Phorbol esters increase [(3)h]-dopamine uptake by human platelets

Dopamine is taken up by platelets by an energy and temperature dependent process that does not involve a known dopamine receptor. Whilst dopamine uptake by platelets has been shown to be altered in several disease states, little is known about factors controlling dopamine uptake by platelets which could cause such changes. As phorbol esters have been shown to affect dopamine functions in other tissues we examined the effect of phorbol esters on dopamine uptake by human platelets. Phorbol 12,13-dibutyrate increased [(3)H]-dopamine uptake by platelets in a dose dependent manner. Similarly, phorbol 12,13-didecanoate increased [(3)H-dopamine uptake by platelets but 4α phorbol 12,13-didecanoate, which does not affect protein kinase, did not. Staurosporin, a protein kinase inhibitor, reversed the effect of phorbol 12,13-dibutyrate. These data suggest protein kinases can modulate dopamine uptake by platelets.     

Combined activation of platelets by cathepsin G and platelet activating factor, two neutrophil-derived agonists.

In this paper we have studied the combined effects on platelet activation, of two polymorphonuclear neutrophil (PMN)-derived agonists, namely platelet-activating factor (PAF) and cathepsin G (Cat.G), used at threshold concentrations. Our results showed that the order of agonist addition was a determinant factor since the addition of Cat.G prior to PAF induced a full platelet activation while the reverse combination had no effects. The successive challenge of platelets by Cat.G and then PAF induced a strong aggregation accompanied by an enhancement of alpha and dense granule secretion. The observed phenomenon was also dependent on the time interval between agonist addition. It was significant at 30 s (P less than 0.05) and plateaued over 1-2 min. Platelet activation resulting from the combination Cat.G-PAF can be described as a function of PAF concentrations, the synergism being significant between 10 nM and 1 microM. The mechanism by which Cat.G primes platelets remains to be elucidated. However, some points have been examined and have led us to conclude that an increase in expression and/or affinity of PAF receptors, [Ca2+]i movements, protein kinase C activation and phospholipase A2 pathway are not involved. Whatever the biochemical mechanism underlying this synergism which involved PMN and platelets, it may constitute a link between the inflammatory and haemostatic processes in response to tissue damage.     

Differentiated reactivity of whole blood neonatal platelets to various agonists

Neonatal platelets have been occasionally reported to show a reduced response to various agonists. The molecular mechanism(s) of such a depressed reactivity remains unclear. To further address this problem we studied neonatal platelet activation with thrombin, TRAP (thrombin receptor activating peptide, Ser-Phe-Leu-Leu-Arg-Asn-Pro-Asn-Asp-Lys-Tyr-Glu-Pro-Phe) and ADP in 42 healthy 1-2 day old neonates using a whole peripheral blood flow cytometry. The neonates did not show an increased fraction of P-selectin-positive circulating platelets, whereas the expression of GPIb (glycoprotein Ib) in resting neonatal platelets was significantly lower compared to adults. Neonatal platelets were significantly less reactive than adult platelets to thrombin and TRAP, especially at lower agonist concentrations, but not to ADP or when incubated for 1 h at room temperature. Activation of neonatal platelets with agonists resulted in a marked alterations in the expression of P-selectin, whereas the internalization of GPIb was not affected. The reduced neonatal platelet sensitivity to thrombin and TRAP was accompanied by significantly reduced ATIII (antithrombin III) and increased prothrombin fragment F(1+2) in neonatal plasma. We conclude that various receptor systems potentially able to bind thrombin are relatively insensitive in neonatal platelets. The novelty of our work is that neonatal platelet hyposensitivity is not a generalized phenomenon, but concerns only selected agonists and selected receptor systems.     

Phorbol esters enhance stretch-induced atrial natriuretic peptide secretion.

Stretching of atrial myocytes stimulates atrial natriuretic peptide (ANP) secretion, but the cellular processes linking mechanical distention to ANP release are unknown. We studied whether or not protein kinase C activation by phorbol ester affects atrial stretch-induced ANP secretion using the modified perfused rat heart preparation that enabled stepwise distention of the right atrium as an experimental model for stretch-stimulated ANP release. The increase in right atrial pressure (2.65 +/- 0.13 mm Hg) was accompanied by an increase in the perfusate immunoreactive ANP (IR-ANP) concentration (from 8.3 +/- 1.1 ng/5 min to 13.9 +/- 2.0 ng/5 min, P less than 0.05, n = 14). During stretch, a slight inotropic response was observed, while heart rate and perfusion pressure remained unchanged. Increase in right atrial pressure in the presence of a phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), known to stimulate protein kinase C activity in heart cells, resulted in a significantly greater increase in the perfusate IR-ANP concentration than after vehicle infusion. The calculated ANP increase corresponding to the 2 mm Hg increase in the right atrial pressure was 1.52-fold in the control group and 1.84-fold when 10 nM TPA was infused (P less than 0.05). Infusion of TPA at a dose of 24 nM further increased the stretch-induced ANP release by causing 2.22-fold (P less than 0.01) increase in IR-ANP secretion. As judged by gel filtration chromatography, abnormal release of the large mol wt stored ANP could not account for the secretory response to phorbol ester. Additionally, a phorbol ester analog, 4 alpha-phorbol 12,13-didecanoate, which is incapable of binding to and activating protein kinase C, was inactive as an ANP secretagogue. In contrast, drugs known to increase the concentration of intracellular Ca2+ in myocytes, Bay K8644 (3 and 6 microns) and forskolin (0.14 microM), significantly inhibited the stretch-stimulated ANP release. This study shows that phorbol ester enhances atrial stretch-stimulated ANP secretion from the isolated perfused heart, suggesting that protein kinase C activity is positively coupled to the stretch-induced ANP release. The results further demonstrate the negative effect of increase in intracellular Ca2+ on stretch-induced ANP release.     

Phorbol esters induce differentiation in human malignant T lymphoblasts

At nanomolar concentrations, phorbol ester, a class of potent tumor promoters, can promote differentiation in the human malignant T-lymphoblastic cell line MOLT-3. The optimal dose for induction, as measured by the increase of the number of cells containing sheep erythrocyte receptors (E-rosette assay), is between 8 and 16 nM 12-O-tetradecanoylphorbol 13-acetate (TPA), although there were significant increases of E-rosette-positive (E⁺) cells at concentrations as low as 1.6 nM TPA. The induction was linear for 4 days, then it reached a plateau. This induction was independent of the cell densities of the cultures, and the viability of the E⁺ cells remained high (95-100%) even after 10 days of culture in the presence of the tumor promoters. The E⁺ cells, when measured with the more stable 2-aminoethylisothiouronium bromide E-rosette assay, indicated that virtually all (75-95%) of the MOLT-3 cells became E⁺ by 4 days in culture. This induction by TPA was also accompanied by a dramatic drop in the plating efficiencies and a reduction in DNA synthesis. Examination of phorbol and other phrobol esters indicated that the ability to induce these cells correlated well with the tumor-promoting activities of these compounds, because only TPA and to a lesser extent phorbol 12,13,-dibenzoate induced E⁺ cells, while phorbol and 4α-phorbol 12,13-didecanoate had no effect. Studies of MOLT-3 cells depleted of E⁺ cells indicated that the induction of E⁺ cells cannot be explained solely on the basis of enrichment or stimulation of the background E⁺ cells in MOLT-3 cultures. Finally, we have shown that TPA also affected another differentiation marker, the loss of the enzyme terminal deoxyribonucleotidyl transferase. Terminal transferase activities and percentages of terminal-transferase-positive cells in these cultures were reduced to as low as 1/10th in 4 days in the presence of 16 nM TPA.     

Phorbol esters potentiate the induction of class I HLA expression by interferon alpha

We have studied the effect of phorbol esters on the induction of class I histocompatibility antigen (HLA) expression by interferons (IFNs) in the T-cell line MOLT-4 and in the MOLT-4 mutant YHHH. Addition of IFN-alpha to phorbol 12,13-dibutyrate-pretreated MOLT-4 cells causes a greater than 20-fold increase in the expression of class I HLA, as compared to a 4- to 7-fold IFN-alpha-induced increase in control cells. Pretreatment with phorbol 12,13-dibutyrate does not alter the class I HLA response to IFN-gamma or the responses of other IFN-induced genes. This effect of phorbol 12,13-dibutyrate reproduces in MOLT-4 cells the phenotype of the mutant YHHH, which also displays a selective enhanced class I HLA response to IFN-alpha. Pretreatment of YHHH with phorbol 12,13-dibutyrate does not affect any of the responses induced by IFN. These findings suggest the existence of a phorbol ester-sensitive factor, inducible in MOLT-4 and constitutively expressed or modified in YHHH, which operates in the pathway of induction of class I HLA by IFN-alpha but not in the pathway used by IFN-gamma.     

Inflammatory changes during the 'common cold' are associated with platelet activation and increased reactivity of platelets to agonists.

Epidemiologic studies have shown increased rates of myocardial infarction after upper respiratory tract infections. We hypothesized that changes in platelet activation and reactivity and inflammation occur during the 'common cold'. Previously healthy individuals with viral upper respiratory tract infections were studied (n = 18). Venous blood samples were obtained during the time of infection and again after 6 weeks. Platelet reactivity was higher during the 'common cold' as measured by low-dose ADP-induced aggregation (46 +/- 28 versus 27 +/- 21% 6 weeks after presentation, P = 0.003) and was higher than control individuals (22 +/- 8%, P = 0.003). Platelet P-selectin expression increased during illness (2.3 +/- 0.2% CD62-positive platelets versus 1.8 +/- 0.1% at 6 weeks after presentation, P = 0.017; and 1.7 +/- 0.2% in the control group, P = 0.03). C-reactive protein (3.7 +/- 1.3 versus 2.2 +/- 1.5 mg/l, P = 0.004) and tumor necrosis factor-alpha (27.6 +/- 28 versus 12.7 +/- 9 pg/ml, P = 0.03) were increased during the 'common cold'. There were no significant differences in levels of soluble P-selectin (P = 0.18), soluble vascular adhesion molecule-1 (P = 0.59) and soluble intercellular adhesion molecule-1 (P = 0.23). Increased platelet reactivity and activation during the 'common cold' are associated with inflammation as measured by increased levels of C-reactive protein and tumor necrosis factor-alpha, but not increased levels of endothelial markers. These findings support a pro-aggregatory state that, in part, explains the thrombotic events shown by epidemiological studies.     

Differentiated reactivity of whole blood neonatal platelets to various agonists

Neonatal platelets have been occasionally reported to show a reduced response to various agonists. The molecular mechanism(s) of such a depressed reactivity remains unclear. To further address this problem we studied neonatal platelet activation with thrombin, TRAP (thrombin receptor activating peptide, Ser–Phe– Leu–Leu–Arg–Asn–Pro–Asn–Asp–LysTyrGlu and ADP in 42 healthy 1–2 day old neonates using a whole peripheral blood flow cytometry. The neonates did not show an increased fraction of P-selectinpositive circulating platelets, whereas the expression of GPIb (glycoprotein Ib) in resting neonatal platelets was significantly lower compared to adults. Neonatal platelets were significantly less reactive than adult platelets to thrombin and TRAP, especially at lower agonist concentrations, but not to ADP or when incubated for 1 h at room temperature. Activation of neonatal platelets with agonists resulted in a marked alterations in the expression of P-selectin, whereas the internalization of GPIb was not affected. The reduced neonatal platelet sensitivity to thrombin and TRAP was accompanied by significantly reduced ATIII (antithrombin III) and increased prothrombin fragment F 1+2 in neonatal plasma. We conclude that various receptor systems potentially able to bind thrombin are relatively insensitive in neonatal platelets. The novelty of our work is that neonatal platelet hyposensitivity is not a generalized phenomenon, but concerns only selected agonists and selected receptor systems.     

Phorbol esters stimulate growth and inhibit differentiation in cultured thyroid cells

The potent tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) has biological effects on cell growth and differentiation similar to the effects of epidermal growth factor (EGF) on a variety of cells. Since EGF has been shown recently to stimulate thyroid cell proliferation and inhibit iodine metabolism, we examined the effects of phorbol esters on primary ovine thyroid cultures. TPA stimulated cell growth in a manner similar to EGF. The growth effects of EGF and TPA in combination were not additive. In contrast, TPA (1.6 X 10(-7) M) was a more potent inhibitor of iodine uptake and incorporation than EGF (10(-9) M) at their maximally effective concentrations. The inhibitory effects of TPA were also more rapid and less reversible than those of EGF. TPA and EGF in combination inhibited iodine metabolism more than either agent alone at its maximally effective concentration. Both TPA and EGF reduced the accumulation of cAMP in TSH-stimulated cells, but (Bu)2cAMP and stimulators of adenylate cyclase failed to overcome TPA's inhibition of iodine metabolism. TPA interacted with EGF by reducing the affinity of membrane receptors for [125I]iodo-EGF. Although the alteration in EGF-receptor interaction induced by TPA may play a role in mediating TPA's biological effects, the additive effects of TPA and EGF on iodine metabolism suggest that TPA does not act solely through the EGF receptor-effector system. Agents other than TSH, including phorbol esters and EGF, are potent modulators of thyroid growth and differentiated function. Despite several similarities in biological activity, TPA and EGF do not modulate differentiated function in an identical manner. Both factors act at least partially through a non-cAMP-dependent pathway, providing indirect evidence of another second messenger(s) in the control of thyroid function.     

Phorbol esters induce multidrug resistance in human breast cancer cells.

Mechanisms responsible for broad-based resistance to antitumor drugs derived from natural products (multidrug resistance) are incompletely understood. Agents known to reverse the multidrug-resistant phenotype (verapamil and trifluoperazine) can also inhibit the activity of protein kinase C. When we assayed human breast cancer cell lines for protein kinase C activity, we found that enzyme activity was 7-fold higher in the multidrug-resistant cancer cells compared with the control, sensitive parent cells. Exposure of drug-sensitive cells to the phorbol ester phorbol 12,13-dibutyrate [P(BtO)2] led to an increase in protein kinase C activity and induced a drug-resistance phenotype, whereas exposure of drug-resistant cells to P(BtO)2 further increased drug resistance. In sensitive cells, this increased resistance was accompanied by a 3.5-fold increased phosphorylation of a 20-kDa particulate protein and a 35-40% decreased intracellular accumulation of doxorubicin and vincristine. P(BtO)2 induced resistance to agents involved in the multidrug-resistant phenotype (doxorubicin and vincristine) but did not affect sensitivity to an unrelated alkylating agent (melphalan). The increased resistance was partially or fully reversible by the calcium channel blocker verapamil and by the calmodulin-antagonist trifluoperazine. These data suggest that stimulation of protein kinase C plays a role in the drug-transport changes in multidrug-resistant cells. This may occur through modulation of an efflux pump by protein phosphorylation.     

Platelet activation and binding of complement components to platelets induced by immune complexes

Clinical disorders such as malignant diseases, infectious diseases or autoimmune diseases are associated with circulating immune complexes. These immune complexes can activate the complement system in the blood or interact with complement or Fc receptors on the surface of cells. Complement activation may cause cytolysis and the immune complex interaction with receptors may cause activation of cells. We have used flow cytometry and labelled chicken antibodies to study the in vitro effects of model immune complexes on platelets and show that such immune complexes activate platelets and deposit Clq, C4 and C5 on them. Either low levels or no C3 could be detected on the platelets by flow cytometry. The immune complexes also induced formation of microparticles from purified platelets. Flow cytometry might become a useful tool in estimation of risk of thrombosis or thrombocytopenia in patients with autoimmune disease. Chicken antibodies are superior to mammalian antibodies for the measurement of platelet bound plasma proteins as they do not induce complement activation or platelet activation.     

Thrombin binding to platelets and their activation in plasma

Summary. The interactions of α-thrombin with platelets are critical in haemostasis and arterial thrombosis. This study established methods for characterizing the binding of α-thrombin to platelets and some of its consequences in platelet-rich plasma. The binding of α-thrombin to platelets and the subsequent platelet activation were quantified by flow cytometry, using affinity purified polyclonal antibodies to human α-thrombin and a monoclonal antibody to GMP-140, respectively. Dose-dependent binding of α-thrombin to platelets and their activation occurred in parallel, both reaching the maxima for each enzyme concentration within 10 s after → 1.0 n m α-thrombin was added to recalcified PRP containing 1 μ m recombinant tick anticoagulant peptide. The tick anticoagulant peptide abrogated prothrombin activation in the platelet-rich plasma. α-Thrombin binding to platelets, and their activation, were abrogated by a monoclonal antibody to the hirudin tail-like domain of the seven transmembrane thrombin receptor on platelets. Therefore this receptor represents an important site for α-thrombin binding to platelets suspended in plasma. d -Phe-Pro-ArgCH₂-α-thrombin only bound to platelets when its concentration was → 100 n m , and it did so without inhibiting platelet activation by α-thrombin. Whereas concentrations of hirudin equimolar to those of α-thrombin failed to abrogate α-thrombin-mediated activation of platelets, a 10-fold molar excesses of hirudin over α-thrombin abrogated α-thrombin binding to platelets. The demonstration that → 1.0 n m α-thrombin can bind to platelets and initiate their activation raises the possibility that the levels of thrombin generated in venous and arterial thrombosis contribute to platelet activation in vivo .     

Phorbol esters potentiate rapid dopamine release from median eminence and striatal synaptosomes

In the present study, we investigated the ability of phorbol esters to potentiate Ca2+-dependent depolarization-induced release of tritium-labeled dopamine ([3H]DA) from median eminence and striatal synaptosomes. Phorbol esters potentiated [3H]DA release in a concentration-dependent manner in both kinds of dopaminergic nerve terminals and with a potency series similar to that reported for stimulation of protein kinase-C (PKC) activity in other cell systems. Evoked [3H]DA release was increased by 12-O-tetradecanoylphorbol-13-acetate (TPA; 10(-7) M) after 1, 3, 5, and 10 sec of depolarization. The effect of TPA was suppressed by sphingosine, a PKC inhibitor. TPA enhanced [3H]DA release evoked by high K+, veratridine or the Ca2+ ionophore A23187. Phorbol ester potentiation was found to be depolarization dependent, as it was present from 30-75 mM, but not at 5-20 mM external K+. Potentiation was seen at all external Ca2+ concentrations studied between 0.01-3 mM. However, in the absence of external free Ca2+ (i.e. with 0.1 mM EGTA), the phorbol effect was not present. These data indicate that an increase in intrasynaptosomal Ca2+ concentration is necessary for the enhancement of [3H]DA release by phorbol esters to occur. The combination of TPA and the Ca2+ ionophore A23187 does not show the marked synergism observed in some other systems, that is maximal release was not reinstated. This suggests that in dopaminergic nerve terminals, activation of PKC has a modulatory, rather than a mediating, effect on release. Recently, we have shown that hyperprolactinemia stimulated [3H]DA release from median eminence synaptosomes by an external Ca2+-independent mechanism which might involve the PKC pathway. However, in the present work we found that the TPA and PRL effects on evoked [3H]DA release were additive, suggesting that two independent mechanisms are involved. A marked difference in the sensitivity of median eminence and striatal synaptosomes to calcium ionophore was discovered. The concentration of A23187 required to support significant [3H]DA release from median eminence synaptosomes was 3-fold greater than that in striatal synaptosomes. This suggests that some difference in calcium homeostatic processes exists, such as a higher resting striatal Ca2+ concentration, in these two kinds of dopaminergic nerve terminals. These data support the hypothesis that PKC activation potentiates the intrasynaptosomal stimulus-secretion coupling mechanism(s) and that nigrostriatal and tuberoinfundibular dopaminergic nerve terminals are affected by phorbol esters in a similar manner.     

Inhibition of PAF-induced activation of human platelets by verapamil.

Verapamil, an inhibitor of calcium channels, was shown to inhibit PAF-induced platelet activation. In the presence of 50 microM Verapamil both thromboxane (Tx) formation and release of ATP from dense granules induced by 100 nM PAF was completely inhibited. This concentration of Verapamil only produced partial inhibition of PAF-induced aggregation. It also reduced the size of the PAF-induced calcium (Ca2+) transient demonstrated in Fura-2 loaded platelets. In the absence of extracellular Ca2+, following chelation by EGTA, PAF was still able to induce a Ca2+ transient confirming the requirement of both intra and extracellular Ca2+ for PAF-induced platelet activation. 100 microM Verapamil was able to completely abolish the calcium transient induced by low doses of PAF. These results further suggest that Verapamil is able to inhibit PAF-induced platelet activation by mechanisms apart from blocking Ca2+ channels.     

Assembly of a fibronectin matrix by adherent platelets stimulated by lysophosphatidic acid and other agonists.

Lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) are agonists of the endothelial differentiation gene (Edg) family of G-protein-coupled receptors. LPA and S1P are generated by platelet activation during blood coagulation. Both lipids induce assembly of exogenous fibronectin (FN) by fibroblasts. This study examined whether LPA and S1P stimulate binding and assembly of fluoresceinated FN (FITC-FN) by adherent platelets. LPA enhanced deposition of FITC-FN into linear arrays overlying platelet surfaces and on edges of platelets adherent to FN or vitronectin (VN). Deposition was greater when platelets were adherent to FN than to VN and was elicited by platelet agonists with the following order of potency: thrombin > LPA = ADP (adenosine diphosphate) > S1P. The linear pattern of FITC-FN deposition was different from the more diffuse pattern of Alexa-fibrinogen (Alexa-FGN) binding to adherent platelets. FITC-FN was deposited by adherent platelets that had dense arrays of cytoskeletal actin when stained with rhodamine-phalloidin. The 70-kd N-terminal fragment of FN or L8 monoclonal antibody to a self-association domain of FN abolished deposition of FITC-FN but had no effect on binding of Alexa-FGN. Conversely, integrilin did not attenuate deposition of FITC-FN but abolished binding of Alexa-FGN. RGDS (Arg-Gly-Asp-Ser) or antibodies to alpha5beta1 or alphaIIbbeta3 integrins caused a partial decrease in LPA-induced deposition of FITC-FN. Correlative electron microscopy with anti-FITC coupled to gold beads revealed linear arrays on platelet surfaces associated with less than 20-nm-diameter filaments. These observations demonstrate that LPA, thrombin, ADP, and S1P induce adherent platelets to bind and assemble FN and suggest that platelets may contribute to early deposition of FN matrix after vascular injury.     

High-density lipoprotein fails to inhibit serotonin-induced activation of blood platelets

High-density lipoprotein (HDL) of 100–400 μg/ml did not prevent morphological alterations of human blood platelets treated with serotonin (1–5 μM). Highly concentrated HDL (1,200 μg/ml) appeared to activate platelets in vitro. These findings indicate that whole HDL may not inhibit agonist-induced platelet activation.     

Phorbol esters stimulate the phosphorylation of receptors for insulin and somatomedin C.

The effect of phorbol esters on the extent of phosphorylation of receptors for insulin and somatomedin C (insulin-like growth factor I) was studied in intact IM-9 cells that were labeled by incubation with H332PO4. The tumor-promoting phorbol esters phorbol tetradecanoate acetate (TPA) and phorbol dibutyrate, but not the inactive 4 alpha-phorbol, enhanced phosphorylation of the beta subunit of both receptors approximately 4-fold; 70 nM TPA maximally stimulated phosphorylation of both receptors, whereas concentrations less than or equal to 0.7 nM had no observable effect. Insulin also enhanced the phosphorylation of the beta subunit of the insulin receptor, and its effects appeared to be additive to those of TPA. Peptide maps indicated that at least some of the residues phosphorylated by these two agents are distinct. These results suggest a possible role of protein kinase C in regulating insulin and somatomedin C receptors.     

Phorbol esters stimulate the phosphorylation of c-Jun but not v-Jun: regulation by the N-terminal delta domain.

c-Jun and its oncogenic counterpart v-Jun are completely conserved within the region from Ser-63 to Ser-73; these serines are sites for phorbol ester-inducible c-Jun phosphorylation. Using a U937 human leukemic cell line stably expressing v-Jun, we have demonstrated that phorbol esters stimulate the in vivo phosphorylation of c-Jun but not v-Jun. We developed an in vitro protein kinase assay to characterize the c-Jun protein kinase and to examine the determinants underlying this differential phosphorylation. Fusion proteins between glutathione S-transferase and the N terminus of c-Jun, v-Jun, or several c-Jun mutants were used as substrates. A c-Jun kinase activity was affinity-purified 5000-fold by using glutathione S-transferase-c-Jun-glutathione-Sepharose beads and was found to phosphorylate the N terminus of c-Jun but not v-Jun or c-Jun containing a 27-amino acid N-terminal deletion found in v-Jun. These effects were also observed in vivo as phorbol 12-myristate 13-acetate did not induce the phosphorylation of v-Jun or the c-Jun deletion mutant in U937 cell lines stably expressing these proteins. These findings indicate that the delta domain of c-Jun (amino acids 34-60), which is deleted in v-Jun, plays a critical role in regulating N-terminal c-Jun phosphorylation.