HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 277, Issue 50, 48372-48378, December 13, 2002

This Article Abstract Full Text (PDF) All Versions of this Article: 277/50/48372    most recent M205485200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Yoshida, K. Articles by Kufe, D. Search for Related Content PubMed PubMed Citation Articles by Yoshida, K. Articles by Kufe, D. Social Bookmarking                      What's this?

Activation of SAPK/JNK Signaling by Protein Kinase C in Response to DNA Damage^*

Kiyotsugu Yoshida^¶, Yoshio Miki, and Donald Kufe^¶§

From the ^¶ Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115 and  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 C (PKC). The functional role of PKC in the DNA damage response is unknown. The present studies demonstrate that PKC is required in part for induction of the stress-activated protein kinase (SAPK/JNK) in cells treated with 1--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 PKC(K-R) mutant, and (iii) down-regulation of PKC by small interfering RNA (siRNA). Coexpression studies demonstrate that PKC activates SAPK by an MKK7-dependent, SEK1-independent mechanism. Previous work has shown that the nuclear Lyn tyrosine kinase activates the MEKK1  MKK7  SAPK pathway but not through a direct interaction with MEKK1. The present results extend those observations by demonstrating that Lyn activates PKC, and in turn, MEKK1 is activated by a PKC-dependent mechanism. These findings indicate that PKC functions in the activation of SAPK through a Lyn  PKC  MEKK1  MKK7  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  MKK7  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 (, , and ), which are calcium-dependent and activated by diacylglycerol, (ii) the novel PKCs (, , , and µ), which are calcium-independent and activated by diacylglycerol, and (iii) the atypical PKCs ( and ), which are calcium-independent and not activated by diacylglycerol (22). The ubiquitously expressed novel PKC, PKC, is tyrosine-phosphorylated and activated by c-Abl in the response to DNA damage (23). As found for c-Abl and Lyn (24, 25), PKC interacts with the nuclear DNA-dependent protein kinase catalytic subunit (DNA-PKcs) (26). Phosphorylation of DNA-PKcs by PKC 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 PKC phosphorylates and inactivates SHPTP1 in the response to DNA damage (29). In cells that respond to genotoxic stress with the induction of apoptosis, PKC is cleaved by caspase-3 to a constitutively active catalytic fragment (PKCCF) (30, 31). The finding that PKCCF induces nuclear condensation and DNA fragmentation has indicated that cleavage of PKC contributes to the apoptotic response (32).

The present studies have addressed the involvement of PKC in the activation of stress signals in response to arrest of DNA replication and to DNA damage. The results demonstrate that PKC is required in part for activation of SAPK. The results also demonstrate that PKC transduces Lyn-mediated signals to MEKK1 in a Lyn  PKC  MEKK1  MKK7  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 PKC(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-PKCCF, pGFP-PKCCF(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 PKC were 5'-GAUGAAGGAGGCGCUCAGTT3' for PKCsiRNA1 and 5'-GGCUGAGUUCUGGCUGGACTT-3' for PKCsiRNA2. 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-PKC (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-PKC 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-PKC, 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 PKC and Lyn by glycerol gradient ultracentrifugation was performed as described (35).

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

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

PKC Functions in Activation of SAPK in Response to DNA Damage-- To investigate whether PKC functions as an upstream effector of the SAPK response to genotoxic agents, U-937 cells were pretreated with PKC 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 PKC and PKC inhibitor, Gö6976 (Fig. 1A). Similar results were obtained with U-937 cells exposed to ionizing radiation (IR) (Fig. 1B). To determine whether PKC 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 PKC in the response to genotoxic agents.

View larger version (51K): [in this window] [in a new window]   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 [-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 PKC Mutant-- To further assess involvement of PKC in SAPK activation, a Myc-tagged kinase-inactive PKC(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-PKC(K-R) (Fig. 2A). Anti-PKC immunoprecipitates from control and araC-treated U-937/neo and U-937/PKC(K-R) cells were analyzed for phosphorylation of histone H1. The results demonstrate that activation of PKC by araC is abrogated in U-937 cells expressing PKC(K-R) (Fig. 2B). To confirm the involvement of PKC in DNA damage-induced activation of SAPK, U-937/neo and U-937/PKC(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 PKC(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/PKC(K-R) cells, as compared with that in U-937/neo cells (data not shown). These findings indicate that PKC is involved in SAPK activation in the cellular response to diverse genotoxic agents.

View larger version (49K): [in this window] [in a new window]   Fig. 2.   Kinase-inactive PKC(K-R) mutant attenuates SAPK activation in response to DNA damage. A, lysates from U-937/neo and U-937/PKC(K-R) (clone 1 and 2) cells were analyzed by immunoblotting (IB) with anti-PKC (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-PKC 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 PKC siRNAs or a Kinase-inactive MKK7(K-R) Mutant-- To further assess the role of PKC in SAPK activation, 293T cells were treated with siRNA duplexes that target PKC. The results demonstrate that the PKCsiRNA1 down-regulates PKC expression (Fig. 3A). Less pronounced results were obtained with PKCsiRNA2 (Fig. 3A). As a control, treatment with a GFPsiRNA had little if any effect (Fig. 3A). Importantly, treatment with PKCsiRNA, but not GFPsiRNA, attenuated araC-induced SAPK activation (Fig. 3B). To determine whether the kinase function of PKC is sufficient for SAPK activation, 293T cells were transfected with PKCCF. Analysis of anti-SAPK immunoprecipitates for phosphorylation of GST-c-Jun demonstrated that expression of PKCCF induces SAPK activity (Fig. 3C). By contrast, expression of kinase-inactive PKCCF(K-R) had little effect on SAPK activity (Fig. 3C). Similar results were obtained in HeLa cells expressing PKCCF or PKCCF(K-R) (data not shown). To define the effectors downstream to PKC in the SAPK pathway, 293T cells were co-transfected with PKCCF 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 PKC (Fig. 3D). These results indicate that PKC induces SAPK activation by an MKK7-dependent, SEK1/MKK4-independent mechanism.

View larger version (35K): [in this window] [in a new window]   Fig. 3.   SAPK activation is attenuated by transfections with PKC siRNAs or a kinase-inactive MKK7(K-R) mutant. A, 293T cells were transfected with GFPsiRNA, PKCsiRNA1, or PKCsiRNA2. Cell lysates were analyzed by immunoblotting (IB) with anti-PKC (upper panel) or anti-tubulin (lower panel). B, 293T cells transfected with GFPsiRNA or PKCsiRNA1 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-PKC (middle panel) or anti-SAPK (lower panel). C, 293T cells were transfected with pGFP, pGFP-PKCCF, or pGFP-PKCCF(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-PKCCF, 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 PKC 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 PKC-mediated SAPK signaling pathway is also MKK7-dependent, we asked whether Lyn is involved in PKC  SAPK signaling. To determine whether PKC binds to Lyn, we incubated glutathione beads containing GST or GST-PKC with purified Lyn. Analysis of the adsorbates demonstrated binding of Lyn with GST-PKC and not GST (Fig. 4A). To determine whether PKC phosphorylates Lyn in vitro, heat-inactivated purified Lyn was incubated with recombinant PKC. Analysis of the reaction products demonstrated no detectable phosphorylation of Lyn by PKC (Fig. 4B, lane 2). By contrast, incubation of heat-inactivated PKC with purified Lyn showed that Lyn phosphorylates PKC (Fig. 4B, lane 5). These findings provide support for a direct interaction between PKC and Lyn.

View larger version (34K): [in this window] [in a new window]   Fig. 4.   PKC interacts directly with Lyn. A, glutathione beads containing GST or GST-PKC 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 PKC was incubated with heat-inactivated purified Lyn (H.I. Lyn) and [-32P]ATP for 15 min. Kinase-active purified Lyn was also incubated with heat-inactivated recombinant PKC (H.I. PKC). The reaction products were analyzed by SDS-PAGE and autoradiography (upper panel) or immunoblotting with anti-PKC and anti-Lyn (lower panel).

To determine whether Lyn contributes to the regulation of PKC in vivo, anti-Lyn immunoprecipitates from control and araC-treated cells were analyzed by immunoblotting with anti-PKC. The results demonstrate that Lyn associates constitutively with PKC and that araC treatment has little if any effect on the extent of the interaction (Fig. 5A). These results were confirmed when anti-PKC immunoprecipitates were subjected to immunoblotting with anti-Lyn (Fig. 5A). A constitutive association between Lyn and PKC 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-PKC demonstrated cosedimentation of Lyn and PKC (Fig. 5B). To extend the analysis, anti-PKC immunoprecipitates from araC-treated U-937/neo and U-937/Lyn(K-R) cells were analyzed for tyrosine phosphorylation of PKC by immunoblotting with anti-phospho-Tyr. The results demonstrate that expression of Lyn(K-R) blocks tyrosine phosphorylation of PKC in response to araC (Fig. 5C). In concert with these findings and the demonstration that PKC is activated by tyrosine phosphorylation (23), Lyn(K-R) also attenuated the induction of PKC activity by araC (Fig. 5D, left). Conversely, to determine whether PKC regulates Lyn in the DNA damage response, anti-Lyn immunoprecipitates from U-937/neo and U-937/PKC(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/PKC(K-R) cells (Fig. 5D, right) suggests that Lyn may function as an upstream effector of PKC.

View larger version (31K): [in this window] [in a new window]   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-PKC. The immunoprecipitates were analyzed by immunoblotting (IB) with anti-Lyn or anti-PKC. 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-PKC 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-PKC immunoprecipitates were subjected to immunoblot analysis with anti-phospho-Tyr (upper panel) or anti-PKC (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-PKC (left panel). Lysates from control and araC-treated U-937/neo and U-937/PKC(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 PKC activity (left panel) and with enolase for Lyn activity (right panel).

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

View larger version (24K): [in this window] [in a new window]   Fig. 6.   MEKK1 is a downstream effector for PKC. 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-PKC (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-PKC (upper panel) or anti-MEKK1 (lower panel). B, kinase-active recombinant PKC was incubated with kinase-inactive GST-MEKK1 (Escherichia coli-derived) and [-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 PKC and ATP. The GST-MEKK1-containing beads were washed again and then incubated with GST-MKK7(K-R) and [-32P]ATP. D, 293T cells were transfected with pGFP, pGFP-PKCCF, 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

PKC Is Activated by Different Mechanisms in DNA Damage Response-- Involvement of PKC 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 PKC activation (23, 29). The available evidence indicates that full-length PKC is activated as an early event within 1 h of exposure to genotoxic agents (23, 29). Phosphorylation of PKC on tyrosine as a mechanism for PKC 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 PKC and that Lyn-mediated tyrosine phosphorylation of PKC contributes to PKC activation.

PKC 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 PKC is required for activation of caspase-3 and thereby PKC cleavage has supported a link between both mechanisms of PKC activation (36). Whereas expression of PKCCF induces characteristics of apoptosis (32), the precise events responsible for this response are unknown but may involve an interaction between PKCCF and DNA-PKcs (26). In contrast to the activation of PKC by tyrosine phosphorylation, cleavage of PKC to the constitutively active catalytic fragment is irreversible and thus may function in the prolonged stimulation of multiple pro-apoptotic pathways.

Role for PKC 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, PKC is also activated by both arrest of DNA replication and DNA damage (23, 29). The present results demonstrate that treatment of cells with the PKC inhibitor, rottlerin, attenuates activation of SAPK in response to genotoxic stress. In concert with these findings, expression of the kinase-inactive PKC(K-R) mutant attenuated activation of both PKC and SAPK in response to araC and IR. Moreover, down-regulation of PKC expression by siRNA was associated with attenuation of DNA damage-induced SAPK activation. These findings provided support for the involvement of PKC as an upstream effector in the regulation of SAPK activation. The results also demonstrate that inhibition of PKC signaling attenuates the early (<1 h) and later periods of SAPK activation. Thus, SAPK is activated by signals transduced by PKC and possibly, after activation of caspase-3, by PKCCF. In this context, the results show that expression of PKCCF 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  SAPK signaling is mediated by MKK7 (8, 20). The respective roles of the c-Abl  SEK1  SAPK and the Lyn  MKK7  SAPK pathways in DNA damage response may vary in different cell types or under different growth conditions. Nonetheless, the finding that inhibition of PKC attenuates SAPK activation in response to araC and IR indicates that PKC affects one or both of the pathways.

Evidence for a Lyn  PKC  MEKK1  MKK7  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 PKC and that tyrosine phosphorylation and activation of PKC is mediated in part by a Lyn-dependent mechanism. The results further demonstrate that PKC associates with MEKK1 and that the interaction between PKC and MEKK1 is increased in response to DNA damage. Moreover, phosphorylation of MEKK1 by PKC stimulated MEKK1 activity. These findings thus collectively support a model in which PKC functions downstream to Lyn and as an upstream effector of the MEKK1  MKK7  SAPK pathway (Fig. 7).

View larger version (13K): [in this window] [in a new window]   Fig. 7.   Model for activation of SAPK in response to genotoxic stress. Effectors are shown for the Lyn  SAPK and c-Abl  SAPK signaling pathways. Cross-talk between the pathways could occur through interactions between (i) PKC 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 PKC as yet another effector of MEKK1 activation. PKC is activated in response to growth factor receptor stimulation (45, 46), DNA-damage (23), and oxidative stress (47). PKC is also activated by caspase-3-mediated cleavage in the apoptotic response. Thus, although the present work has focused on involvement of PKC in DNA damage-induced signaling, the findings do not exclude the possibility that PKC or PKCCF 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--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

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				A. Doller, E.-S. Akool, A. Huwiler, R. Muller, H. H. Radeke, J. Pfeilschifter, and W. Eberhardt Posttranslational Modification of the AU-Rich Element Binding Protein HuR by Protein Kinase C{delta} Elicits Angiotensin II-Induced Stabilization and Nuclear Export of Cyclooxygenase 2 mRNA Mol. Cell. Biol., April 15, 2008; 28(8): 2608 - 2625. [Abstract] [Full Text] [PDF]

				H. Liu, Z.-G. Lu, Y. Miki, and K. Yoshida Protein Kinase C {delta} Induces Transcription of the TP53 Tumor Suppressor Gene by Controlling Death-Promoting Factor Btf in the Apoptotic Response to DNA Damage Mol. Cell. Biol., December 15, 2007; 27(24): 8480 - 8491. [Abstract] [Full Text] [PDF]

				N. M. Mhaidat, R. F. Thorne, X. D. Zhang, and P. Hersey Regulation of Docetaxel-Induced Apoptosis of Human Melanoma Cells by Different Isoforms of Protein Kinase C Mol. Cancer Res., October 1, 2007; 5(10): 1073 - 1081. [Abstract] [Full Text] [PDF]

				D. Zhang, V. Anantharam, A. Kanthasamy, and A. G. Kanthasamy Neuroprotective Effect of Protein Kinase C{delta} Inhibitor Rottlerin in Cell Culture and Animal Models of Parkinson's Disease J. Pharmacol. Exp. Ther., September 1, 2007; 322(3): 913 - 922. [Abstract] [Full Text] [PDF]

				R. Ginnan, B. J. Guikema, H. A. Singer, and D. Jourd'heuil PKC-{delta} mediates activation of ERK1/2 and induction of iNOS by IL-1beta in vascular smooth muscle cells Am J Physiol Cell Physiol, June 1, 2006; 290(6): C1583 - C1591. [Abstract] [Full Text] [PDF]

				K. Yoshida, T. Yamaguchi, H. Shinagawa, N. Taira, K. I. Nakayama, and Y. Miki Protein Kinase C {delta} Activates Topoisomerase II{alpha} To Induce Apoptotic Cell Death in Response to DNA Damage. Mol. Cell. Biol., May 1, 2006; 26(9): 3414 - 3431. [Abstract] [Full Text] [PDF]

				M. J. Humphries, K. H. Limesand, J. C. Schneider, K. I. Nakayama, S. M. Anderson, and M. E. Reyland Suppression of Apoptosis in the Protein Kinase C{delta} Null Mouse in Vivo J. Biol. Chem., April 7, 2006; 281(14): 9728 - 9737. [Abstract] [Full Text] [PDF]

				K. Yoshida, H. Liu, and Y. Miki Protein Kinase C {delta} Regulates Ser46 Phosphorylation of p53 Tumor Suppressor in the Apoptotic Response to DNA Damage J. Biol. Chem., March 3, 2006; 281(9): 5734 - 5740. [Abstract] [Full Text] [PDF]

				K.-W. Zhao, D. Li, Q. Zhao, Y. Huang, R. H. Silverman, P. J. Sims, and G.-Q. Chen Interferon-{alpha}-induced Expression of Phospholipid Scramblase 1 through STAT1 Requires the Sequential Activation of Protein Kinase C{delta} and JNK J. Biol. Chem., December 30, 2005; 280(52): 42707 - 42714. [Abstract] [Full Text] [PDF]

				T. Panaretakis, E. Laane, K. Pokrovskaja, A.-C. Bjorklund, A. Moustakas, B. Zhivotovsky, M. Heyman, M. C. Shoshan, and D. Grander Doxorubicin Requires the Sequential Activation of Caspase-2, Protein Kinase C{delta}, and c-Jun NH2-terminal Kinase to Induce Apoptosis Mol. Biol. Cell, August 1, 2005; 16(8): 3821 - 3831. [Abstract] [Full Text] [PDF]

				O. H. Voss, S. Kim, M. D. Wewers, and A. I. Doseff Regulation of Monocyte Apoptosis by the Protein Kinase C{delta}-dependent Phosphorylation of Caspase-3 J. Biol. Chem., April 29, 2005; 280(17): 17371 - 17379. [Abstract] [Full Text] [PDF]

				L. Catley, Y.-T. Tai, R. Shringarpure, R. Burger, M. T. Son, K. Podar, P. Tassone, D. Chauhan, T. Hideshima, L. Denis, et al. Proteasomal Degradation of Topoisomerase I Is Preceded by c-Jun NH2-Terminal Kinase Activation, Fas Up-Regulation, and Poly(ADP-Ribose) Polymerase Cleavage in SN38-Mediated Cytotoxicity against Multiple Myeloma Cancer Res., December 1, 2004; 64(23): 8746 - 8753. [Abstract] [Full Text] [PDF]

				T. A. DeVries, R. L. Kalkofen, A. A. Matassa, and M. E. Reyland Protein Kinase C{delta} Regulates Apoptosis via Activation of STAT1 J. Biol. Chem., October 29, 2004; 279(44): 45603 - 45612. [Abstract] [Full Text] [PDF]

				R. Ginnan, P. J. Pfleiderer, K. Pumiglia, and H. A. Singer PKC-{delta} and CaMKII-{delta}2 mediate ATP-dependent activation of ERK1/2 in vascular smooth muscle Am J Physiol Cell Physiol, June 1, 2004; 286(6): C1281 - C1289. [Abstract] [Full Text] [PDF]

				T. Abbas, D. White, L. Hui, K. Yoshida, D. A. Foster, and J. Bargonetti Inhibition of Human p53 Basal Transcription by Down-regulation of Protein Kinase C{delta} J. Biol. Chem., March 12, 2004; 279(11): 9970 - 9977. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 277/50/48372    most recent M205485200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Yoshida, K. Articles by Kufe, D. Search for Related Content PubMed PubMed Citation Articles by Yoshida, K. Articles by Kufe, D. Social Bookmarking                      What's this?