Interaction between protein kinase C delta and the c-Abl tyrosine kinase in the cellular response to oxidative stress.

Protein kinase C (PKC) isoforms are phosphorylated on tyrosine in the response of cells to oxidative stress. The present studies demonstrate that treatment of cells with hydrogen peroxide (H(2)O(2)) induces binding of the PKCdelta isoform and the c-Abl protein-tyrosine kinase. The results show that c-Abl phosphorylates PKCdelta in the H(2)O(2) response. We also show that PKCdelta phosphorylates and activates c-Abl in vitro. In cells, induction of c-Abl activity by H(2)O(2) is attenuated by the PKCdelta inhibitor, rottlerin, and by overexpression of the regulatory domain of PKCdelta. These findings support a functional interaction between PKCdelta and c-Abl in the cellular response to oxidative stress.

The protein kinase C (PKC) 1 family of serine/threonine kinases consists of at least 12 isoforms that possess conserved catalytic domains (1,2). The isoforms have been divided into conventional PKC, novel PKC, and atypical PKC subgroups on the basis of differences in their regulatory domains. The ubiquitously expressed PKC␦ is a member of the nPKC subgroup and is activated by phorbol esters/diacylglycerol in a calciumindependent manner (3)(4)(5). Phorbol ester treatment of cells overexpressing PKC␦ is associated with growth arrest (6,7). Other studies have demonstrated that PKC␦ inhibits growth by suppressing cyclin G1 expression (8). PKC␦ is cleaved by caspase 3 at the third variable region (V3) to a 38-kDa regulatory domain (RD) and a 40-kDa catalytically active fragment in cells induced to undergo apoptosis (9 -11). The finding that overexpression of the PKC␦ catalytic fragment is associated with chromatin condensation, nuclear fragmentation, and lethality has supported a role for cleavage of PKC␦ in induction of apoptosis (11). The association of PKC␦ with growth arrest and apoptosis is in concert with findings that PKC␦ confers a potential tumor suppressor function (12).
Diverse signals have been associated with phosphorylation of PKC␦ on tyrosine. Activation of the platelet-derived growth factor or epidermal growth factor receptors induces tyrosine phosphorylation of PKC␦ (13,14). Transformation by Ras or v-Src also results in phosphorylation of PKC␦ on tyrosine (15,16). Other studies have demonstrated that PKC␦ is phosphorylated on tyrosine and activated in cells treated with hydrogen peroxide (H 2 O 2 ) (17,18). In vitro, PKC␦ is tyrosine-phosphorylated by c-Src (14,16,19), c-Fyn (13,14), and growth factor receptors (13,14). The effects of tyrosine phosphorylation on the serine/threonine kinase activity of PKC␦, however, have been conflicting. Moreover, little information is available regarding the tyrosine kinases that directly phosphorylate PKC␦ in cells.
Recent work has demonstrated that the c-Abl tyrosine kinase phosphorylates and activates PKC␦ in the response of cells to DNA damage (20). In the present studies, we show that treatment of cells with H 2 O 2 induces binding of PKC␦ and c-Abl. The results also show that PKC␦ contributes to the activation of c-Abl in the oxidative stress response.

In Vitro Kinase Assays
Phosphorylation of PKC␦ by c-Abl-Kinase-active c-Abl prepared from baculovirus-infected insect cells (23) was incubated with heatinactivated purified PKC␦ (Calbiochem) and [␥-32 P]ATP in kinase buffer for 20 min at 30°C. Phosphorylated proteins were separated by 6% SDS-PAGE and analyzed by autoradiography. The PKC␦ fragment encoding amino acids 473-537 was generated by polymerase chain reaction and ligated into the GST pGEX4T3 vector. The Tyr 512 and Tyr 523 sites were mutated to Phe by site-directed mutagenesis (Stratagene). The wild-type and mutant GST-PKC␦-(473-537) fusion proteins were incubated with kinase-active c-Abl and [␥-32 P]ATP. The products were analyzed by 12% SDS-PAGE and autoradiography.
Activation of PKC␦ by c-Abl-Kinase-active PKC␦ was incubated with kinase-active c-Abl, histone H1, and [␥-32 P]ATP in PKC kinase buffer. Phosphorylated proteins were separated by 12% SDS-PAGE and analyzed by autoradiography.
Phosphorylation of c-Abl by PKC␦-Kinase-active PKC␦ was incubated with heat-inactivated c-Abl and [␥-32 P]ATP in kinase buffer. Phosphorylated proteins were separated by 6% SDS-PAGE and analyzed by autoradiography.

RESULTS AND DISCUSSION
Previous work has demonstrated that c-Abl interacts with PKC␦ in the cellular response to genotoxic stress (20). To assess whether the oxidative stress response involves an interaction between c-Abl and PKC␦, we cotransfected COS7 cells with FLAG-tagged PKC␦ and c-Abl. Analysis of anti-FLAG immunoprecipitates by immunoblotting with anti-c-Abl demonstrated little association of PKC␦ and c-Abl in control cells (Fig.  1A). By contrast, treatment with H 2 O 2 resulted in the coprecipitation of FLAG-PKC␦ and c-Abl (Fig. 1A). To confirm these findings, anti-c-Abl immunoprecipitates from the transfected cells were analyzed by immunoblotting with anti-PKC␦. The results demonstrate that H 2 O 2 treatment induces the binding of PKC␦ and c-Abl (Fig. 1B). Similar studies were performed on nontransfected cells to assess the interaction between endogenous PKC␦ and c-Abl. Analysis of anti-PKC␦ immunoprecipitates by immunoblotting with anti-c-Abl demonstrated binding of PKC␦ and c-Abl in H 2 O 2 -treated, but not untreated, cells (Fig. 1C). Further support for H 2 O 2 -induced binding of PKC␦ and c-Abl was obtained by immunoblot analysis of anti-c-Abl immunoprecipitates with anti-PKC␦ (Fig. 1D). These results demonstrate that H 2 O 2 induces the association of PKC␦ and c-Abl.
To determine whether the kinase functions of PKC␦ or c-Abl are involved in H 2 O 2 -induced binding of these proteins, we cotransfected cells with c-Abl and kinase-active FLAG-PKC␦ or kinase-inactive FLAG-PKC␦(K-R). The results demonstrate that, although H 2 O 2 treatment is associated with binding of c-Abl to PKC␦, the interaction is substantially decreased with PKC␦(K-R) ( Fig. 2A). Likewise, H 2 O 2 -induced binding of FLAG-PKC␦ to wild-type c-Abl was greater than that found with kinase-inactive c-Abl(K-R) (Fig. 2B). These results indicate that activation of both PKC␦ and c-Abl contributes to the association of these proteins in the response to H 2 O 2 . To determine whether H 2 O 2 induces c-Abl-mediated tyrosine phosphorylation of PKC␦, we cotransfected cells with FLAG-PKC␦ and empty vector (pSR), wild-type c-Abl, or kinase-inactive c-Abl(K-R). Analysis of anti-FLAG immunoprecipitates by immunoblotting with anti-Tyr(P) demonstrated no detectable tyrosine phosphorylation of PKC␦ in the transfectants not exposed to H 2 O 2 (Fig. 2C). Treatment with H 2 O 2 , however, was associated with tyrosine phosphorylation of PKC␦ and this effect was increased in cells cotransfected with c-Abl, but not c-Abl(K-R) (Fig. 2C). These results indicated that PKC␦ is phosphorylated by c-Abl in H 2 O 2 -treated cells.
As the findings support a role for c-Abl-mediated phosphorylation in the regulation of PKC␦, we asked if PKC␦ can, in turn, phosphorylate c-Abl. Incubation of purified c-Abl with [␥-32 P]ATP and analysis of the products by SDS-PAGE and autoradiography demonstrated c-Abl autophosphorylation (Fig. 3A). By contrast, there was no detectable phosphorylation observed with heat-inactivated c-Abl (Fig. 3A). Incubation of heat-inactivated c-Abl with kinase-active PKC␦ was associated with autophosphorylation of PKC␦ and phosphorylation of c-Abl (Fig. 3A). These findings demonstrate that PKC␦ phospho-

FIG. 1. Association of PKC␦ and c-Abl is induced by H 2 O 2 . A, COS7 cells were cotransfected with FLAG-tagged PKC␦ and c-Abl.
After 36 h, cells were incubated in the absence (Ϫ) or presence (ϩ) of 3 mM H 2 O 2 for 15 min. Cell lysates were subjected to immunoprecipitation (IP) with mouse IgG or anti-FLAG. The immunoprecipitates and lysate not subjected to IP were analyzed by immunoblotting (IB) with anti-c-Abl and anti-FLAG. B, COS7 cells were treated as described in the legend to A. Cell lysates were subjected to IP with IgG or anti-c-Abl. The immunoprecipitates and lysate were analyzed by IB with anti-FLAG and anti-c-Abl. C, COS7 cells (nontransfected) were treated with 3 mM H 2 O 2 for 15 min. Anti-PKC␦ or IgG immunoprecipitates were analyzed by IB with anti-c-Abl and anti-PKC␦. Lysate not subjected to IP was used as a control. D, COS7 cells were treated as described in the legend to C. Anti-c-Abl or IgG immunoprecipitates were analyzed by IB with anti-PKC␦ and anti-c-Abl. Lysate was used as a control. Anti-c-Abl immunoprecipitates or lysate not subjected to IP were analyzed by IB with anti-FLAG. C, cells were cotransfected with FLAG-PKC␦ and empty pSR vector, c-Abl, or c-Abl(K-R). As indicated, cells were left untreated or exposed to H 2 O 2 . Anti-FLAG immunoprecipitates were analyzed by IB with anti-P-Tyr and anti-FLAG.
Interaction between PKC␦ and c-Abl Tyrosine Kinase 7471 rylates c-Abl. As controls and in concert with findings in H 2 O 2treated cells, PKC␦ served as a substrate for c-Abl (Fig. 3A).
Previous studies have demonstrated that H 2 O 2 induces tyrosine phosphorylation of Tyr 512 and Tyr 523 in the PKC␦ catalytic domain (17). Incubation of kinase-active c-Abl with GST-PKC␦-(473-537) and [␥-32 P]ATP was associated with PKC␦ phosphorylation (data not shown). In addition, mutation of Tyr 512 , but not Tyr 523 , to Phe resulted in abrogation of c-Abl-mediated phosphorylation. To extend the analysis, kinase-active c-Abl and kinase-active PKC␦ were incubated in the presence of [␥-32 P]ATP. The results demonstrate that phosphorylation of both c-Abl and PKC␦ was increased compared with that obtained with either alone (Fig. 3B). As these results demonstrate that PKC␦ phosphorylates and potentially activates c-Abl, we assayed c-Abl activity in the presence of PKC␦. Of note, the c-Abl substrate GST-Crk-(120 -225) is not phosphorylated by PKC␦ and the PKC␦ substrate histone H1 is not phosphorylated by c-Abl (Fig. 3C). Incubation of c-Abl and PKC␦ resulted in increased phosphorylation of GST-Crk-(120 -225) compared with that found with c-Abl alone (Fig. 3D). In addition, phosphorylation of histone H1 was increased by incubating PKC␦ with c-Abl compared with that obtained with PKC␦ alone (Fig.  3D). These findings collectively support an interaction between c-Abl and PKC␦ that involves activation of both kinases.
To determine whether PKC␦ activates c-Abl in vivo, we treated cells with H 2 O 2 and asked if c-Abl is activated by a PKC␦-dependent mechanism. Analysis of anti-PKC␦ immunoprecipitates for phosphorylation of histone H1 demonstrated H 2 O 2 -induced increases in PKC␦ activity (Fig. 4A). In addition, analysis of anti-c-Abl immunoprecipitates for phosphorylation of GST-Crk-(120 -225) demonstrated that the constitutive level of c-Abl activity found in control cells is increased by H 2 O 2 exposure (Fig. 4B). To determine whether PKC␦ contributes to the activation of c-Abl in the response to H 2 O 2 , cells were treated with the selective PKC␦ inhibitor, rottlerin (24). While there was no effect of rottlerin on constitutive c-Abl activity, this agent inhibited H 2 O 2 -induced c-Abl activation (Fig. 4B). To further assess involvement of PKC␦ in c-Abl activation, we prepared a vector to express a FLAG-tagged PKC␦ regulatory domain (RD) (amino acids 1-330). Immunoblot analysis with anti-FLAG demonstrated expression of FLAG-PKC␦RD in the stably transfected cells and not in those expressing the empty vector (Fig. 4C). Analysis of anti-c-Abl immunoprecipitates for phosphorylation of GST-Crk-(120 -225) showed that c-Abl activity is induced to a similar extent by H 2 O 2 in wild-type cells and those expressing the empty vector (Fig. 4D). By contrast, H 2 O 2 -induced c-Abl activity was attenuated in cells expressing PKC␦RD (Fig. 4D). These results provide evidence for activation of c-Abl by a PKC␦-dependent mechanism in the response to H 2 O 2 .
Reactive oxygen species have been implicated in the regulation of cell growth and apoptosis (25,26). The intracellular signals that are activated by reactive oxygen species, however, are largely unknown. Certain insights have been made available from the finding that H 2 O 2 treatment is associated with phosphorylation of PKC␦ on tyrosine (17). The present studies indicate that c-Abl is in part responsible for tyrosine phosphorylation of PKC␦ in the response to H 2 O 2 . In this context, H 2 O 2 induces the binding c-Abl and PKC␦. In addition, H 2 O 2 -induced tyrosine phosphorylation of PKC␦ is attenuated in cells expressing the kinase-inactive c-Abl(K-R). The findings also demonstrate that c-Abl-mediated phosphorylation of PKC␦ activates the PKC␦ kinase function and that this effect occurs in the absence of lipid cofactors. The phosphorylation of PKC␦ on Tyr 512 and Tyr 523 , has been shown to be important for H 2 O 2induced activation (17). Our findings demonstrate that c-Abl phosphorylates PKC␦ on Tyr 512 , but not Tyr 523 . Taken together, these findings indicate that PKC␦ is phosphorylated by c-Abl and at least one other tyrosine kinase.
Previous studies have demonstrated that the c-Abl kinase is activated by DNA-damage (22). The present work demon- Anti-PKC␦ immunoprecipitates were incubated with histone H1 and [␥-32 P]ATP. The reaction products were analyzed by 12% SDS-PAGE and autoradiography. B, cells were pretreated with 10 M rottlerin for 10 min and then exposed as indicated to 3 mM H 2 O 2 for an additional 15 min. Anti-c-Abl immunoprecipitates were incubated with GST-Crk-(120 -225) and [␥-32 P]ATP. Reaction products were analyzed by 12% SDS-PAGE and autoradiography. C, COS7 cells were stably transfected to express the empty pcDNA3-FLAG vector or FLAG-PKC␦RD. Lysates were subjected to IB with anti-FLAG and, as a control, anti-PCNA. D, the indicated cells were treated with 3 mM H 2 O 2 for 15 min. Anti-c-Abl immunoprecipitates were incubated with GST-Crk-(120 -225) and [␥-32 P]ATP. Reaction products were analyzed by 12% SDS-PAGE and autoradiography.
Interaction between PKC␦ and c-Abl Tyrosine Kinase 7472 strates that c-Abl is also activated by H 2 O 2 and that this response is dependent on the PKC␦ kinase function. The results support a model in which c-Abl is activated by PKC␦ in response to oxidative stress, and in a potential feedback loop, c-Abl phosphorylates and thereby further activates PKC␦. PKC␦, like c-Abl, has been linked to the induction of apoptosis in the response of cells to genotoxic stress (9 -11, 27). Other studies have shown that PKC␦ is activated by serum stimulation and that phosphorylation of the PKC␦ activation loop is mediated by PDK1 (28). Thus, PKC␦ may be functional in both pro-and anti-apoptotic pathways. In this context, PKC␦ could represent a switch that determines cell fate, such that PKC␦ confers anti-apoptotic signals following PDK1-mediated phosphorylation and pro-apoptotic signals as a consequence of c-Abl-mediated phosphorylation.