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Originally published In Press as doi:10.1074/jbc.M205485200 on October 10, 2002

J. Biol. Chem., Vol. 277, Issue 50, 48372-48378, December 13, 2002
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Activation of SAPK/JNK Signaling by Protein Kinase Cdelta in Response to DNA Damage*

Kiyotsugu YoshidaDagger , Yoshio MikiDagger , and Donald Kufe§

From the  Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115 and Dagger  Department of Molecular Genetics, Medical Research Institute, Tokyo Medical and Dental University, Tokyo 113-8510, Japan

Received for publication, June 3, 2002, and in revised form, October 9, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The cellular response to genotoxic stress includes activation of protein kinase Cdelta (PKCdelta ). The functional role of PKCdelta in the DNA damage response is unknown. The present studies demonstrate that PKCdelta is required in part for induction of the stress-activated protein kinase (SAPK/JNK) in cells treated with 1-beta -D-arabinofuranosylcytosine (araC) and other genotoxic agents. DNA damage-induced SAPK activation was attenuated by (i) treatment with rottlerin, (ii) expression of a kinase-inactive PKCdelta (K-R) mutant, and (iii) down-regulation of PKCdelta by small interfering RNA (siRNA). Coexpression studies demonstrate that PKCdelta activates SAPK by an MKK7-dependent, SEK1-independent mechanism. Previous work has shown that the nuclear Lyn tyrosine kinase activates the MEKK1 right-arrow MKK7 right-arrow SAPK pathway but not through a direct interaction with MEKK1. The present results extend those observations by demonstrating that Lyn activates PKCdelta , and in turn, MEKK1 is activated by a PKCdelta -dependent mechanism. These findings indicate that PKCdelta functions in the activation of SAPK through a Lyn right-arrow PKCdelta right-arrow MEKK1 right-arrow MKK7 right-arrow SAPK signaling cascade in response to DNA damage.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The mechanisms by which DNA damage is converted into intracellular signals that control the mammalian genotoxic stress response are largely unknown. Certain insights were derived from the finding that cells respond to agents that arrest DNA replication or damage DNA with induction of c-jun and other early response genes (1-5). Subsequent work showed that DNA damage is associated with activation of the stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK)1 (6-12). Phosphorylation of the c-Jun N terminus by SAPK activates the c-Jun transcription function and thereby autoinduction of the c-jun gene (13, 14). SAPK also activates early response gene expression by phosphorylation of the ATF2 and Elk1 transcription factors (15-17). The available evidence indicates that genotoxic stress activates a nuclear complex of the c-Abl and Lyn protein-tyrosine kinases (18, 19) and that this complex regulates SAPK activation (7, 20). c-Abl functions directly upstream to MEKK1 and activates SAPK by a SEK1-dependent mechanism (8, 21). By contrast, whereas Lyn activates the MEKK1 right-arrow MKK7 right-arrow SAPK pathway, the direct downstream effector of Lyn is not known (20).

The protein kinase C (PKC) family of serine/threonine kinases has been subdivided into the following: (i) the conventional PKCs (alpha , beta , and gamma ), which are calcium-dependent and activated by diacylglycerol, (ii) the novel PKCs (delta , epsilon , theta , and µ), which are calcium-independent and activated by diacylglycerol, and (iii) the atypical PKCs (zeta  and lambda ), which are calcium-independent and not activated by diacylglycerol (22). The ubiquitously expressed novel PKC, PKCdelta , is tyrosine-phosphorylated and activated by c-Abl in the response to DNA damage (23). As found for c-Abl and Lyn (24, 25), PKCdelta interacts with the nuclear DNA-dependent protein kinase catalytic subunit (DNA-PKcs) (26). Phosphorylation of DNA-PKcs by PKCdelta inhibits the function of DNA-PKcs to form complexes with DNA and to phosphorylate downstream targets (26). Other studies have demonstrated that the nuclear complex of c-Abl and Lyn includes the protein-tyrosine phosphatase, SHPTP1 (27, 28), and that PKCdelta phosphorylates and inactivates SHPTP1 in the response to DNA damage (29). In cells that respond to genotoxic stress with the induction of apoptosis, PKCdelta is cleaved by caspase-3 to a constitutively active catalytic fragment (PKCdelta CF) (30, 31). The finding that PKCdelta CF induces nuclear condensation and DNA fragmentation has indicated that cleavage of PKCdelta contributes to the apoptotic response (32).

The present studies have addressed the involvement of PKCdelta in the activation of stress signals in response to arrest of DNA replication and to DNA damage. The results demonstrate that PKCdelta is required in part for activation of SAPK. The results also demonstrate that PKCdelta transduces Lyn-mediated signals to MEKK1 in a Lyn right-arrow PKCdelta right-arrow MEKK1 right-arrow MKK7 right-arrow SAPK pathway.

    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell Culture-- Human U-937 myeloid leukemia cells were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 100 units/ml penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine. MCF-7 cells, HeLa cells, and 293T embryonal kidney cells were grown in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and antibiotics. Cells were treated with araC (Sigma-Aldrich), rottlerin (Calbiochem), or Gö6976 (Calbiochem). Irradiation was performed at room temperature with a Gammacell 1000 (Atomic Energy of Canada) and a 137Cs source emitting at a fixed rate of 0.21 gray/min.

Cell Transfections-- The PKCdelta (K-R) cDNA (29) was cloned into a pcDNA3.1/myc-His vector (Invitrogen) and introduced stably into U-937 cells by electroporation (Gene Pulser; Bio-Rad) and selection in geneticin (Roche Molecular Biochemicals). U-937/Lyn(K-R) cells were prepared as described previously (20, 28). 293T cells were transiently transfected with pGFP, pGFP-PKCdelta CF, pGFP-PKCdelta CF(K-R) (29), pFLAG, pFLAG-SEK1(K-R), pFLAG-MKK7(K-R), or pFLAG-MEKK1(K-M) (20) by the calcium phosphate method. DNA concentrations were adjusted with empty vector.

siRNA Transfections-- siRNA duplexes (siRNAs) were synthesized and purified by Japan Bio Service (Saitama, Japan). The siRNA sequences for targeting PKCdelta were 5'-GAUGAAGGAGGCGCUCAGTT3' for PKCdelta siRNA1 and 5'-GGCUGAGUUCUGGCUGGACTT-3' for PKCdelta siRNA2. GFPsiRNA was used as a negative control (33). Transfection of siRNAs was performed as described (34).

Immunoprecipitations-- Cell lysates were prepared as described (20, 28) and cleared by centrifugation at 14000 rpm for 15 min. Soluble proteins were incubated with anti-JNK1 (sc-474; Santa Cruz Biotechnology, Inc.) (SCBT), anti-ERK2 (sc-154; SCBT), anti-p38 MAPK (sc-535; SCBT), anti-PKCdelta (sc-937; SCBT), anti-Lyn (sc-15; SCBT), or anti-MEKK1 (21) antibodies for 2-6 h at 4 °C followed by 1 h of incubation with protein A/G-Sepharose beads (SCBT). The immune complexes were washed three times with lysis buffer and separated by SDS-PAGE.

In Vitro Kinase Assays-- In vitro kinase assays for SAPK, ERK, and p38 MAPK were performed as described (20, 29). Anti-PKCdelta immunoprecipitates were incubated with histone H1 as a substrate (29).

Immunoblot Analyses-- Proteins were separated by SDS-PAGE and transferred to nitrocellulose filters. After blocking with 5% dry milk in phosphate-buffered saline containing 0.05% Tween 20, the filters were incubated with anti-JNK1/SAPK, anti-ERK2, anti-p38 MAPK, anti-PKCdelta , anti-Lyn (Transduction Laboratories), anti-MEKK1, anti-tubulin (Sigma-Aldrich), anti-GFP (Roche Molecular Biochemicals), anti-GST (Upstate Biotechnology Inc.), anti-FLAG (Sigma-Aldrich), or anti-phospho-Tyr (4G10; Upstate Biotechnology, Inc.) antibodies for 1-4 h at room temperature. The antigen-antibody complexes were visualized by chemiluminescence (PerkinElmer Life Sciences).

Glycerol Gradient Sedimentation Analysis-- Cosedimentation analysis of PKCdelta and Lyn by glycerol gradient ultracentrifugation was performed as described (35).

Direct Binding Assays-- Assays for direct binding of PKCdelta and Lyn in vitro were performed as described previously (35).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

PKCdelta Functions in Activation of SAPK in Response to DNA Damage-- To investigate whether PKCdelta functions as an upstream effector of the SAPK response to genotoxic agents, U-937 cells were pretreated with PKCdelta inhibitor, rottlerin, followed by exposure to araC. Anti-SAPK immunoprecipitates were subjected to in vitro kinase assays by phosphorylating GST-c-Jun. The results demonstrate that pretreatment with rottlerin attenuates araC-induced activation of SAPK (Fig. 1A). By contrast, there was no detectable effect of pretreatment with the PKCalpha and PKCbeta inhibitor, Gö6976 (Fig. 1A). Similar results were obtained with U-937 cells exposed to ionizing radiation (IR) (Fig. 1B). To determine whether PKCdelta is required for the induction of other MAPK family members, ERK and p38 MAPK, cells exposed to rottlerin and then araC or IR were subjected to immunoprecipitation with anti-ERK2 and anti-p38 MAPK. Analysis of the immunoprecipitates for phosphorylation of myelin basic protein (MBP) or ATF-2 demonstrated that rottlerin has less of an inhibitory effect on araC- or IR-induced p38 MAPK activity and no apparent effect on ERK activation (Fig. 1, C and D and data not shown). These results suggest that activation of SAPK is at least in part dependent on PKCdelta in the response to genotoxic agents.


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  		Fig. 1.   DNA damage-induced activation of SAPK, but not ERK or p38 MAPK, is attenuated by rottlerin. A and B, U-937 cells were pretreated with 5 µM rottlerin or 50 nM Gö6978 for 30 min. The cells were then treated with 10 µM araC (A) or 15 gray of IR (B), harvested at the indicated times, lysed, and subjected to immunoprecipitation (IP) with anti-SAPK. The immunoprecipitates were incubated with GST-c-Jun and [gamma -32P]ATP. GST-c-Jun phosphorylation was assessed by SDS-PAGE and autoradiography (upper panels). The lysates were also subjected to immunoblot analysis (IB) with anti-SAPK (lower panel). C and D, cells were pretreated with rottlerin and then exposed 10 µM araC. Anti-ERK (C) or anti-p38 MAPK (D) immunoprecipitates were subjected to immune complex kinase assays with MBP or GST-ATF2, respectively, as substrate (upper panels). The lysates were also subjected to immunoblot analysis with anti-ERK (C) or anti-p38 MAPK (D) (lower panels).

Attenuation of SAPK Activation in Cells Expressing a Kinase-inactive PKCdelta Mutant-- To further assess involvement of PKCdelta in SAPK activation, a Myc-tagged kinase-inactive PKCdelta (K-R) mutant was introduced stably in U-937 cells. In contrast to U-937 cells expressing the empty vector, two separate clones of transfected cells expressed Myc-PKCdelta (K-R) (Fig. 2A). Anti-PKCdelta immunoprecipitates from control and araC-treated U-937/neo and U-937/PKCdelta (K-R) cells were analyzed for phosphorylation of histone H1. The results demonstrate that activation of PKCdelta by araC is abrogated in U-937 cells expressing PKCdelta (K-R) (Fig. 2B). To confirm the involvement of PKCdelta in DNA damage-induced activation of SAPK, U-937/neo and U-937/PKCdelta (K-R) cells exposed to araC were analyzed for SAPK activity. The results demonstrate that activation of SAPK is attenuated, but not completely inhibited, in araC-treated cells expressing kinase-negative PKCdelta (K-R) (Fig. 2C). Similar results were obtained in IR-treated cells (Fig. 2D). By contrast, DNA damage-induced ERK and p38 MAPK activation were unaffected in U-937/PKCdelta (K-R) cells, as compared with that in U-937/neo cells (data not shown). These findings indicate that PKCdelta is involved in SAPK activation in the cellular response to diverse genotoxic agents.


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  		Fig. 2.   Kinase-inactive PKCdelta (K-R) mutant attenuates SAPK activation in response to DNA damage. A, lysates from U-937/neo and U-937/PKCdelta (K-R) (clone 1 and 2) cells were analyzed by immunoblotting (IB) with anti-PKCdelta (upper panel), anti-Myc (middle panel), or anti-tubulin (lower panel). B, cells were left untreated or treated with 10 µM araC for 1 h. Immunoprecipitates (IP) prepared with the anti-PKCdelta antibody (sc-937) were assayed for phosphorylation of histone H1. C and D, cells were treated with 10 µM araC (C) or 15 gray of IR (D) and harvested at the indicated times. Anti-SAPK immunoprecipitates were assayed for phosphorylation of GST-c-Jun (upper panels). Lysates were also subjected to immunoblot analysis with anti-SAPK (lower panels).

Attenuation of SAPK Activation in Cells Transfected with PKCdelta siRNAs or a Kinase-inactive MKK7(K-R) Mutant-- To further assess the role of PKCdelta in SAPK activation, 293T cells were treated with siRNA duplexes that target PKCdelta . The results demonstrate that the PKCdelta siRNA1 down-regulates PKCdelta expression (Fig. 3A). Less pronounced results were obtained with PKCdelta siRNA2 (Fig. 3A). As a control, treatment with a GFPsiRNA had little if any effect (Fig. 3A). Importantly, treatment with PKCdelta siRNA, but not GFPsiRNA, attenuated araC-induced SAPK activation (Fig. 3B). To determine whether the kinase function of PKCdelta is sufficient for SAPK activation, 293T cells were transfected with PKCdelta CF. Analysis of anti-SAPK immunoprecipitates for phosphorylation of GST-c-Jun demonstrated that expression of PKCdelta CF induces SAPK activity (Fig. 3C). By contrast, expression of kinase-inactive PKCdelta CF(K-R) had little effect on SAPK activity (Fig. 3C). Similar results were obtained in HeLa cells expressing PKCdelta CF or PKCdelta CF(K-R) (data not shown). To define the effectors downstream to PKCdelta in the SAPK pathway, 293T cells were co-transfected with PKCdelta CF and kinase-inactive SEK1(K-R) or MKK7(K-R). Analysis of anti-SAPK immunoprecipitates demonstrated that expression of MKK7(K-R), but not SEK1(K-R), is associated with attenuation of SAPK activity by PKCdelta (Fig. 3D). These results indicate that PKCdelta induces SAPK activation by an MKK7-dependent, SEK1/MKK4-independent mechanism.


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  		Fig. 3.   SAPK activation is attenuated by transfections with PKCdelta siRNAs or a kinase-inactive MKK7(K-R) mutant. A, 293T cells were transfected with GFPsiRNA, PKCdelta siRNA1, or PKCdelta siRNA2. Cell lysates were analyzed by immunoblotting (IB) with anti-PKCdelta (upper panel) or anti-tubulin (lower panel). B, 293T cells transfected with GFPsiRNA or PKCdelta siRNA1 were treated with 10 µM araC at the indicated times. Anti-SAPK immunoprecipitates were subjected to immune complex kinase assays using GST-c-Jun as a substrate (upper panel). Lysates were analyzed by immunoblotting with anti-PKCdelta (middle panel) or anti-SAPK (lower panel). C, 293T cells were transfected with pGFP, pGFP-PKCdelta CF, or pGFP-PKCdelta CF(K-R). At 48 h post-transfection, anti-SAPK immunoprecipitates were subjected to immune complex kinase assays using GST-c-Jun as a substrate (upper panel). Lysates were analyzed by immunoblotting with anti-GFP (middle panel) or anti-SAPK (lower panel). D, 293T cells were transfected with pGFP, pGFP-PKCdelta CF, pFLAG, pFLAG-SEK1(K-R), or pFLAG-MKK7(K-R). Anti-SAPK immunoprecipitates were analyzed for phosphorylation of GST-c-Jun (top panel). Lysates were analyzed by immunoblotting with anti-GFP (second panel), anti-FLAG (third panel), or anti-SAPK (bottom panel).

Lyn Tyrosine Kinase Is an Upstream Effector of PKCdelta in Response to Genotoxic Stress-- Previous studies have demonstrated that the Lyn tyrosine kinase is activated in the response to genotoxic stress and that Lyn regulates SAPK activation by an MKK7-dependent pathway (20). Whereas the present results indicate that the PKCdelta -mediated SAPK signaling pathway is also MKK7-dependent, we asked whether Lyn is involved in PKCdelta right-arrow SAPK signaling. To determine whether PKCdelta binds to Lyn, we incubated glutathione beads containing GST or GST-PKCdelta with purified Lyn. Analysis of the adsorbates demonstrated binding of Lyn with GST-PKCdelta and not GST (Fig. 4A). To determine whether PKCdelta phosphorylates Lyn in vitro, heat-inactivated purified Lyn was incubated with recombinant PKCdelta . Analysis of the reaction products demonstrated no detectable phosphorylation of Lyn by PKCdelta (Fig. 4B, lane 2). By contrast, incubation of heat-inactivated PKCdelta with purified Lyn showed that Lyn phosphorylates PKCdelta (Fig. 4B, lane 5). These findings provide support for a direct interaction between PKCdelta and Lyn.


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  		Fig. 4.   PKCdelta interacts directly with Lyn. A, glutathione beads containing GST or GST-PKCdelta were incubated with purified Lyn. The adsorbates were analyzed by immunoblotting (IB) with anti-Lyn (upper panel) or anti-GST (lower panel). B, kinase-active recombinant PKCdelta was incubated with heat-inactivated purified Lyn (H.I. Lyn) and [gamma -32P]ATP for 15 min. Kinase-active purified Lyn was also incubated with heat-inactivated recombinant PKCdelta (H.I. PKCdelta ). The reaction products were analyzed by SDS-PAGE and autoradiography (upper panel) or immunoblotting with anti-PKCdelta and anti-Lyn (lower panel).

To determine whether Lyn contributes to the regulation of PKCdelta in vivo, anti-Lyn immunoprecipitates from control and araC-treated cells were analyzed by immunoblotting with anti-PKCdelta . The results demonstrate that Lyn associates constitutively with PKCdelta and that araC treatment has little if any effect on the extent of the interaction (Fig. 5A). These results were confirmed when anti-PKCdelta immunoprecipitates were subjected to immunoblotting with anti-Lyn (Fig. 5A). A constitutive association between Lyn and PKCdelta was also observed in MCF-7 and HeLa cells (data not shown). To confirm these findings with another approach, lysates from U-937 cells were separated by sedimentation in a glycerol gradient. Analysis of the gradient fractions by immunoblotting with anti-Lyn and anti-PKCdelta demonstrated cosedimentation of Lyn and PKCdelta (Fig. 5B). To extend the analysis, anti-PKCdelta immunoprecipitates from araC-treated U-937/neo and U-937/Lyn(K-R) cells were analyzed for tyrosine phosphorylation of PKCdelta by immunoblotting with anti-phospho-Tyr. The results demonstrate that expression of Lyn(K-R) blocks tyrosine phosphorylation of PKCdelta in response to araC (Fig. 5C). In concert with these findings and the demonstration that PKCdelta is activated by tyrosine phosphorylation (23), Lyn(K-R) also attenuated the induction of PKCdelta activity by araC (Fig. 5D, left). Conversely, to determine whether PKCdelta regulates Lyn in the DNA damage response, anti-Lyn immunoprecipitates from U-937/neo and U-937/PKCdelta (K-R) cells were analyzed for Lyn activity by assessing autophosphorylation and transphosphorylation of enolase. The finding that araC-induced activation of Lyn is comparable in U-937/neo and U-937/PKCdelta (K-R) cells (Fig. 5D, right) suggests that Lyn may function as an upstream effector of PKCdelta .


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  		Fig. 5.   Lyn is an upstream effector of PKCd. A, U-937 cells were left untreated or treated with 10 µM araC for 1 h. Cell lysates were subjected to immunoprecipitation with anti-Lyn, pre-immune rabbit serum (PIRS), or anti-PKCdelta . The immunoprecipitates were analyzed by immunoblotting (IB) with anti-Lyn or anti-PKCdelta . B, lysates from U-937 cells were layered onto a 10 to 35% glycerol gradient. After centrifugation, the indicated fractions (fr.) were subjected to SDS-PAGE and immunoblotting with anti-PKCdelta and anti-Lyn. C, U-937/neo and U-937/Lyn(K-R) cells were treated with 10 µM araC for 1 and 2 h. Anti-PKCdelta immunoprecipitates were subjected to immunoblot analysis with anti-phospho-Tyr (upper panel) or anti-PKCdelta (lower panel). D, lysates from control and araC-treated U-937/neo and U-937/Lyn(K-R) cells were subjected to immunoprecipitation with anti-PKCdelta (left panel). Lysates from control and araC-treated U-937/neo and U-937/PKCdelta (K-R) cells were subjected to immunoprecipitation with anti-Lyn (right panel). Immune complex kinase assays were performed by incubating the precipitates with histone H1 for PKCdelta activity (left panel) and with enolase for Lyn activity (right panel).

PKCdelta Is an Upstream Effector of MEKK1-- Our previous studies showed that Lyn-induced SAPK activation is MEKK1-dependent (20). To investigate the possibility that PKCdelta interacts with MEKK1, anti-MEKK1 immunoprecipitates were analyzed by immunoblotting with anti-PKCdelta . The results show that complexes of PKCdelta and MEKK1 are detectable in control and araC-treated cells (Fig. 6A, left panel). araC-induced increases in the association of PKCdelta and MEKK1 were detectable at 0.5 h and maximal at 2 h of treatment (Fig. 6A, right panel). To further assess the interaction between PKCdelta and MEKK1, we first performed in vitro kinase assays by incubating recombinant PKCdelta with GST or GST-MEKK1 in the presence of [gamma -32P]ATP. Analysis of the products demonstrated that PKCdelta phosphorylates MEKK1 in vitro (Fig. 6B). To further define whether PKCdelta activates MEKK1, GST-MKK7(K-R) was co-incubated with or without recombinant PKCdelta and kinase-active MEKK1 (yeast-derived) in the presence of [gamma -32P]ATP. The results demonstrate that MEKK1 phosphorylates GST-MKK7(K-R) and that the addition of recombinant PKCdelta increases MEKK1-mediated MKK7(K-R) phosphorylation (Fig. 6C, lane 3). As a control, there was no detectable phosphorylation of MKK7(K-R) by PKCdelta (Fig. 6C, lane 1). To extend these findings, anti-SAPK immunoprecipitates from 293T cells expressing PKCdelta CF and FLAG-tagged kinase-inactive MEKK1(K-M) were analyzed for phosphorylation of GST-c-Jun. The results demonstrate that expression of MEKK1(K-M) decreases PKCdelta -induced SAPK activation in a dose-dependent manner (Fig. 6D). These results collectively indicate that MEKK1 is a downstream effector of PKCdelta in the SAPK signaling pathway.


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  		Fig. 6.   MEKK1 is a downstream effector for PKCdelta . A, left, U-937 cells were left untreated or treated with 10 µM araC for 1 h. Cell lysates were subjected to immunoprecipitation with pre-immune rabbit serum (PIRS) or anti-MEKK1. The immunoprecipitates were analyzed by immunoblotting (IB) with anti-PKCdelta (upper panel) or anti-MEKK1 (middle panel). As a loading control, lysates used for immunoprecipitation were also subjected to immunoblot analysis with anti-tubulin (lower panel). Right, lysates from U-937 cells treated with 10 µM araC for the indicated times were subjected to immunoprecipitation with anti-MEKK1. The immunoprecipitates (IP) were analyzed by immunoblotting with anti-PKCdelta (upper panel) or anti-MEKK1 (lower panel). B, kinase-active recombinant PKCdelta was incubated with kinase-inactive GST-MEKK1 (Escherichia coli-derived) and [gamma -32P]ATP for 30 min. The reaction products were analyzed by SDS-PAGE and autoradiography. C, kinase-active GST-MEKK1 (yeast-derived) bound to glutathione beads was incubated with alkaline phosphatase. After washing, the beads were incubated in the absence or presence of recombinant PKCdelta and ATP. The GST-MEKK1-containing beads were washed again and then incubated with GST-MKK7(K-R) and [gamma -32P]ATP. D, 293T cells were transfected with pGFP, pGFP-PKCdelta CF, pFLAG, or pFLAG-MEKK1(K-M). Anti-SAPK immunoprecipitates were subjected to in vitro kinase assays using GST-Jun as substrate (upper panel). Lysates were also subjected to immunoblot analysis with anti-GFP (middle panel) or anti-FLAG (lower panel).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

PKCdelta Is Activated by Different Mechanisms in DNA Damage Response-- Involvement of PKCdelta in the genotoxic stress response has been supported by the finding that both arrest of DNA replication and induction of DNA lesions are associated with PKCdelta activation (23, 29). The available evidence indicates that full-length PKCdelta is activated as an early event within 1 h of exposure to genotoxic agents (23, 29). Phosphorylation of PKCdelta on tyrosine as a mechanism for PKCdelta activation by DNA-damaging agents is mediated in part by the c-Abl kinase (23, 36). The results of the present study demonstrate that Lyn also phosphorylates PKCdelta and that Lyn-mediated tyrosine phosphorylation of PKCdelta contributes to PKCdelta activation.

PKCdelta is also activated as a later event (3-6 h) in the DNA damage response by caspase-3-mediated proteolytic cleavage (23, 30, 31, 37). The resulting C-terminal 40-kDa fragment contains the catalytic domain, which, in the absence of the N-terminal regulatory domain, is constitutively active (30-32). The finding that tyrosine phosphorylation of PKCdelta is required for activation of caspase-3 and thereby PKCdelta cleavage has supported a link between both mechanisms of PKCdelta activation (36). Whereas expression of PKCdelta CF induces characteristics of apoptosis (32), the precise events responsible for this response are unknown but may involve an interaction between PKCdelta CF and DNA-PKcs (26). In contrast to the activation of PKCdelta by tyrosine phosphorylation, cleavage of PKCdelta to the constitutively active catalytic fragment is irreversible and thus may function in the prolonged stimulation of multiple pro-apoptotic pathways.

Role for PKCdelta in SAPK Activation-- SAPK is activated in diverse cell types by agents, such as araC, that block DNA replication and by others, such as IR, that induce DNA lesions (7-12). Like SAPK, PKCdelta is also activated by both arrest of DNA replication and DNA damage (23, 29). The present results demonstrate that treatment of cells with the PKCdelta inhibitor, rottlerin, attenuates activation of SAPK in response to genotoxic stress. In concert with these findings, expression of the kinase-inactive PKCdelta (K-R) mutant attenuated activation of both PKCdelta and SAPK in response to araC and IR. Moreover, down-regulation of PKCdelta expression by siRNA was associated with attenuation of DNA damage-induced SAPK activation. These findings provided support for the involvement of PKCdelta as an upstream effector in the regulation of SAPK activation. The results also demonstrate that inhibition of PKCdelta signaling attenuates the early (<1 h) and later periods of SAPK activation. Thus, SAPK is activated by signals transduced by PKCdelta and possibly, after activation of caspase-3, by PKCdelta CF. In this context, the results show that expression of PKCdelta CF is also associated with SAPK activation.

Previous studies have shown that nuclear c-Abl is an upstream effector of the SAPK response to both arrest of DNA replication and DNA damage (7, 8). Other work has demonstrated that activated forms of Abl induce SAPK activity (5, 38, 39). Lyn forms a nuclear complex with c-Abl and, like c-Abl, also contributes to SAPK activation in response to genotoxic stress (20). Whereas c-Abl activates SAPK by a SEK1-dependent mechanism, Lyn right-arrow SAPK signaling is mediated by MKK7 (8, 20). The respective roles of the c-Abl right-arrow SEK1 right-arrow SAPK and the Lyn right-arrow MKK7 right-arrow SAPK pathways in DNA damage response may vary in different cell types or under different growth conditions. Nonetheless, the finding that inhibition of PKCdelta attenuates SAPK activation in response to araC and IR indicates that PKCdelta affects one or both of the pathways.

Evidence for a Lyn right-arrow PKCdelta right-arrow MEKK1 right-arrow MKK7 right-arrow SAPK Cascade-- c-Abl interacts directly with MEKK1 and activates MEKK1 in response to DNA damage (21). In turn, MEKK1 transduces c-Abl-mediated signals to SEK1 and SAPK (8, 21) (Fig. 7). The finding that kinase-inactive MEKK1(K-M) blocks Lyn-mediated activation of SAPK provided support for the involvement of MEKK1 in both the c-Abl and Lyn pathways (Fig. 7). However, unlike studies with c-Abl (21), the available evidence failed to support a direct interaction between Lyn and MEKK1 (20). The present findings demonstrate that Lyn associates with PKCdelta and that tyrosine phosphorylation and activation of PKCdelta is mediated in part by a Lyn-dependent mechanism. The results further demonstrate that PKCdelta associates with MEKK1 and that the interaction between PKCdelta and MEKK1 is increased in response to DNA damage. Moreover, phosphorylation of MEKK1 by PKCdelta stimulated MEKK1 activity. These findings thus collectively support a model in which PKCdelta functions downstream to Lyn and as an upstream effector of the MEKK1 right-arrow MKK7 right-arrow SAPK pathway (Fig. 7).


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  		Fig. 7.   Model for activation of SAPK in response to genotoxic stress. Effectors are shown for the Lyn right-arrow SAPK and c-Abl right-arrow SAPK signaling pathways. Cross-talk between the pathways could occur through interactions between (i) PKCdelta and c-Abl and/or (ii) MEKK1 and SEK1/MKK7.

SAPK is activated in the response of cells to environmental stress (40). MKK7 is a specific activator of SAPK, whereas SEK1 (also known as MKK4) activates both SAPK and p38 MAPK. In turn and depending on the type of stress, upstream effectors of MKK7 and SEK1 include MEKK1-4 and the mixed-lineage protein kinases, ASK1-2, TAK1, and TPL2 (40). The available evidence indicates that MEKK1 is activated by the Rho family GTPases (41), TRAF family members (42), the ECSIT adapter protein (43), protein kinase G (44), and c-Abl (21). The results of the present studies provide support for PKCdelta as yet another effector of MEKK1 activation. PKCdelta is activated in response to growth factor receptor stimulation (45, 46), DNA-damage (23), and oxidative stress (47). PKCdelta is also activated by caspase-3-mediated cleavage in the apoptotic response. Thus, although the present work has focused on involvement of PKCdelta in DNA damage-induced signaling, the findings do not exclude the possibility that PKCdelta or PKCdelta CF contributes to SAPK activation in response to other types of stress.

    ACKNOWLEDGEMENTS

We acknowledge Kamal Chauhan and Tomoko Yamaguchi for excellent technical assistance.

    FOOTNOTES

* This work was supported by NCI, National Institutes of Health Grants CA29431 and CA55241.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. Tel.: 617-632-3141; Fax: 617-632-2934; E-mail: Donald-Kufe@dfci.harvard.edu.

Published, JBC Papers in Press, October 10, 2002, DOI 10.1074/jbc.M205485200

    ABBREVIATIONS

The abbreviations used are: SAPK/JNK, stress-activated protein kinase; PKC, protein kinase C; araC, 1-beta -D-arabinofuranosylcytosine; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; CF, catalytic fragment; GST, glutathione S-transferase; GFP, green fluorescence protein; MEKK, MAPK kinase kinase; SEK, SAPK/ERK kinase; MKK, MAPK kinase; PKcs, protein kinase catalytic subunit; siRNA, small interfering RNA; IR, ionizing radiation; MBP, myelin basic protein; SCBT, Santa Cruz Biotechnology, Inc.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1. Sherman, M., Datta, R., Hallahan, D., Weichselbaum, R., and Kufe, D. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 5663-5666[Abstract/Free Full Text]
2. Datta, R., Kharbanda, S., and Kufe, D. (1990) Mol. Pharmacol. 38, 435-439[Abstract]
3. Rubin, E., Kharbanda, S., Gunji, H., and Kufe, D. (1991) Mol. Pharm. 39, 697-701[Abstract]
4. Kharbanda, S., Datta, R., and Kufe, D. (1991) Biochemistry 30, 7947-7952[CrossRef][Medline] [Order article via Infotrieve]
5. Sanchez, I., Hughes, R. T., Mayer, B. J., Yee, K., Woodgett, J. R., Avruch, J., Kyriakis, J. M., and Zon, L. I. (1994) Nature 372, 794-798[Medline] [Order article via Infotrieve]
6. Saleem, A., Datta, R., Kharbanda, S., and Kufe, D. (1995) Cell Growth Differ. 6, 1651-1658[Abstract]
7. Kharbanda, S., Ren, R., Pandey, P., Shafman, T. D., Feller, S. M., Weichselbaum, R. R., and Kufe, D. W. (1995) Nature 376, 785-788[CrossRef][Medline] [Order article via Infotrieve]
8. Kharbanda, S., Pandey, P., Ren, R., Feller, S., Mayer, B., Zon, L., and Kufe, D. (1995) J. Biol. Chem. 270, 30278-30281[Abstract/Free Full Text]
9. Chen, Y. R., Meyer, C. F., and Tan, T. H. (1996) J. Biol. Chem. 271, 631-634[Abstract/Free Full Text]
10. Chen, Y.-R., Wang, X., Templeton, D., Davis, R. J., and Tan, T.-H. (1996) J. Biol. Chem. 271, 31929-31936[Abstract/Free Full Text]
11. Verheij, M., Bose, R., Lin, X. H., Yhao, B., Jarvis, W. D., Grant, S., Birrer, M. J., Szabo, E., Zon, L. I., Kyriakis, J. M., Haimovitz-Friedman, A., Fuks, Z., and Kolesnick, R. N. (1996) Nature 380, 75-79[CrossRef][Medline] [Order article via Infotrieve]
12. Zanke, B. W., Boudreau, K., Rubie, E., Winnett, E., Tibbles, L. A., Zon, L., Kyriakis, J., Liu, F.-F., and Woodgett, J. R. (1996) Curr. Biol. 6, 606-613[CrossRef][Medline] [Order article via Infotrieve]
13. Derijard, B., Hibi, M., Wu, I. H., Barrett, T., Su, B., Deng, T., Karin, M., and Davis, R. J. (1994) Cell 76, 1025-1037[CrossRef][Medline] [Order article via Infotrieve]
14. Kyriakis, J. M., Banerjee, P., Nikolakaki, E., Dai, T., Rubie, E. A., Ahmad, M. F., Avruch, J., and Woodgett, J. R. (1994) Nature 369, 156-160[CrossRef][Medline] [Order article via Infotrieve]
15. Gupta, S., Campbell, D., Dérijard, B., and Davis, R. J. (1995) Science 267, 389-393[Abstract/Free Full Text]
16. Raingeaud, J., Whitmarsh, A. J., Barrett, T., Derijard, B., and Davis, R. J. (1996) Mol. Cell. Biol. 16, 1247-1255[Abstract]
17. Whitmarsh, A. J., Shore, P., Sharrocks, A. D., and Davis, R. J. (1995) Science 269, 403-407[Abstract/Free Full Text]
18. Kharbanda, S., Yuan, Z. M., Rubin, E., Weichselbaum, R., and Kufe, D. (1994) J. Biol. Chem. 269, 20739-20743[Abstract/Free Full Text]
19. Yuan, Z., Kharbanda, S., and Kufe, D. (1995) Biochemistry 34, 1058-1063[CrossRef][Medline] [Order article via Infotrieve]
20. Yoshida, K., Weichselbaum, R., Kharbanda, S., and Kufe, D. (2000) Mol. Cell. Biol. 20, 5370-5380[Abstract/Free Full Text]
21. Kharbanda, S., Pandey, P., Yamauchi, T., Kumar, S., Kaneki, M., Kumar, V., Bharti, A., Yuan, Z., Ghanem, L., Rana, A., Weichselbaum, R., Johnson, G., and Kufe, D. (2000) Mol. Cell. Biol. 20, 4979-4989[Abstract/Free Full Text]
22. Nishizuka, Y. (1988) Nature 334, 661-665[CrossRef][Medline] [Order article via Infotrieve]
23. Yuan, Z.-M., Utsugisawa, T., Ishiko, T., Nakada, S., Huang, Y., Kharbanda, S., Weichselbaum, R., and Kufe, D. (1998) Oncogene 16, 1643-1648[CrossRef][Medline] [Order article via Infotrieve]
24. Kharbanda, S., Pandey, P., Jin, S., Inoue, S., Bharti, A., Yuan, Z.-M., Weichselbaum, R., Weaver, D., and Kufe, D. (1997) Nature 386, 732-735[CrossRef][Medline] [Order article via Infotrieve]
25. Kumar, S., Pandey, P., Bharti, A., Jin, S., Weichselbaum, R., Weaver, D., Kufe, D., and Kharbanda, S. (1998) J. Biol. Chem. 273, 25654-25658[Abstract/Free Full Text]
26. Bharti, A., Kraeft, S.-K., Gounder, M., Pandey, P., Jin, S., Yuan, Z.-M., Lees-Miller, S. P., Weichselbaum, R., Weaver, D., Chen, L. B., Kufe, D., and Kharbanda, S. (1998) Mol. Cell. Biol. 18, 6719-6728[Abstract/Free Full Text]
27. Kharbanda, S., Bharti, A., Pei, D., Wang, J., Pandey, P., Ren, R., Weichselbaum, R., Walsh, C. T., and Kufe, D. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 6898-6901[Abstract/Free Full Text]
28. Yoshida, K., Kharbanda, S., and Kufe, D. (1999) J. Biol. Chem. 274, 34663-34668[Abstract/Free Full Text]
29. Yoshida, K., and Kufe, D. (2001) Mol. Pharmacol. 60, 1431-1438[Abstract/Free Full Text]
30. Emoto, Y., Manome, G., Meinhardt, G., Kisaki, H., Kharbanda, S., Robertson, M., Ghayur, T., Wong, W. W., Kamen, R., Weichselbaum, R., and Kufe, D. (1995) EMBO J. 14, 6148-6156[Medline] [Order article via Infotrieve]
31. Emoto, Y., Kisaki, H., Manome, Y., Kharbanda, S., and Kufe, D. (1996) Blood 87, 1990-1996[Abstract/Free Full Text]
32. Ghayur, T., Hugunin, M., Talanian, R. V., Ratnofsky, S., Quinlan, C., Emoto, Y., Pandey, P., Datta, R., Kharbanda, S., Allen, H., Kamen, R., Wong, W., and Kufe, D. (1996) J. Exp. Med. 184, 2399-2404[Abstract/Free Full Text]
33. Hirai, I., and Wang, H. G. (2002) J. Biol. Chem. 277, 25722-25727[Abstract/Free Full Text]
34. Elbashir, S. M., Harborth, J., Weber, K., and Tuschl, T. (2002) Methods 26, 199-213[CrossRef][Medline] [Order article via Infotrieve]
35. Yoshida, K., Komatsu, K., Wang, H.-G., and Kufe, D. (2002) Mol. Cell. Biol. 22, 3292-3300[Abstract/Free Full Text]
36. Blass, M., Kronfeld, I., Kazimirsky, G., Blumberg, P., and Brodie, C. (2002) Mol. Cell. Biol. 22, 182-195[Abstract/Free Full Text]
37. Koriyama, H., Kouchi, Z., Umeda, T., Saido, T., Mamoi, T., Ishiura, S., and Suzuki, K. (1999) Cell. Signal. 11, 831-838[CrossRef][Medline] [Order article via Infotrieve]
38. Raitano, A. B., Halpern, J. R., Hambuch, T. M., and Sawyers, C. L. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 11746-11750[Abstract/Free Full Text]
39. Renshaw, M. W., McWhirter, J. R., and Wang, J. Y. J. (1995) Mol. Cell. Biol. 15, 1286-1293[Abstract]
40. Davis, R. (2000) Cell 103, 239-252[CrossRef][Medline] [Order article via Infotrieve]
41. Fanger, G., Schlesinger, T., and Johnson, G. (2000)
42. Baud, V., Liu, Z. G., Bennett, B., Suzuki, N., Xia, Y., and Karin, M. (1999) Genes Dev. 13, 1297-1308[Abstract/Free Full Text]
43. Kopp, E., Medzhitov, R., Carothers, J., Xiao, C., Douglas, I., Janeway, C. A., and Ghosh, S. (1999) Genes Dev. 13, 2059-2071[Abstract/Free Full Text]
44. Soh, J. W., Mao, Y., Liu, L., Thompson, W. J., Pamukcu, R., and Weinstein, I. B. (2001) J. Biol. Chem. 276, 16406-16410[Abstract/Free Full Text]
45. Li, W., Yu, J. C., Michieli, P., Beeler, J. F., Ellmore, N., Heidaran, M. A., and Pierce, J. H. (1994) Mol. Cell. Biol. 14, 6727-6735[Abstract/Free Full Text]
46. Denning, M. F., Dlugosz, A. A., Threadgill, D. W., Magnuson, T., and Yuspa, S. H. (1996) J. Biol. Chem. 271, 5325-5331[Abstract/Free Full Text]
47. Konishi, H., Tanaka, M., Takemura, Y., Matsuzaki, H., Ono, Y., Kikkawa, U., and Nishizuka, Y. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 11223-11237

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