proteins, including two cytosolic proteins, p93 and p70, which are maximally phosphorylated 5-15 min after addition of growth factor to factor-deprived cells. GM-CSF-induced proliferation of MO7 cells was found to be inhibited by two activators of protein kinase C, phorbol 12-myristate 13-acetate (PMA) and bryostatin-1. PMA did not affect surface expression or affinity of the GM-CSF receptor but significantly inhibited GM-CSF- or IL-3-induced tyrosine phosphorylation of p93 and p70. In contrast, PMA augmented GM-CSF-induced tyrosine phosphorylation of another protein, p42. Pretreatment of cells with sodium orthovanadate to inhibit protein tyrosine phosphatases (PTPase) partially reversed the inhibitory effects of PMA. These results suggest that one aspect of GM-CSF and IL-3 signal transduction, protein tyrosine phosphorylation, can be inhibited by a mechanism which does not involve receptor down-regulation, and may involve either receptor down-regulation, and may involve either inhibition of a receptor-activated tyrosine kinase, activation of a protein tyrosine phosphatase, or both. This mechanism could be important in exerting control of proliferation of some types of hematopoietic cells.     

c-Kit-kinase induces a cascade of protein tyrosine phosphorylation in normal human melanocytes in response to mast cell growth factor and stimulates mitogen-activated protein kinase but is down-regulated in melanomas.

The proto-oncogene c-Kit, a transmembrane receptor tyrosine kinase, is an important regulator of cell growth whose constitutively active oncogenic counterpart, v-kit, induces sarcomas in cats. Mutations in murine c-kit that reduce the receptor tyrosine kinase activity cause deficiencies in the migration and proliferation of melanoblasts, hematopoietic stem cells, and primordial germ cells. We therefore investigated whether c-Kit regulates normal human melanocyte proliferation and plays a role in melanomas. We show that normal human melanocytes respond to mast cell growth factor (MGF), the Kit-ligand that stimulates phosphorylation of tyrosyl residues in c-Kit and induces sequential phosphorylation of tyrosyl residues in several other proteins. One of the phosphorylated intermediates in the signal transduction pathway was identified as an early response kinase (mitogen-activated protein [MAP] kinase). Dephosphorylation of a prominent 180-kDa protein suggests that MGF also activates a phosphotyrosine phosphatase. In contrast, MGF did not induce proliferation, the cascade of protein phosphorylations, or MAP kinase activation in the majority of cells cultured from primary nodular and metastatic melanomas that grow independently of exogenous factors. In the five out of eight human melanoma lines expressing c-kit mRNAs, c-Kit was not constitutively activated. Therefore, although c-Kit-kinase is a potent growth regulator of normal human melanocytes, its activity is not positively associated with malignant transformation.