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J. Biol. Chem., Vol. 275, Issue 30, 22868-22875, July 28, 2000

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12-O-Tetradecanoylphorbol-13-acetate (TPA)-induced c-Jun N-terminal Kinase (JNK) Phosphatase Renders Immortalized or Transformed Epithelial Cells Refractory to TPA-inducible JNK Activity^*

Heng Zhou, Anning Lin^§, Zhennan Gu^¶, Sam Chen^¶, No-Hee Park, and Robert Chiu^¶**

From the  Department of Oral Biology and Medicine, Dental Research Institute, UCLA School of Dentistry, and the ^¶ Department of Surgical Oncology, UCLA School of Medicine, and the  Jonsson Comprehensive Cancer Center, Los Angeles, California 90095-1668 and the ^§ Ben May Institute for Cancer Research, University of Chicago, Chicago, Illinois 60637

Received for publication, November 16, 1999, and in revised form, April 5, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

c-Jun N-terminal kinase (JNK) regulates gene expression in response to various extracellular stimuli. JNK can be activated by the tumor promoting agent, 12-O-tetradecanoylphorbol-13-acetate (TPA) in normal human oral keratinocytes but not in human keratinocytes that have been immortalized (HOK-16B and HaCaT) or transformed (HOK-16B-Bap-T) nor in a cervical carcinoma cell line (HeLa). The refractory JNK activation response to TPA is not due a defect in the JNK pathway, because JNK can be activated by other stimuli, e.g. UV irradiation and an alkylating agent N-methyl-N'-nitrosoguanidine in these immortalized or transformed cells. More importantly, the refractory JNK and JNKK activation response to TPA can be restored by treatment of the cells with a combination of TPA and a protein-tyrosine phosphatase inhibitor, sodium orthovanadate. Furthermore, pretreatment of cells with TPA partially inhibited UV- or N-methyl-N'-nitrosoguanidine-induced JNK activity. These results suggest that a TPA-inducible, orthovanadate-sensitive protein-tyrosine phosphatase may specifically down-regulate JNK signaling pathway in these immortalized/transformed epithelial cells. In contrast, ERK and p38/Mpk2 are not regulated by this TPA-induced phosphatase. This putative protein-tyrosine phosphatase appears to be JNK pathway-specific.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Members of the mitogen-activated protein kinase (MAPK)¹ superfamily are proline-directed serine/threonine protein kinases that play pivotal roles in transducing various extracellular signals to the nucleus. They consist of three major subfamilies: MAPK/extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK)/stress-activated protein kinase (SAPK), and p38/Mpk2. MAPK/ERK is activated mainly by growth factors and phorbol esters and is associated with cellular proliferation and differentiation (1-3). JNK/SAPK and p38/Mpk2 are activated by various extracellular stimuli, including growth factors, phorbol esters, UV irradiation and ionizing radiation, heat shock, hyperosmolarity, and cytotoxic drugs (3-6). Activation of these protein kinases leads to various responses, including gene expression, cell proliferation, differentiation, cell cycle arrest, apoptosis, early development, etc., depending on the cell type (7-11).

MAPKs phosphorylate diverse target proteins in the membrane or cytosol as well as a number of nuclear transcription factors, indicating their critical role in orchestrating many short and long term changes in cell function (11, 12). This has been confirmed recently using specific chemical inhibitors or by expressing mutant versions of different MAPKs or their upstream activators. These studies show that ERKs play a pivotal role in mediating neuronal differentiation in PC12 cells, as well as growth factor-stimulated proliferation and oncogenic transformation in fibroblasts (13, 14). Similar approaches have yielded results supporting the view that JNK/SAPK and p38/Mpk2 are critical in processes mediating platelet aggregation and secretion, in generation of inflammatory cytokines, and in pathways leading to apoptotic death in a number of cell types (15-18).

Full activation of MAPKs requires phosphorylation of critical tyrosine and threonine residues. Several upstream dual specificity kinases catalyzing this modification have now been identified (1, 2, 9). Once activated, MAPKs phosphorylate and regulate several cellular proteins, including other protein kinases, cytoskeletal elements, stathmin, phospholipase A₂, and transcription factors, notably Myc, Elk-1, Jun, and ATF-2 (12, 19, 20). The broad range of substrates indicates a pivotal role for MAPKs in cellular signal transduction, suggesting that the extent and duration of their activation play a key role in controlling cell function.

A precise balance of the activities of protein kinases and protein phosphatases (PPs) plays a major role in receptor-mediated signaling pathways and cell cycle control. Several components of the ERK1/ERK2 pathway are subjected to regulation by PPs such as PP1 and PP2A. Protein-tyrosine phosphatases (PTPs) also play an important role in the attenuation of signals generated by protein-tyrosine kinases involved in mitogenesis. The extent and duration of protein-tyrosine kinase-mediated tyrosine phosphorylation can be greatly enhanced by treatment with the PTP inhibitor, sodium orthovanadate, or its peroxy derivatives (21, 22). It has also been shown that overexpression of dual specificity MAPK-phosphatase 1 (MKP-1) inhibits ERK2 activity and relieves the inhibitory effects of mitogens on muscle-specific gene expression (23).

It is clear that PPs and PTPs play crucial roles in controlling cellular activities. MAPK/ERK, SAPK/JNK, and p38/Mpk2 are regulated by dual phosphorylation and dephosphorylation within the motifs TEY, TPY, and TGY, respectively, by several upstream dual specificity kinases (MAPK kinases) and several types of phosphatases (1, 10). Among the large number of PTPs currently identified (24-26), an emerging class of dual specificity PTPs may directly and specifically regulate MAPK family members through dephosphorylation of both threonine and tyrosine residues crucial for enzymatic activity (27). These dual specificity PTPs, termed MKPs, harbor distinct substrate preferences for the various MAPK family members (28-31).

Currently, 10 distinct dual specificity phosphatases have been identified, including MKP-1/CL100 (3CH134) (32, 33), VHR (34), PAC1 (35, 36), hVH-2 (MKP-2, TYP-1) (37-39), hVH-3 (B23) (34, 40), hVH-5 (M3/6) (41), MKP-3 (rVH6, Pyst1) (28, 29, 42), MKP-X (Pyst2) (28, 29, 42), MKP-4 (43), and MKP-5 (44). Although they share sequence homology, each has distinct properties concerning substrate specificity, tissue distribution, subcellular localization, and inducibility by extracellular stimuli. These phosphatases all posses a characteristic extended active site motif, VXVHCXXGXSRSXTXXXAYLM (where X is any amino acid) and two N-terminal CH2 domains displaying homology to the cell cycle regulator Cdc25 phosphatase.

We report here that activation of JNKs by TPA is repressed in the human papilloma virus-immortalized keratinocytes or their tumorigenic derivatives, cervical carcinoma cell line, HeLa, and spontaneously immortalized keratinocyte, HaCaT. This repression can be restored by treatment of these cells with a combination of TPA and sodium orthovanadate. We provide evidence that there may well be a TPA-inducible dual specificity phosphatase involved in the refractory effect of the TPA-induced JNK activity. This TPA-inducible phosphatase is specific in inactivating kinase activities in the JNK pathway and associates only with immortalized or transformed epithelial cells.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- Protein phosphatase inhibitor (okadaic acid), protein-tyrosine phosphatase inhibitor (sodium orthovanadate), and tumor promoters (sodium arsenite and TPA) were purchased from Sigma. GST-Sepharose was obtained from Amersham Pharmacia Biotech. [-³²P]ATP (4500 Ci/mmol) is a product of ICN Biochemicals, Inc. (Costa Mesa, CA). UV lamp (GATES MR-4) is a product of George W. Gates & Co., Inc. Rabbit polyclonal anti-MKP-1 antibody (C-19) was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). All other chemicals were of analytical grade.

Cell Culture and Preparation of Cell Extracts-- Normal human oral keratinocytes (NHOKs) were isolated from normal human oral tissue as described previously (45). NHOKs were immortalized by transfection with recombinant human papilloma virus-16 DNA. These cells (HOK-16B) were subsequently exposed to benzo(a)pyrene for 6 months, at which point the resulting cells (HOK-16B-Bap-T) were tumorigenic, i.e. they developed tumors in nude mice (46). Cell culture conditions for NHOK, HOK-16B, and HOK-16B-Bap-T were the same as described by Kim et al. (45). HeLa and HaCaT cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. Cells were grown at 37 °C in a humidified incubator with 5% CO₂. Whole cell extracts were prepared in WCE buffer (25 mM HEPES, pH 7.7, 0.3 M NaCl, 1.5 mM MgCl₂, 0.2 mM EDTA, 0.1% Triton X-100, 0.5 mM dithiothreitol, 20 mM -glycerolphosphate, 0.1 mM Na₃VO₄, 5 µg/ml of leupeptin, 30 µg/ml of phenylmethylsulfonyl flouride, 5 µg/ml of pepstatin A, 5 µg/ml of aprotinin). The cell suspension was centrifuged at 4 °C for 30 min, and the extract was cleared by centrifugation at 10,000 × g for 10 min. Protein concentration was estimated using the Bio-Rad protein assay.

Protein Kinase Assays-- JNK activity was measured by a solid state kinase assay with GST-c-Jun (1-79) as a ligand and a substrate. Cell extracts were mixed with glutathione-agarose beads, to which GST-c-Jun (1-79) was bound. After incubation at 4 °C for several hours, the beads were washed extensively and incubated in kinase buffer (20 mM HEPES, pH 7.6, 20 mM MgCl₂, 10 mM -glycerolphosphate, 20 mM p-nitrophenylphosphate, 0.5 mM Na₃VO₄, 2 mM dithiothreitol, 50 µCi of [-³²P]ATP at 30 °C for 20 min. Phosphorylated GST-c-Jun (1-79) fusion proteins was eluted with 30 µl of 1.5× Laemilli sample buffer and resolved on a 10% SDS-polyacrylamide gel, followed by autoradiography.

Immunocomplex Kinase Assays-- The kinase activity of ERK was assessed by an immunocomplex kinase assays using myelin basic protein (MBP) as a substrate. Soluble cell lysates containing 400 µg of protein were incubated with 1 µg of rabbit polyclonal anti-ERK2 antibody (Santa Cruz Biotech) and 20 µl of protein A-Sepharose beads (Sigma) at 4 °C for 16 h with gentle rotation. The immunoprecipitates were then washed three times with wash buffer (500 mM LiCl, 100 mM Tris-HCl, pH 7.6, 0.1% Triton X-100, 1 mM dithiothreitol), and three times with kinase assay buffer (20 mM MOPS, pH 7.2, 2 mM EGTA, 10 mM MgCl₂, 1 mM dithiothreitol, 0.1% Triton X-100). The kinase reactions were carried out at 30 °C for 20 min in 55 µl of kinase assay buffer containing 10 µM ATP, 10 µCi of [-³²p]ATP, 20 mM MgCl_2, and 6 µg of MBP (Sigma).

Electrophoretic Mobility Shift Assays-- Oligonucleotides corresponding to the NFB-binding site sequence were purchased from Promega. Oligonucleotides were labeled at their 5' ends using [-³²P]ATP (4500 Ci/mmol; ICN Biochemicals, Inc.) and T4 polynucleotide kinase. Radiolabeled double-stranded oligonucleotides were purified through a Sephadex G-25 spin column. The specific activities of oligonucleotide probes were typically 10⁵ cpm/ng of DNA. Electrophoretic mobility shift assays were performed as described previously (47).

Western Blot Analysis of MKP1/2-- Cell extracts (100 µg) were subjected to electrophoretic separation in 10% SDS-polyacrylamide gels. The gel was transferred to nitrocellulose membrane, which was incubated in 5% nonfat dry milk in 1× phosphate-buffered saline with 0.1% Tween-20 for 1 h. The membrane was then incubated with primary antibody specific for MKP-1 (C-19, Santa Cruz) for at least 1 h at room temperature. The membrane was rinsed and incubated with second antibody (anti-rabbit IgG, Sigma) for at least 1 h at room temperature. The membrane was then developed with 44 µl of NBT and 33 µl of BCIP in 10 ml of developing buffer (100 mM Tris-HCl, pH 9.5, 100 mM NaCl, 50 mM MgCl₂) at room temperature with agitation.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

TPA Induces JNK Activity in Normal Human Keratinocytes but Not in Immortalized Keratinocytes and Transformed Epithelial Cell Lines-- Many of the stimuli that activate MAPKs are also stimulators of cell proliferation and involved in cell cycle regulation. MAPKs are critical for the transmission of stimuli to the nucleus, resulting in modification of cell growth-related transcription factors such as c-Jun, ATF-2, and Elk. To determine whether JNK can be activated by the tumor promoter agent TPA in human keratinocytes, normal or immortalized/transformed keratinocytes were treated with or without TPA (100 ng/ml, 1 h), and the cell extracts were used to assay JNK activity in a solid phase JNK assay with GST-c-Jun (1-79) fusion proteins as a substrate. As shown in Fig. 1, treatment with TPA resulted in activation of JNK only in NHOK (Fig. 1A, upper panel, lane 4), but not in other immortalized/transformed epithelial cell lines (Fig. 1A, upper panel, compare lanes 8 and 12 with lanes 5 and 9; Fig. 1B, upper panel, compare lanes 2 and 6 with lanes 1 and 5). The refractory JNK activation response to TPA treatment was not due to a defect in the JNK signaling pathway in these cell types, because JNK was activated by both UV irradiation (30 J/m²) and MNNG (80 µM) treatment (Fig. 1A, upper panel, lanes 2, 3, 6, 7, 10, and 11; Fig. 1B, upper panel, lanes 3, 4, 7, and 8).

View larger version (52K): [in this window] [in a new window]   Fig. 1.   UV irradiation-, MNNG-, or TPA-induced MAPKs activities. A, regulation of JNK and ERK activities in human oral keratinocytes. Equal numbers of cells treated for 1 h with UV irradiation (30 J/m2) (lanes 2, 6, and 10), MNNG (80 µM) (lanes 3, 7, and 11) or TPA (100 ng/ml) (lanes 4, 8, and 12) were lysed, and cell extracts were mixed with 10 µg of GST-c-Jun(1-79) or MBP to determine JNK or ERK activity, respectively. Cell lysates prepared from untreated cells were used as controls (lanes 1, 5, and 9). After incubation at 4 °C for 1 h, 5 µCi of [-32P]ATP was added to the reaction mixture and allowed to incubate for 20 min at 30 °C. The activity of transiently expressed JNK was determined by resolution of the phosphorylated substrate on a 10% SDS-polyacrylamide gel, followed by autoradiography. B, regulation of JNK and ERK activities in HeLa and HaCaT cells. Equal numbers of cells treated with UV irradiation (lanes 3 and 7), MNNG (lanes 4 and 8) or TPA (lanes 2 and 6) were lysed, and cell extracts were mixed with 10 µg of GST-c-Jun(1-79) or 3 µg of MBP to determine JNK or ERK activity, respectively. Cell lysates prepared from untreated cells were used as controls (lanes 1 and 5). The experiment conditions are the same as above.

We also examined ERK activities in response to TPA in these cell types, using MBP as substrates. Interestingly, TPA weakly induced ERK in NHOK but not in HOK-16B and HOK-16B-Bap-T (Fig. 1A, lower panel, lanes 4, 8, and 12). In contrast, TPA strongly activated ERK in HeLa and HaCaT cells (Fig. 1B, lower panel, lanes 2 and 6). On other hand, UV irradiation weakly or moderately induced ERK activities in all tested cells except HaCaT cells (Fig. 1A, lower panel, lanes 2, 6, and 10; Fig. 1B, lanes 3 and 7), whereas MNNG induced ERK activities to a greater or lesser degree (Fig. 1A, lower panel, lanes 3, 7, and 11; Fig. 1B, lower panel, lanes 4 and 8).

Induction of NF-B DNA Binding Activity by TPA-- It is known that the transcription factor NFB translocates to the nucleus following the exposure of cells to a variety of extracellular stimuli including TPA, leading to activation and transcription of its target genes. To determine whether NFB can be activated by TPA in the immortalized/transformed cells, the cells were treated with or without TPA and NFB DNA binding activity was measured by the electrophoretic mobility shift assay. As shown in Fig. 2, TPA induced NFB DNA binding activity to a similar extent in both normal and immortalized/transformed cells, suggesting that TPA-induced downstream signaling pathways maintain their integrity in immortalized/transformed epithelial cells.

View larger version (42K): [in this window] [in a new window]   Fig. 2.   TPA-induced NFB DNA binding activity. Nuclear extracts (10 µg) prepared from untreated (C) or TPA-treated (T) NHOK, HOK-16B, HOK-16B-Bap-T, HeLa, and HaCaT cells were used for electrophoretic mobility shift assays. An NFB-binding site specific oligonucleotide (5'-AGTTGAGGGGACTTTCCCAGGC-3') was used as probe. Complementary oligonucleotides were annealed and labeled at their 5' ends, using [-32P]ATP (4, 500 Ci/mmol; ICN) and T4 polynucleotide kinase (New England Biolabs). To confirm the specificity of binding, a 100-fold molar excess of unlabeled oligonucleotides was added to the reaction mixture prior to the addition of radiolabeled probe.

Restoration of JNK Activation in Response to TPA by Treatment of Cells with a Combination of TPA and Sodium Orthovanadate in Immortalized Keratinocytes and Transformed Epithelial Cell Lines-- Previous studies reported that in activated Raf-transformed cells, a putative OV-sensitive protein phosphatase was able to negatively regulate ERK activity (48). To determine whether such a phosphatase activity was responsible for the repression of JNK activation by TPA in immortalized/transformed cells, cells were pretreated with or without the protein-tyrosine phosphatase inhibitor OV for 30 min and then either treated with TPA for additional 30 min or left untreated. As expected, TPA alone induced JNK activity only in NHOK (Fig. 3A). The combination of TPA and OV, however, synergistically induced JNK activity in the immortalized/transformed epithelial cell lines (HOK-16B, HeLa, and HaCaT) (Fig. 3A). Similar results also observed in squamous cell carcinoma cell lines, i.e. SCC-4, SCC-9, CAL 27, and CAL 33 (data not shown). In contrast, TPA alone induced ERK activity in all cells tested, except HOK-16B (Fig. 3B). In addition, ERK activity was not synergistically induced by TPA and OV in HOK-16B or the other cells tested (Fig. 3B). These results suggest that TPA may induce an orthovanadate-sensitive protein-tyrosine phosphatate that specifically down-regulates JNK activity in certain immortalized/transformed epithelial cell lines.

View larger version (33K): [in this window] [in a new window]   Fig. 3.   Induction of JNK or ERK activity by TPA in the presence or absence of protein-tyrosine phosphatase inhibitor. A, induction of JNK activity by TPA, OV, or combination of TPA and OV. NHOK, HOK-16B, HeLa, and HaCaT cells were untreated (C) or treated with TPA (T, 100 ng/ml), OV (1 mM) or pretreatment of cells with OV for 30 min and then incubation with TPA for an additional hour. JNK activity was measured by the solid phase kinase assay with GST-c-Jun(1-79) as a substrate. B, induction of ERK activity by TPA, OV, or both TPA and OV. NHOK, HOK-16B, HeLa, and HaCaT cells were treated as described above. ERK activity was measured by immune complex kinase assay with MBP as a substrate.

To determine whether the repression of JNK activation by TPA in the immortalized/transformed cells can be released by okadaic acid (OA), a serine/threonine protein phosphatase inhibitor, HOK-16B cells were pretreated with or without OA for 30 min, followed by treatment with TPA for an additional 30 min or left untreated. Cell lysates were used to determine JNK activity, as measured by protein kinase assays with GST-c-Jun as substrate. As shown in Fig. 4, OA alone was able to activate JNK activity. Treatment of cells with a combination of TPA and OA did not further enhance the activation (Fig. 4). These results further demonstrate that a OV-sensitive, not an OA-sensitive, JNK-specific phosphatase is likely involved in repression of JNK activity in response to TPA.

View larger version (23K): [in this window] [in a new window]   Fig. 4.   Induction of JNK activity in the presence of protein phosphatase inhibitor. JNK activity was measured by solid phase kinase assays with GST-c-Jun as a substrate. Lane 1, control (C); lane 2, TPA treatment for 1 h (T); lane 3, treatment cells with OA for 1 h; lane 4, pretreatment of cells with OA for 30 min and then TPA for 30 min.

TPA-induced JNK Phosphatase Partially Inactivated UV- or MNNG-induced JNK Activity-- To test the hypothesis that TPA-induced phosphatase is specific for JNK, we measured its effect on UV irradiation or MNNG-induced JNK activity. HOK-16B cells were pretreated with or without TPA for various period of times (15 min, 30 min, or 1 h), followed by either UV irradiation (5 J/m²), MNNG (40 µM) treatment, or no treatment. Cell lysates were prepared to measure the JNK activity as compared with cell lysates prepared from TPA-, UV-, or MNNG-treated cells. As shown in Fig. 5, pretreatment of cells with TPA for 15 min dramatically decreased UV- or MNNG-induced JNK activity (lane 6). A significant effect was also observed at 30 min after TPA treatment (lane 5). These results suggested that an early TPA-induced phosphatase may play an important role in the regulation of JNK activity in certain immortalized/transformed cells.

View larger version (13K): [in this window] [in a new window]   Fig. 5.   TPA-induced JNK-specific phosphatase partially inactivated UV- or MNNG-induced JNK activity. A, TPA-induced phosphatase partially inactivates UV-induced JNK activity in HOK-16B cells. Lane 1, control; lane 2, cells treated with UV irradiation (5 J/m2) and incubated for 1 h; lane 3, cells treated with TPA (100 ng/ml) for 1 h; lanes 4-6, cells pretreated with TPA for 15 min, 30 min, and 1 h, respectively, followed by UV irradiation (5 J/m2). The cells were lysed, and JNK activity was measured in the solid phase kinase assay with GST-c-Jun(1-79) as substrate. B, TPA-induced phosphatase partially inactivates MNNG-induced JNK activity in HOK-16B. Lane 1, control; lane 2, cells treated with MNNG (40 µM) for 1 h; lane 3, cells treated with TPA (100 ng/ml) for 1 h; lanes 4-6, cells treated with TPA for 15 min, 30 min, and 1 h, respectively, followed by exposure of cells to MNNG (40 µM) for 1 h. The cells were lysed, and JNK activity was measured in the solid phase kinase assay with GST-c-Jun(1-79) as substrate.

TPA-induced Phosphatase Also Regulates JNKK Activity-- Because JNKK (also known as SEK, MKK4/7) is a dual specificity kinase that regulates JNK activity, it was of interest to compare the activities of JNKK in HOK-16B, HeLa, and HaCaT cells with NHOK after TPA stimulation. Therefore, we examined the JNKK activity in response to TPA, as well as to UV irradiation, MNNG, OV, and a combination of TPA and OV. JNKK activity was measured by anti-phospho-JNK antibody in Western blots. The results clearly demonstrated that TPA moderately induced JNKK activity only in NHOK (Fig. 6, lane 2) but not in the other test cells (Fig. 6, lane 2). UV irrradiation strongly activated JNKK activity in all tested cells (Fig. 6, lane 3), whereas MNNG strongly activated JNKK activity in NHOK, HOK-16B, and HaCaT cells but only weakly induced JNKK activity in HeLa cells (Fig. 6, lane 4). OV has a strong or moderate stimulatory effect on JNKK activity in NHOK (Fig. 6, lane 5) but has little stimulatory effect on JNKK activity in HOK-16B, HeLa and HaCaT cells (Fig. 6, lane 5). Interestingly, a combination of TPA and OV also synergistically activated JNKK activity in HOK-16B, HeLa, and HaCaT cells (Fig. 6, lane 6), whereas there was only an additive effect in NHOK (Fig. 6, lane 6). This clearly indicated that the TPA-induced, OV-sensitive phosphatase also negatively regulates JNKK activity in certain immortalized/transformed cells.

View larger version (33K): [in this window] [in a new window]   Fig. 6.   A combination of TPA and sodium OV synergistically activates JNKK activity. Combination of TPA and OV additively activates JNKK activity in NHOK cells only, whereas synergistically activates JNKK activity in HOK-16B, HeLa and HaCaT cells. Cell lysates were prepared for measurement of JNKK activity and JNK expression by Western blot analyses with anti-phospho-JNK antibody and anti-JNK antibody, respectively. Cells were untreated (lane 1, C, control) or treated with TPA (lane 2), UV irradiation (lane 3), MNNG (lane 4), OV (lane 5), or a combination of TPA and OV (lane 6).

The TPA-induced JNK Phosphatase Involved in Regulation of JNK Activity Is Not MKP-1 or MKP-2-- MKP-1 and MKP-2 are dual specificity phosphatases involved in the dephosphorylation of MAP kinases. To test whether TPA-induced JNK phosphatase is one of these MKPs, we performed a Western blot analysis, using a polyclonal antibody against both MKP-1 and MKP-2. 100 µg of cell extracts prepared from TPA-treated, UV-irradiated, MNNG-treated, OV-treated, or both OV- and TPA-treated NHOK, HOK-16B, HeLa, and HaCaT cells were subjected to 10% SDS-polyacrylamide gel electrophoresis, followed by Western blot analyses. The results showed that MKP-2 was not induced by these stimuli in any of the epithelial cells tested (Fig. 7). In contrast, MKP-1 was weakly induced in NHOK, HOK-16B, and HaCaT cells and strongly induced in HeLa cells by TPA treatment (Fig. 7). However, our previous data showed that JNK activity was strongly induced in NHOK, weakly induced in the HaCaT, and not induced in the HOK-16B or HeLa cells by TPA treatment (Fig. 1). Moreover, OV alone or a combination of TPA and OV did not affect the basal level of MKP-1 or induce MKP-1 in NHOK, suggesting that the JNK activity induced by the indicated conditions above (Fig. 3A) is not due to the inhibition of MKP-1. Similarly, the MKP-1 level in HOK-16B, HeLa, and HaCaT cells (Fig. 7) did not correlate with JNK activity induced by OV or the combination of TPA and OV treatment (Fig. 3A). Taken together, the TPA-induced, OV-sensitive phosphatase reported here appears to be neither MKP-1 nor MKP-2.

View larger version (39K): [in this window] [in a new window]   Fig. 7.   Western blot analyses of the dual specificity of MAPK phosphatases, MKP-1 and MPK-2. NHOK, HOK-16B, HeLa, and HaCaT cells were treated with various stimuli as indicated. 100 µg of cell lysates were subjected to 10% SDS-polyacrylamide gel electrophoresis. Western blots were hybridized with anti-MKP-1 polyclonal antibody (C-19, Santa Cruz Biotechnology), which is reactive with both MKP-1 and MKP-2. C, control.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

It has been reported previously that the tumor promoter agent, TPA, is able to strongly induce ERK activity (49, 50) and had little effect on JNK activity in fibroblasts and epithelial cells (4, 51). However, JNK activity can be rapidly stimulated by UV irradiation, but not by TPA, in human 293 (transformed primary embryonal kidney cells) and HeLa (cervical carcinoma) cells (5, 52). Similarly, JNK is activated in NIH3T3 and CCD-18Co colon fibroblasts by cellular stress and tumor necrosis factor- but is poorly activated by TPA (6). It was speculated that the difference in JNK activation resulted from the differences in cell types. Recently, it was reported that retinoic acid inhibited serum-induced JNK activity by increasing the activity of dual specificity phosphatase in human bronchial epithelial cell (53). Other studies also demonstrated that intestinal trefoil factor decreases ERK activity by activating the dual specificity phosphatase in intestinal epithelial cell (54). Intestinal trefoil factor also blocked activation of ERK activity induced by transforming growth factor- or TPA (54).

In this study, we found that TPA induced JNK activity only in NHOK and not in the immortalized and transformed epithelial cells. This refractory JNK activation response to TPA treatment was not due to a defect in the JNK pathway, because JNK activity and NFB binding activity were strongly activated by UV irradiation or MNNG treatment and TPA treatment, respectively, in all cell types examined. In contrast, ERK activity was weakly induced by TPA in NHOK, whereas refractory to TPA treatment in transformed/immortalized cells, except in HeLa and HaCaT cells. The p38/Mpk2 pathway was also refractory to TPA treatment (data not shown). Although all three MAPKs were refractory to TPA treatment in certain immortalized/transformed cells, only JNK activation could be restored by a combination of TPA and OV treatment. These results suggest that a TPA-induced, OV-sensitive protein phosphatase specifically down-regulates JNK activity in response to TPA treatment in certain immortalized/transformed epithelial cells. In addition, TPA may also induce different protein phosphatases that down-regulate ERK or p38 activity.

One possible explanation for the refractory JNK activation response to TPA in certain immortalized and transformed epithelial cells is that these cells may have delayed kinetics of JNK activation. To test this possibility, we measured JNK activity after exposure of HOK-16B cells to TPA, UV irradiation, or MNNG at different time points. Both UV irradiation and MNNG significantly activated JNK, with the peak at 1 h and a return to the background level after three hours (data not shown). In contrast, TPA did not activate JNK even at a much longer time period or higher TPA concentration of exposure (data not shown). Similar results were also obtained when HeLa or HaCaT cells were used. These results suggested that repression of JNK activation by TPA in these cell lines is not due to delayed kinetics of activation or insufficiency of TPA required for JNK activation.

The finding that JNK activation by TPA can be restored in the immortalized/transformed cells by a combination of sodium OV and TPA provides an explanation, for the first time, for the differential regulation of JNK activity by TPA in different cell types. Our results suggest that TPA may induce an OV-sensitive phosphatase, which is a negative regulator of JNK, and is able to override activation of JNK by TPA in certain immortalized/transformed cells. It appears that the TPA-induced, OV-sensitive JNK phosphatase is a dual specificity phosphatase. First, the phosphatase activity can be induced by TPA, while most members of the MKP group are inducible (29, 33). Second, OV, but not okadaic acid, can inhibit the phosphatase activity, consistent with the sensitivity of the dual specificity phosphatases to the low molecular weight protein-tyrosine phosphatase inhibitors. Third, other tyrosine phosphatase inhibitors such as benzylphosphonic acid-(AM)₂ and L-p-bromotetramisole oxalate (BIOMOL Research Lab., Plymouth Meeting, PA) have no inhibitory effect on TPA-induced phosphatase.² Therefore, this putative TPA-induced, dual specificity phosphatase may play a critical role in suppressing TPA-induced JNK activity in immortalized and transformed epithelial cells.

MKPs such as MKP-1, MKP-2, MKP-5, and M3/6 are able to target JNK. Our data showed that JNK activity was strongly induced in NHOK, very weakly in the HaCaT, and not at all in the HOK-16B or HeLa cells in response to TPA treatment (Fig. 1). Moreover, OV alone or a combination of TPA and OV neither affected the basal level of MKP-1 nor induced MKP-1 in NHOK. These data clearly demonstrated that MKP-1 may not be involved in the suppression of JNK activity in NHOK, although MKP-1 level was increased in response to TPA treatment. On the other hand, MKP-2 was not induced in any of the epithelial cells tested. Collectively, our results suggest that this TPA-induced phosphatase is neither MKP-1 nor MKP-2. The most recently cloned dual specificity phosphatase, MKP-5, was able to inactivate p38/Mpk2 and SAPK/JNK, but not ERK. In addition, inactivation of p38/Mpk2 was greater than that of JNK/SAPK (44). It has been reported that the dual specificity phosphatase M3/6, which is expressed predominantly in the adult brain, heart, and skeletal muscle, is highly selective for inactivation of both JNK/SAPK and p38/Mpk2 and is induced by nerve growth factor and insulin in PC12 cells (41). In contrast, the TPA-inducible phosphatase in the immortalized and transformed epithelial cells does not affect ERK and p38/Mpk2 activity. Based on these characteristics, the TPA-inducible phosphatase described in this report is not likely to be any one of above MKPs.

Other dual specificity phosphatases may also contribute to the refractory JNK activation response to TPA treatment. TPA may increase the activity of MKPs through mechanisms other than an increase of their expression, such as post-translational modification by protein phosphorylation or farnesylation, which are important regulators of phosphatase activity (55, 56). This is unlikely, because treatment with cyclohexamide, protein synthesis inhibitor, results in the reduction or elimination of the synergistic activation of JNK by TPA or a combination of OV and TPA, respectively.³ These results suggested that de novo protein synthesis is required to inhibit TPA-induced JNK activity, as well as the synergistic activation of JNK by the combination of OV and TPA.

The TPA-induced JNK phosphatase seems to be a TPA-induced immediate early gene as pretreatment of cells with TPA for 15-30 min led to partial inhibition of UV- or MNNG-induced JNK activity. Similarly, it has been reported that pretreatment of PC12 cells with nerve growth factor blunted JNK activation by UV irradiation (31). Our results further demonstrated that TPA-induced phosphatase is a JNK-specific phosphatase. However, we also observed a similar effect on the JNKK activity in HOK-16B, HeLa, and HaCaT cells, but not in NHOK with a combination of OV and TPA. At the present time, we do not know that both JNK and JNKK activities were inhibited by a single or by two different TPA-induced phosphatases. MEKK (JNKKK) activity was also reduced by ectopic expressing MEKK in HeLa cells in the presence of TPA (data not shown). This suggests that TPA-induced phosphatase(s) can override or down-modulate the JNK signaling pathway at different levels along the pathway.

Most members of the MKP group of dual specificity phosphatases, if not all, are nuclear proteins (29), whereas MKP-3, MKP-4, and M3/6 are localized in cytoplasm. Our data suggested that the TPA-inducible phosphatase is a cytoplasmic protein because this phosphatase can target MAPK at different levels. The specific subcellular localization of this TPA-induced phosphatase remains to be elucidated. The mechanisms for inactivation of MAK kinases by MAPK phosphatases are highly conserved in species ranging from yeast to humans. However, specific questions such as the function of MAPK phosphatases in a pathological state remain to be answered. To answer these important questions, purification and cloning of this TPA-inducible phosphatase is essential, and the process has been undertaken.

	ACKNOWLEDGEMENTS

We thank Michael Karin for several GST constructs, Dennis McCance for HaCaT cell line, and Junko Nishitani and David Le for technical assistance and valuable discussion. We thank Sharon Hunt Gerardo for valuable comments and for critically reviewing the manuscript.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grants CA66746 (to R. C.) and CA73740 (to A. L.) and by American Heart Association Grant SDG9639261N (to A. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^** To whom correspondence should be addressed.

Published, JBC Papers in Press, May 11, 2000, DOI 10.1074/jbc.M909273199

² S. Chen and R. Chiu, unpublished results.

³ H. Zhou and R. Chiu, unpublished results.

	ABBREVIATIONS

The abbreviations used are: MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; SAPK, stress-activated protein kinase; PP, protein phosphatase; PTP, protein-tyrosine phosphatase; MKP, MAPK-phosphatase; TPA, 12-O-tetradecanoylphorbol-13-acetate; GST, glutathione S-transferase; NHOK, normal human oral keratinocyte; MBP, myelin basic protein; MOPS, 4-morpholinepropanesulfonic acid; OV, orthovanadate; OA, okadaic acid; MNNG, N-methyl-N'-nitrosoguanidine.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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