A short N-terminal sequence of PTEN controls cytoplasmic localization and is required for suppression of cell growth.

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is an important negative regulator of cell growth and a tumor suppressor. Its growth-attenuating activity is based on the dephosphorylation of phosphatidylinositol 3,4,5-trisphosphate (PIP3), an essential second messenger for the phosphoinositide 3-kinase/Akt signaling pathway. This activity may require localization of PTEN to cytoplasmic membranes. Yet PTEN can also localize to the cell nucleus where its functions remain unclear. Here we present data that define a short sequence in the N-terminal region of PTEN required for cytoplasmic localization. We will refer to this sequence as cytoplasmic localization signal (CLS). It could function as a non-canonical signal for nuclear export or as a cytoplasmic retention signal of PTEN. Mutations within the CLS induce nuclear localization and impair growth suppressive activities of PTEN while preserving lipid phosphatase activity. We propose that nuclear localization of PTEN is not compatible with plasma membrane-targeted growth suppressive functions of PTEN.

Plasminogen activator inhibitor-1 promotes angiogenesis by stimulating endothelial cell migration toward fibronectin.

Increased expression of plasminogen activator inhibitor-1 (PAI-1) in cancer patients is associated with unfavorable outcome, and the reason for this paradox has been poorly understood. We have previously reported elevated levels of PAI-1 in primary tumors of advanced neuroblastomas (Y. Sugiura et al., Cancer Res., 59: 1327-1336, 1999). Here we demonstrate that PAI-1 is coexpressed with the angiogenesis marker alpha(v)beta3 integrin in blood vessels of primary neuroblastoma tumors, suggesting that PAI-1 plays a role in angiogenesis. Using human brain microvascular endothelial cells (HBMECs), we found that PAI-1 inhibits alpha(v)beta3 integrin-mediated cell adhesion to vitronectin but promotes alpha5beta1-mediated migration from vitronectin toward fibronectin. Inhibition of vitronectin adhesion by PAI-1 did not induce HBMEC apoptosis. PAI-1 also inhibited endothelial tube formation on Matrigel in the presence of vitronectin but had a stimulatory effect in the presence of fibronectin. This effect of PAI-1 on microvascular endothelial cells is primarily related to the ability of PAI-1 to bind to vitronectin via its NH2-terminal domain and to interfere with cell adhesion to vitronectin. We propose that PAI-1 acts as a positive switch for angiogenesis by promoting endothelial cell migration away from their vitronectin-containing perivascular space toward fibronectin-rich tumor tissue. These observations provide a novel explanation for the enhancing effect of PAI-1 in cancer progression.

Integrins alpha(v)beta3 and alpha(v)beta5 are expressed by endothelium of high-risk neuroblastoma and their inhibition is associated with increased endogenous ceramide.

Inhibition of the RGD-binding integrins, alpha(v)beta3 and alpha(v)beta5, prevents endothelial cell anchorage and induces endothelial apoptosis, which results in disruption of tumor angiogenesis and inhibition of tumor growth in animal models. In this study, we demonstrate by immunohistochemical analysis that integrin alpha(v)beta3 was expressed by 61% (mean) of microvessels in high-risk neuroblastomas (stage IV and MYCN-amplified stage III; n = 28) but only by 18% (mean) of microvessels in low-risk tumors (stages I and II and non-MYCN-amplified stage III; n = 12). Integrin alpha(v)beta5 was found on 60% (mean) of microvessels in 21 Stage IV tumors. These data suggest that neuroblastomas may be targeted for antiangiogenic treatment directed against endothelial integrins alpha(v)beta3 and alpha(v)beta5. In cell culture, inhibition of integrin-dependent endothelial cell anchorage to vitronectin by RGDfV, an RGD function-blocking cyclic peptide, induced apoptosis in bovine brain endothelial cells compared with the control peptide, RADfV (37.5% versus 8.7%, respectively), as detected by chromatin condensation and nuclear fragmentation. Treatment with RGDfV but not with RADfV, which prevented attachment of endothelial cells to vitronectin or fibronectin, was associated with up to a 50% increase in endogenous ceramide, a lipid second messenger that can mediate cell death. Furthermore, exogenous C2-ceramide was cytotoxic to bovine brain endothelial cells and induced activation of C-jun N-terminal kinase (JNK), a MAP kinase that can be activated in stress-induced apoptosis pathways. This suggests that ceramide may function in detachment-induced endothelial cell apoptosis, originating from inhibition of vitronectin binding to integrins such as alpha(v)beta3 and alpha(v)beta5. This is the first report to demonstrate expression of integrins alpha(v)beta3 and alpha(v)beta5 by microvascular endothelium of a childhood tumor and association of their expression with neuroblastoma aggressiveness. Furthermore, our data provide the first suggestion that inhibition of endothelial cell anchorage, resulting from specific blockade of RGD-binding integrins, increases endogenous ceramide, which may contribute to endothelial cell death.

Cbl functions downstream of Src kinases in Fc gamma RI signaling in primary human macrophages.

Cbl is a cytosolic protein that is rapidly tyrosine phosphorylated in response to Fc receptor activation and binds to the adaptor proteins Grb2, CrkL, and Nck. A few reports describe Cbl interactions in primary human hematopoietic cells. We show evidence that Cbl participates in signaling initiated by Fc gammaRI receptor cross-linking in human primary macrophages, and functions downstream of Src family kinases in this pathway. Fc gammaRI stimulation in human macrophages was associated with rapid and transient tyrosine phosphorylation of the Cbl adaptor protein. Immunoprecipitated Cbl was complexed with several tyrosine phosphorylated proteins, the most prominent of which was a 38-kDa band identified as the CrkL adaptor protein. CrkL associated with tyrosine-phosphorylated Cbl and itself became tyrosine phosphorylated after Fc gammaRI cross-linking. SLP-76, a recently cloned Grb2-associated protein, was strongly tyrosine phosphorylated after Fc gammaRI stimulation and was associated with both Cbl and Grb2. Grb2 and Cbl binding to SLP-76 were inducible after Fc gammaRI stimulation of the macrophages. Nck was inducibly bound to Cbl after Fc gammaRI stimulation, whereas Grb2 was constitutively associated with it. Shc was also inducibly tyrosine phosphorylated and bound to Grb2 after Fc gammaRI stimulation of the macrophages. PP1, a specific inhibitor of Src kinases, inhibited the Fc gammaRI-induced respiratory burst, as well as the tyrosine phosphorylation of Cbl and its inducible association with CrkL. These results suggest a fundamental role for the tyrosine phosphorylation of Cbl, CrkL, SLP-76, and Shc and the association of Cbl with CrkL, SLP-76, and Nck in Fc gammaRI signaling in human macrophages. Experiments performed with PP1, the specific Src kinase inhibitor, demonstrate the first evidence that Cbl and the Cbl-Crkl interaction are downstream targets for myeloid Src kinases required for the activation of myeloid NADPH oxidase activity.

Treatment of collagen induced arthritis in DBA/1 mice with L-asparaginase.

OBJECTIVE: To evaluate the safety and efficacy of L-asparaginase as an immunosuppressive agent in a mouse model of rheumatoid arthritis. METHODS: Male DBA/1 mice with collagen-induced arthritis (CIA) were treated at different intervals with various doses of native and pegylated L-asparaginase from E. coli. The mice were observed for 4 weeks during which time arthritis was scored. Outcome parameters included effect on severity and progression of established arthritis as well as prevention of disease. In addition, X-rays from the affected joints were obtained for comparison. RESULTS: Both native L-asparaginase at a dose of 50 IU/injection intraperitoneally three days a week and pegylated asparaginase (PEG-L-asparaginase) at a dose of 25 IU/injection twice a week, significantly reduced the mean arthritic score (MAS) in mice with established arthritis (p < 0.001 for PEG-L-asparaginase). When native L-asparaginase was administered before the onset of arthritis (days 14-post immunization) the number of mice developing arthritis as well as the number of arthritic paws and the severity of arthritis in the treatment group were significantly decreased (p < 0.0001). Significant differences were found in the X-ray evaluation between treated and control mice. None of the animals died due to drug related events or showed signs of asparaginase induced toxicity. CONCLUSION: Our data provide the first direct evidence that L-asparaginase is a potent antiarthritic agent and may represent an effective second line agent for future treatment studies in juvenile and adult rheumatoid arthritis.

Distinct roles for fibroblast growth factor signaling in cerebellar development and medulloblastoma.

Cerebellar granule neurons are the most abundant neurons in the brain, and a critical element of the circuitry that controls motor coordination and learning. In addition, granule neuron precursors (GNPs) are thought to represent cells of origin for medulloblastoma, the most common malignant brain tumor in children. Thus, understanding the signals that control the growth and differentiation of these cells has important implications for neurobiology and neurooncology. Our previous studies have shown that proliferation of GNPs is regulated by Sonic hedgehog (Shh), and that aberrant activation of the Shh pathway can lead to medulloblastoma. Moreover, we have demonstrated that Shh-dependent proliferation of GNPs and medulloblastoma cells can be blocked by basic fibroblast growth factor (bFGF). But while the mitogenic effects of Shh signaling have been confirmed in vivo, the inhibitory effects of bFGF have primarily been studied in culture. Here, we demonstrate that mice lacking FGF signaling in GNPs exhibit no discernable changes in GNP proliferation or differentiation. In contrast, activation of FGF signaling has a potent effect on tumor growth: treatment of medulloblastoma cells with bFGF prevents them from forming tumors following transplantation, and inoculation of tumor-bearing mice with bFGF markedly inhibits tumor growth in vivo. These results suggest that activators of FGF signaling may be useful for targeting medulloblastoma and other Shh-dependent tumors.

Phase I pharmacokinetic and pharmacodynamic study of the pan-PI3K/mTORC vascular targeted pro-drug SF1126 in patients with advanced solid tumours and B-cell malignancies.

BACKGROUND: SF1126 is a peptidic pro-drug inhibitor of pan-PI3K/mTORC. A first-in-human study evaluated safety, dose limiting toxicities (DLT), maximum tolerated dose (MTD), pharmacokinetics (PK), pharmacodynamics (PD) and efficacy of SF1126, in patients with advanced solid and B-cell malignancies. PATIENTS AND METHODS: SF1126 was administered IV days 1 and 4, weekly in 28day-cycles. Dose escalation utilised modified Fibonacci 3+3. Samples to monitor PK and PD were obtained. RESULTS: Forty four patients were treated at 9 dose levels (90-1110 mg/m(2)/day). Most toxicity was grade 1 and 2 with a single DLT at180 mg/m(2) (diarrhoea). Exposure measured by peak concentration (C(max)) and area under the time-concentration curve (AUC(0-)(t)) was dose proportional. Stable disease (SD) was the best response in 19 of 33 (58%) evaluable patients. MTD was not reached but the maximum administered dose (MAD) was 1110 mg/m(2). The protocol was amended to enrol patients with CD20+ B-cell malignancies at 1110 mg/m(2). A CLL patient who progressed on rituximab [R] achieved SD after 2 months on SF1126 alone but in combination with R achieved a 55% decrease in absolute lymphocyte count and a lymph node response. PD studies of CLL cells demonstrated SF1126 reduced p-AKT and increased apoptosis indicating inhibition of activated PI3K signalling. CONCLUSION: SF1126 is well tolerated with SD as the best response in patients with advanced malignancies.

PI-3 kinase-PTEN signaling node: an intercept point for the control of angiogenesis.

Angiogenesis is tightly regulated by opposing mechanisms in mammalian cells and is controlled by the angiogenic switch. Other review articles have described a central role for the PTEN/PI-3 kinase/AKT signaling node in the coordinate control of cell division, tumor growth, apoptosis, invasion and cellular metabolism [1, 2]. In this review, we focus on literature that supports the PTEN/PI-3 kinase/AKT signaling node as a major control point for the angiogenic switch in both the on and off positions. We also discuss the rationale for designing small molecule drugs that target the PTEN/PI-3 kinase/AKT signaling node for therapeutic intervention. Our hypothesis is that, instead of inhibiting one cell surface receptor, such as VEGFR2 with bevacizumab (Avastin), thereby leaving a significant number of receptors free to pulse angiogenic signals, a more effective strategy may be to regulate signaling through an intercept node where redundant cell surface receptor signals converge to transmit important signaling events within the cell. This therapeutic configuration brings coordinate control over multiple cell surface receptors in concert with a physiologic response which may combine arrest of cell cycle progression with growth inhibition and the induction of genes involved in specialized functions such as movement, which are all required for the complex process of angiogenesis to occur in a temporal-spatial paradigm.

Protein-tyrosine kinase p72syk in Fc gamma RI receptor signaling.

In this report we show that gamma-interferon (IFN) induces the expression of the nonreceptor protein tyrosine kinase, p72syk, and that cross-linking the Fc gamma RI receptor in IFN-differentiated U937 cells (U937IF cells) results in the activation of syk kinase. We show that syk is tyrosine phosphorylated (12-fold increase) after Fc gamma RI cross-linking. In vitro kinase assays demonstrate that the specific kinase activity of syk increased eightfold after Fc gamma RI cross-linking. The activation of signal transduction through the Fc gamma RI receptor, as measured by the respiratory burst, is associated with the tyrosine phosphorylation and catalytic activation of the syk kinase. We show that syk coprecipitates with the gamma subunit of the Fc gamma RI, Fc gamma RI gamma. The data suggest that p72syk is involved in signal transduction through the Fc gamma RI receptor, involving the Fc gamma RI gamma subunit.

Serine/threonine phosphorylation of the gamma-subunit after activation of the high-affinity Fc receptor for immunoglobulin G.

In this report we show that interferon gamma treatment of U937 cells induces increased expression of the gamma-subunit of the high-affinity Fc receptor for IgG (Fc gamma RI). Interferon treatment results in a 10-fold increased expression of the gamma-subunit and induces expression of a phosphorylated form (gamma 1). The increased expression of the gamma-subunit correlates with its ability to transmit a signal via Fc gamma R, as measured by activation of the respiratory burst using insoluble immune complexes. During Fc gamma R activation, a mobility shift occurs in the phosphorylated form of this gamma 1-subunit. Phosphoamino acid analysis demonstrates that this gamma 1 subunit is threonine phosphorylated in resting differentiated U937 cells and becomes predominantly serine phosphorylated on Fc receptor activation. The mobility shift in the gamma-subunit can be induced by treating U937 cells with phorbol 12-myristate 13-acetate or by monoclonal antibody cross-linking of Fc gamma RI. Hence the gamma-subunit is serine phosphorylated in response to Fc gamma RI and protein kinase C activation. Therefore the gamma-subunit, initially described as a subunit of Fc epsilon RI, now appears to be involved in signal transduction via Fc gamma RI. The data also suggest that the gamma-subunit, in contrast with the zeta-subunit of the T-cell receptor-CD3 complex, is a substrate for serine/threonine kinase(s) in the cell. The serine phosphorylation of the gamma-subunit suggests a divergence of structure and function between the gamma-subunit and its homologue, the zeta-subunit of the T-cell receptor. Phosphorylation of the gamma-subunit on serine may play some regulatory role in Fc gamma RI signal transduction in myeloid cells.

A role for Shc, Grb2, and Raf-1 in FcgammaRI signal relay.

The activation of the serine/threonine kinase, Raf-1, serves to connect upstream protein tyrosine kinases to downstream signaling events. We previously reported that FcgammaRI stimulation of interferon gamma-differentiated U937 cells (termed U937IF cells) induces a mobility shift in Erk2. Herein, we report that cross-linking of FcgammaRI receptor in U937IF cells induces a marked tyrosine phosphorylation of Raf-1 (10-fold increase). Tyrosine phosphorylation of Raf-1 is induced by FcgammaRI activation and not by PMA (1 microg/ml), N-formyl-Met-Leu-Phe (1 microM), calcium ionophore (1 microM), thrombin (0.05 unit/ml), FcgammaRII, or FcgammaRIII stimulation. The kinetics of Raf-1 tyrosine phosphorylation is rapid, reaching peak levels 1-2 min after FcgammaRI activation, and the tyrosine phosphorylation of Raf-1 precedes the activation of the respiratory burst. FcgammaRI cross-linking induces the tyrosine phosphorylation of Shc; tyrosine-phosphorylated Shc binds to Grb2 forming a Shc-Grb2 complex. The data provide evidence that the FcgammaRI receptor signals via the upstream activation of nonreceptor protein tyrosine kinases, which leads to the subsequent activation of Ras family GTPases and serine/threonine kinases, Raf-1 and mitogen-activated protein kinase.

Functions of the major tyrosine phosphorylation site of the PDGF receptor beta subunit.

Two tyrosine phosphorylation sites in the human platelet-derived growth factor receptor (PDGFR) beta subunit have been mapped previously to tyrosine (Y)751, in the kinase insert, and Y857, in the kinase domain. Y857 is the major site of tyrosine phosphorylation in PDGF-stimulated cells. To evaluate the importance of these phosphorylations, we have characterized the wild-type (WT) and mutant human PDGF receptor beta subunits in dog kidney epithelial cells. Replacement of either Y751 or Y857 with phenylalanine (F) reduced PDGF-stimulated DNA synthesis to approximately 50% of the WT level. A mutant receptor with both tyrosines mutated was unable to initiate DNA synthesis, as was a kinase-inactive mutant receptor. Transmodulation of the epidermal growth factor receptor required Y857 but not Y751. We also tested the effects of phosphorylation site mutations on PDGF-stimulated receptor kinase activity. PDGF-induced tyrosine phosphorylation of two cellular proteins, phospholipase C gamma 1 (PLC gamma 1) and the GTPase activating protein of Ras (GAP), was assayed in epithelial cells expressing each of the mutant receptors. Tyrosine phosphorylation of GAP and PLC gamma 1 was reduced markedly by the F857 mutation but not significantly by the F751 mutation. Reduced kinase activity of F857 receptors was also evident in vitro. Immunoprecipitated WT receptors showed a two- to fourfold increase in specific kinase activity if immunoprecipitated from PDGF-stimulated cells. The F751 receptors showed a similar increase in activity, but F857 receptors did not. Our data suggest that phosphorylation of Y857 may be important for stimulation of kinase activity of the receptors and for downstream actions such as epidermal growth factor receptor transmodulation and mitogenesis.

Protein tyrosine phosphatase inhibitors in Fc gamma RI-induced myeloid oxidant signaling.

Fc-receptor stimulation in myeloid cells results in increased oxygen consumption, termed the respiratory burst, which is coupled to a rapid and transient increase in tyrosine phosphorylation of cellular proteins. In a previous paper in this journal we showed that the protein tyrosine phosphatase (PTPase) inhibitors sodium orthovanadate and phenylarsine oxide (PAO) block the Fc gamma RI-induced respiratory burst in interferon-gamma-differentiated U937 cells (U937IF) while augmenting the Fc gamma RI-induced tyrosine phosphorylation of cellular proteins. Herein we examine the effects of PTPase inhibitors on specific molecules involved in Fc gamma RI signaling. We show that orthovanadate and PAO augmented the Fc gamma RI-induced tyrosine phosphorylation of the adaptor protein CBL. CBL interactions with other phosphoproteins, among them SHC and CRKL, were also augmented in response to pretreatment with the PTPase inhibitors. SHC was tyrosine phosphorylated in response to Fc gamma RI stimulation of U937IF cells and bound to the SH2 domain of GRB2 in a stimulation-dependent manner. In fusion protein pull-down experiments the interaction of SHC with the SH2 domain of GRB2 was increased in PTPase inhibitor pretreated U937IF cells in response to Fc gamma RI stimulation. Our data support the hypothesis that a tyrosine dephosphorylation event is required for effective transmission of the Fc gamma RI signal to result in activation of the myeloid respiratory burst response.

Mouse mammary tumor virus (MMTV) antigen(s) are present on B lymphocytes of BALB/c mice.

Immunofluorescence studies using a polyvalent anti-MMTV serum revealed the presence of MMTV antigen(s) on the surface of spleen cells from the low mammary tumor incidence mouse strain BALB/cCrgl. Upon separation of the splenocytes on nylon-wool columns the populations of cells exhibiting positive fluorescence were nylon-adherent. These cells were considered to be B lymphocytes by the following criteria: presence of surface immunoglobulin (Slg), absence of Thy-1 antigens, enhanced responses to B-cell mitogens and decreased reactivity to T-cell mitogens. In order to ascertain the specificity of the cell surface immunofluorescence reaction, blocking studies were performed. Immunofluorescence was blocked by preincubation of the antiserum or with purified MMTV, but not by R-MuLV. Non-specific binding of the anti-MMTV to Fc receptors present on B lymphocytes was ruled out by the use of Fab'2 fragments of IgG from the various sera. Double label immunofluorescent studies with rhodamine-conjugated anti-mouse Slg Fab'2 fragments and rabbit anti-MMTV Fab'2 with fluorescein-conjugated goat anti-rabbit Fab'2 showed the simultaneous appearance of both labels in the nylon-adherent splenocytes. These results suggest the expression of viral polypeptide(s) encoded by an endogenous MMTV on the surface of B-type splenocytes in mice devoid of intact viral particles.

CBL-GRB2 interaction in myeloid immunoreceptor tyrosine activation motif signaling.

In this study, we provide the first evidence for role of the CBL adapter protein interaction in Fc gammaRI receptor signal transduction. We study the Fc gammaRI receptor, an immunoreceptor tyrosine activation motif (ITAM)-linked signaling pathway, using IFN-gamma-differentiated U937 myeloid cells, termed U937IF cells. CBL is constitutively associated with both GRB2 and the ITAM-containing receptor subunit, Fc gammaRIgamma of Fc gammaRI, providing direct evidence that CBL functions in myeloid ITAM signaling. Fc gammaRI cross-linking of U937IF cells induces the tyrosine phosphorylation of CBL that is associated with an altered CBL-GRB2 interaction. Both GRB2-SH3 and SH2 domains bind CBL in resting cell lysates; upon Fc gammaRI stimulation, phosphorylated CBL binds exclusively to the GRB2-SH2 domain. Glutathione-S-transferase fusion protein data demonstrate that the constitutive interaction of CBL with GRB2 and CRKL is mediated via two discrete regions of the CBL C terminus. The proximal C terminus (residues 461-670) binds to GRB2 constitutively, and under conditions of receptor activation binds to the tyrosine-phosphorylated SHC adapter molecule. The distal C terminus of CBL (residues 671-906) binds the CRKL adapter protein. The data demonstrate that the CBL-GRB2 and GRB2-SOS protein complexes are distinct and mutually exclusive in U937IF cells, supporting a model by which the CBL-GRB2 and GRB2-SOS complexes function in separate pathways for myeloid Fc gammaRI signaling.

Protein tyrosine phosphatase inhibitors block myeloid signal transduction through the Fc gamma RI receptor.

Fc-receptor stimulation in certain myeloid cells results in an increase in oxygen consumption termed the respiratory burst. In this report we examine the effects of protein tyrosine phosphatase inhibitors on the Fc gamma receptor-induced myeloid respiratory burst. Antiphosphotyrosine immunoblotting of neutrophils stimulated with opsonized oil particles shows that Fc-receptor stimulation is associated with the tyrosine phosphorylation of cellular proteins. Pretreatment of neutrophils for 10 min with vanadate or phenylarsine oxide (PAO), protein tyrosine phosphatase inhibitors, augments tyrosine phosphorylation in response to Fc-receptor stimulation. Vanadate and PAO inhibit the respiratory burst in a dose-dependent fashion, but have no effect on Fc gamma receptor-mediated phagocytosis, suggesting that the inhibition of the respiratory burst is not due to a general inhibition of Fc gamma-receptor signaling. Neutrophil phagolysosomal membranes were isolated from vanadate-treated and control neutrophils after Fc-receptor stimulation show a reduction in protein tyrosine phosphatase activity and a reduction in the NADPH-dependent oxidase activity and contain greater amounts of phosphotyrosine, relative to control membranes. Vanadate did not inhibit the NADPH-oxidase directly or interfere with the superoxide assay. Vanadate and PAO also inhibited the respiratory burst of interferon-differentiated U937 cells in response to immune complex and Fc gamma RI crosslinking. Pretreatment of U937 cells with PAO completely blocks the serine phosphorylation of the gamma subunit of the Fc gamma R, a response that is associated with Fc gamma RI-receptor activation. The data supports the recent observation that CD45 modulates signal transduction through the Fc gamma RI receptor, suggesting that protein tyrosine phosphatases play a positive modulatory role in the signal relay pathway(s) involving the myeloid Fc gamma RI receptor, resulting in the phosphorylation of the gamma subunit and the activation of the NADPH-oxidase complex.

The Fc gamma RI receptor signals through the activation of hck and MAP kinase.

U937 cells differentiated with IFN-gamma (termed U937IF cells) were used to study Fc gamma RI signaling. IFN induces a functional Fc gamma RI receptor signaling pathway in U937 cells, leading to the activation of the respiratory burst. IFN induces the expression of the nonreceptor protein tyrosine kinase, hck, and cross-linking the Fc gamma RI receptor in U937IF cells results in the activation of hck kinase as evidenced by the three- to fivefold increased tyrosine phosphorylation of hck. In vitro kinase assays demonstrate that the specific kinase activity of hck is increased 10-fold after Fc gamma RI stimulation. hck is observed to associate with two prominent tyrosine-phosphorylated proteins, p72 and p95, after Fc gamma RI-activation. Fc gamma RI cross-linking also results in mobility shift in MAP kinase in U937IF cells, suggesting that the Fc gamma RI receptor signals through the activation of MAP kinase. The data suggest that hck, p72, p95, and MAP kinase are involved in signal transduction through the Fc gamma RI receptor.