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Ionizing Radiation Stimulates a Grb2-mediated Association of the Stress-activated Protein Kinase with Phosphatidylinositol 3-Kinase (∗)

Open AccessPublished:August 11, 1995DOI:https://doi.org/10.1074/jbc.270.32.18871
      The stress-activated protein (SAP) kinases are induced by tumor necrosis factor, oncoproteins, and UV light. The present studies demonstrate that ionizing radiation (IR) activates p54 SAP kinase. IR-induced activation of SAP kinase is associated with binding to the SH2/SH3-containing adaptor protein Grb2. This interaction is mediated by the SH3 domains of Grb2 and the proline-rich sequence PPPKIP in the carboxyl-terminal region of SAP kinase. We also demonstrate that SAP kinase and the p85α-subunit of phosphatidylinositol (PI) 3-kinase form a complex in irradiated cells. The results indicate that this complex involves binding of the p85α subunit of PI 3-kinase to the SH2 domain of Grb2. The functional role of linking SAP kinase to PI 3-kinase is further supported by the finding that wortmannin, an inhibitor of PI 3-kinase, stimulates SAP kinase activity. These results suggest that the cellular response to IR may include regulation of SAP kinase by a PI 3-kinase-dependent signaling pathway.

      INTRODUCTION

      The c-jun early response gene is induced by diverse mitogenic stimuli. Although the role of c-Jun in mitogenesis remains unclear, studies have shown that this protein is involved in the G0/G1 transition and progression through late G1/S(
      • Ryseck R.P.
      • Hirai S.I.
      • Yaniv M.
      • Bravo R.
      ,
      • Carter R.H.
      • Park D.J.
      • Rhee S.G.
      • Fearon D.T.
      ). Other studies have indicated that c-Jun is involved in induction of cellular differentiation(
      • Sherman M.
      • Stone R.
      • Datta R.
      • Bernstein S.
      • Kufe D.
      ). Moreover, the finding that transcription of c-jun is rapidly induced in cells exposed to UV light (
      • Devary Y.
      • Gottleib R.A.
      • Lau L.F.
      • Karin M.
      ) and other DNA-damaging agents (
      • Kharbanda S.
      • Sherman M.L.
      • Kufe D.
      ,
      • Rubin E.
      • Kharbanda S.
      • Gunji H.
      • Kufe D.
      ) has supported a role for c-Jun in the response to genotoxic stress.
      The cellular response to ionizing radiation (IR)
      The abbreviations used are: IR
      ionizing radiation
      SAP
      stress-activated protein
      PI
      phosphatidylinositol
      MAP
      mitogen-activated protein
      GST
      glutathione S-transferase
      PAGE
      polyacrylamide gel electrophoresis
      Grb2
      growth factor receptor binding protein
      PMSF
      phenylmethylsulfonyl fluoride
      pp90
      pp90 ribosomal S6 kinase.
      includes activation of DNA repair, cell cycle arrest, and cell death(
      • Hall E.J.
      ). Recent studies have shown that exposure of eukaryotic cells to IR is also associated with activation of c-jun transcription. These findings have provided support for the transduction of early nuclear signals to longer term changes in gene expression in the response to radiation(
      • Sherman M.
      • Stone R.
      • Datta R.
      • Bernstein S.
      • Kufe D.
      ). Other studies have demonstrated that the pp44/42 mitogen-activated protein (MAP) kinases are activated in irradiated cells and that this event is temporally related to the activation of pp90rsk and early response gene expression(
      • Kharbanda S.
      • Saleem A.
      • Shafman T.
      • Emoto Y.
      • Weichselbaum R.
      • Kufe D.
      ). The involvement of MAP kinase in the regulation of c-Jun (
      • Pulverer B.J.
      • Kyriakis J.M.
      • Avruch J.
      • Nikolakaki E.
      • Woodgett J.R.
      ) has suggested that this pathway may contribute to IR-induced c-jun expression.
      The stress-activated protein (SAP) kinases, also known as c-Jun amino-terminal kinases, are a MAP kinase-related family of p54/p46 serine/threonine kinases that phosphorylate c-Jun(
      • Kyriakis J.M.
      • Banerjee P.
      • Nikolakaki E.
      • Dai T.
      • Rubie E.A.
      • Ahmad M.F.
      • Avruch J.
      • Woodgett J.R.
      ,
      • Derijard B.
      • Hibi M.
      • Wu I.H.
      • Barrett T.
      • Su B.
      • Deng T.
      • Karin M.
      • Davis R.J.
      ,
      • Hibi M.
      • Lin A.
      • Smeal T.
      • Minden A.
      • Karin M.
      ). Two serine residues (Ser63 and Ser73) in the amino-terminal transactivation domain of c-Jun that are phosphorylated in response to mitogens, Ras, and phorbol esters have been identified as substrates for SAP kinase(
      • Devary Y.
      • Gottleib R.A.
      • Lau L.F.
      • Karin M.
      ,
      • Derijard B.
      • Hibi M.
      • Wu I.H.
      • Barrett T.
      • Su B.
      • Deng T.
      • Karin M.
      • Davis R.J.
      ,
      • Hibi M.
      • Lin A.
      • Smeal T.
      • Minden A.
      • Karin M.
      ). SAP kinases are activated by tumor necrosis factor, sphingomyelinase, and UV light(
      • Kyriakis J.M.
      • Banerjee P.
      • Nikolakaki E.
      • Dai T.
      • Rubie E.A.
      • Ahmad M.F.
      • Avruch J.
      • Woodgett J.R.
      ,
      • Derijard B.
      • Hibi M.
      • Wu I.H.
      • Barrett T.
      • Su B.
      • Deng T.
      • Karin M.
      • Davis R.J.
      ,
      • Hibi M.
      • Lin A.
      • Smeal T.
      • Minden A.
      • Karin M.
      ). The demonstration that Ha-Ras is also involved in the activation of SAP kinase has supported the involvement of this pathway in protection against UV-induced damage to cellular components other than DNA(
      • Derijard B.
      • Hibi M.
      • Wu I.H.
      • Barrett T.
      • Su B.
      • Deng T.
      • Karin M.
      • Davis R.J.
      ). Recent studies have demonstrated that SAP kinase is activated by a pathway involving MAP kinase and SAP kinase-activating kinase 1(
      • Sanchez I.
      • Hughes R.T.
      • Mayer B.J.
      • Yee K.
      • Woodgett J.R.
      • Avruch J.
      • Kyriakis J.M.
      • Zon L.I.
      ,
      • Yan M.
      • Dai T.
      • Deak J.C.
      • Kyriakis J.M.
      • Zon L.I.
      • Woodgett J.R.
      • Templeton D.J.
      ).
      The present work has examined involvement of the SAP kinases in IR-induced signaling. The results demonstrate that IR exposure is associated with the activation of SAP kinase and the formation of a Grb2-mediated complex involving SAP kinase and phosphatidylinositol (PI) 3-kinase.

      MATERIALS AND METHODS

      Cell Culture

      Human U-937 myeloid leukemia cells (ATCC) were grown in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 100 units/ml penicillin, 100 μg/ml streptomycin, and 2 mML-glutamine. Irradiation was performed at room temperature using a Gammacell 1000 (Atomic Energy of Canada, Ottawa, Ontario) under aerobic conditions with a 137-centistoke source emitting at a fixed dose rate of 13.3 grays/min as determined by dosimetry. U-937 cells were also treated with 20 ng/ml human recombinant tumor necrosis factor (specific activity, 6.60 × 106 units/mg protein), provided by BASF, Inc. (Worcester, MA), 1 ng/ml rapamycin (provided by the National Cancer Institute, Bethesda, MD), 50 μM H-7 (Seikagaku America, Inc., St. Petersburg, FL), 50 μM HA1004 (Seikagaku), or 250 nM wortmannin (Sigma).

      Antibodies and Fusion Proteins

      Anti-p85α was purchased from Transduction Laboratories (Lexington, KY). Anti-Grb2 was obtained from Santa Cruz Biotechnology (San Diego, CA). Anti-p54 SAP kinase antibodies were prepared as described(
      • Kyriakis J.M.
      • Banerjee P.
      • Nikolakaki E.
      • Dai T.
      • Rubie E.A.
      • Ahmad M.F.
      • Avruch J.
      • Woodgett J.R.
      ). Anti-pp90rsk and anti-Erk 1/2 were obtained from J. Blenis (Harvard Medical School, Boston, MA). c-Jun fusion protein was prepared as described(
      • Saleem A.
      • Yuan Z.-M.
      • Kufe D.W.
      • Kharbanda S.M.
      ). GST-Grb2 (full-length), GST-Grb2 SH2, GST-Grb2 N-SH3 (amino-terminal SH3 domain), and GST-Grb2 C-SH3 (carboxyl-terminal SH3 domain) fusion proteins were purchased from Santa Cruz Biotechnology. The GST-Grb2 N-SH3 and GST-Grb2 C-SH3 include amino acids 1-68 and 156-210, respectively, of the Grb2 protein.

      Preparation of Cell Lysates and Kinase Assays

      Cells (2-3 × 107) were washed with phosphate-buffered saline and lysed in 1 ml of lysis buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 1 mM sodium vanadate, 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, and 10 μg/ml of leupeptin and aprotinin). After incubation on ice for 30 min, insoluble material was removed by centrifugation at 14,000 rpm for 10 min at 4°C. Lysates were incubated with 5 μg of immobilized GST or GST-Jun for 2 h at 4°C. The protein complexes were washed with lysis buffer and then incubated in kinase buffer (20 mM HEPES, pH 7.0, 10 mM MgCl2, 10 mM MnCl2, 2 mM dithiothreitol, and 0.1 mM sodium vanadate) containing [γ-32P]ATP for 15 min at 30°C. Reactions were terminated by the addition of SDS-PAGE sample buffer and boiling. Phosphorylated proteins were resolved by SDS-PAGE and autoradiography.

      Immune Complex Kinase Assays

      Lysates were precleared by incubation with 5 μg/ml rabbit anti-mouse IgG for 1 h at 4°C and then for an additional 30 min after the addition of protein A-Sepharose. After preclearing of the cell lysates, the supernatant was incubated with preimmune rabbit serum, anti-p54 SAP kinase, or anti-Grb2 for 1 h at 4°C and then for 30 min after the addition of protein A-Sepharose. The supernatant was also incubated with GST-Grb2 for 1 h at 4°C, and the complexes were recovered by the addition of glutathione beads. The protein complexes were washed three times with lysis buffer and once with kinase buffer and resuspended in kinase buffer containing 1 μCi/μl [γ-32P]ATP (3000 Ci/mmol; DuPont NEN) and GST-Jun. The reaction mixture was incubated for 15 min at 30°C and terminated by the addition of SDS sample buffer. The proteins were analyzed by SDS-PAGE and autoradiography.

      Immunoprecipitation and Immunoblot Analysis

      Immunoprecipitation was performed as described(
      • Kharbanda S.
      • Yuan Z.-M.
      • Rubin E.
      • Weichselbaum R.
      • Kufe D.
      ). In brief, soluble proteins (200 μg) were incubated with anti-p85α (Transduction Laboratories), anti-Grb2 (Santa Cruz Biotechnology), or anti-SAP kinase antibodies for 1 h and precipitated with protein A-Sepharose for an additional 30 min. The resulting immune complexes were washed three times with lysis buffer, separated by electrophoresis on 7.5 or 10% SDS-polyacrylamide gels, and then transferred to nitrocellulose paper. The residual binding sites were blocked by incubating the filters with 5% dry milk in phosphate-buffered saline and 0.05% Tween 20 for 1 h at room temperature. The filters were incubated with anti-Grb2 or anti-p85α for 1 h with shaking. After washing twice with phosphate-buffered saline and 0.05% Tween 20, the blots were incubated with anti-rabbit IgG peroxidase conjugate (Amersham Corp.). The antigen antibody complexes were visualized by chemiluminescence (ECL detection system, Amersham Corp.).

      Fusion Protein Binding Assays and Peptide Competition Assays

      The fusion proteins GST, GST-Grb2, GST-Grb2 SH2, GST-Grb2 N-SH3, and GST-Grb2 C-SH3 were purified by affinity chromatography using glutathione-Sepharose beads and equilibirated in lysis buffer. Cell lysates were incubated with 2 μg of immobilized GST, GST-Grb2, GST-Grb2 SH2, GST-Grb2 N-SH3, or GST-Grb2 C-SH3 fusion proteins for 2 h at 4°C. The resulting protein complexes were washed three times with lysis buffer containing 0.1% detergent and boiled for 5 min in SDS sample buffer. The complexes were then separated by 7.5% SDS-PAGE and subjected to silver staining or immunoblot analysis with anti-SAP kinase antibody. Peptide was synthesized using Fmoc (N-(9-fluorenyl)methoxycarbonyl)) and subsequently purified by ether precipitation and preparative reverse-phase high pressure liquid chromatography. Amino acid analysis was used to confirm the sequence of the SAP kinase-derived peptide: AEAPPPKIPDKQ. GST-Grb2 N-SH3 protein was incubated in the presence or absence of 50 μM synthetic peptide for 1 h at 4°C. The fusion proteins were incubated with lysates from control and irradiated cells, and the adsorbates were then analyzed by immunoblotting with anti-SAP kinase antibody.

      RESULTS AND DISCUSSION

      Human U-937 myeloid leukemia cells were exposed to 20 grays IR and harvested at 1 h. Lysates were subjected to affinity chromatography with a GST-Jun fusion protein. Adsorbates from IR-treated cells exhibited an increase in Jun kinase activity compared with that of control cells (Fig. 1A). IR-induced stimulation of Jun kinase activity was comparable with that obtained in tumor necrosis factor- (Fig. 1A) or sphingomyelinase-treated cells (data not shown). In order to determine whether the IR-induced Jun kinase activity is due to stimulation of SAP kinase, we assayed anti-p54 SAP kinase immunoprecipitates for phosphorylation of GST-Jun. The finding that IR activates SAP kinase to phosphorylate GST-Jun (Fig. 1B) and not GST (data not shown) supports the involvement of this kinase in IR-induced signaling.
      Figure thumbnail gr1
      Figure 1:IR induces activation of SAP kinase. A, lysates from control (U-937) and IR-treated cells were incubated with glutathione-Sepharose beads containing 5 μg of GST-Jun amino acids(2-100) for 2 h. The beads were washed and then incubated in kinase buffer containing [γ-32P]ATP for 15 min at 30°C. Reactions were terminated by addition of 6 × SDS-PAGE sample buffer. The phosphorylated proteins were resolved by 10% SDS-PAGE, dried, and analyzed by autoradiography. B, lysates from control and IR-treated cells were subjected to immunoprecipitation with anti-p54 antibody. Immune complex kinase assays were performed by the addition of GST-Jun fusion protein and [γ-32P]ATP and incubation for 15 min at 30°C. The proteins were analyzed by 10% SDS-PAGE and autoradiography. TNF, tumor necrosis factor.
      Analysis of anti-p54 immunoprecipitates suggested that other proteins associate with p54 after IR exposure. For example, the finding that a 26-kDa protein coprecipitates with SAP kinase raised the possibility of an interaction with Grb2. Grb2, also known as Ash, Sem-5, or Drk (
      • Lowenstein E.J.
      • Daly R.J.
      • Batzer A.G.
      • Li W.
      • Margolis B.
      • Lammers R.
      • Ullrich A.
      • Skolnik E.Y.
      • Bar-Sagi D.
      • Schlessinger J.
      ,
      • Clark S.G.
      • Stern M.J.
      • Horowitz H.R.
      ,
      • Matuoka K.
      • Shibata M.
      • Yamakawa A.
      • Takenawa T.
      ,
      • Simon M.A.
      • Dodson G.S.
      • Rubin G.M.
      ), is an SH2- and SH3-containing adaptor protein that links activated protein-tyrosine kinase receptors to the Ras activator protein Sos. Immunoblot analysis of anti-p54 immunoprecipitates with anti-Grb2 revealed increased reactivity with a 26-kDa protein following IR exposure (Fig. 2A, left panel). To provide additional support for an association between Grb2 and SAP kinase, anti-Grb2 immunoprecipitates were analyzed for phosphorylation of GST-Jun. The results demonstrate that Grb2 associates with a Jun kinase activity that is induced by IR treatment (Fig. 2A, middle panel). The interaction between Grb2 and SAP kinase was further examined by incubating lysates from control and IR-treated cells with a GST-Grb2 (full-length) fusion protein. Analysis of the adsorbates to GST-Grb2 supported binding of a kinase that phosphorylates GST-Jun (Fig. 2A, right panel). Analysis of the adsorbates by immunoblotting with anti-p54 also revealed increased reactivity with a 54-kDa protein in the irradiated cells (Fig. 2B). Lysates were similarly incubated with GST fusion proteins prepared from the SH2 and SH3 (carboxyl- and amino-terminal) domains of Grb2. Adsorbates obtained with the GST-Grb2 SH2 fusion protein exhibited no detectable anti-p54 reactivity (Fig. 2B). In contrast, adsorbates from GST-Grb2 N-SH3 or GST-Grb2 C-SH3 revealed binding of SAP kinase when assaying lysates from irradiated but not control cells (Fig. 2B). Lysates from irradiated cells were also incubated with GST-Lyn SH3 and GST-Fyn SH3 fusion proteins. The results demonstrate little if any binding of SAP kinase to these SH3-containing proteins (data not shown). These findings indicated that the IR-induced association between Grb2 and SAP kinase is specifically mediated through the SH3 domains of Grb2.
      Figure thumbnail gr2
      Figure 2:Association of p54 SAP kinase and Grb2. A, lysates from control and IR-treated cells were immunoprecipitated with anti-p54 antibody (left panel) or anti-Grb2 antibody (middle panel) or subjected to affinity chromatography with GST-Grb2 (full-length) (right panel). The immune complexes were analyzed by immunoblotting with anti-Grb2. Anti-Grb2 immune complexes and GST-Grb2 protein complexes were incubated in kinase buffer containing [γ-32P]ATP and 5 μg of GST-Jun fusion protein for 15 min at 30°C. The proteins were resolved by 10% SDS-PAGE, dried, and analyzed by autoradiography. B, lysates from control and IR-treated cells were incubated with GST-Grb2 (full-length), GST-Grb2 SH2, GST-Grb2 C-SH3, GST-Grb2 N-SH3, or GST-Grb2 N-SH3 in the absence(-) or presence (+) of competitor peptide (AEAPPPKIPDKQ). The bound proteins were resolved by 7.5% SDS-PAGE and immunoblotted with anti-p54 antibody. C, lysates from control and IR-treated cells were incubated with GST-Grb2 N-SH3. Lysates from IR-treated cells were also immunoprecipitated with anti-pp90rsk or anti-Erk1/2 antibodies. The adsorbed or immunoprecipitated proteins were resolved by 7.5% SDS-PAGE and analyzed by immunoblotting with anti-pp90rsk or anti-MAP kinase antibodies.
      Alignment of the Grb2, Crk, Nck, and Abl SH3-binding sites has identified two proline residues spaced by two nonconserved amino acids. The consensus PXXP has also been observed for certain other SH3-binding sites(
      • Egan S.E.
      • Giddings B.W.
      • Brooks M.W.
      • Buday L.
      • Sizeland A.M.
      • Weinberg R.A.
      ,
      • Rozakis-Adcock M.
      • Fernley R.
      • Wade J.
      • Pawson P.
      • Bowtell D.
      ,
      • Li N.
      • Batzer A.
      • Daly R.
      • Yajnik V.
      • Skolnik E.
      • Chardin P.
      • Bar-Sagi D.
      • Margolis B.
      • Schlessinger J.
      ). These findings suggested that a proline-rich candidate sequence (PPPKIP) in SAP kinase may be responsible for the association with Grb2 SH3 domains. A synthetic peptide corresponding to amino acids 330-341 (AEAPPPKIPDKQ) of SAP kinase was used in competition assays. Preincubation of GST-Grb2 N-SH3 with the peptide inhibited binding of SAP kinase by 50% in lysates of irradiated cells (Fig. 2B). In contrast, there was no detectable inhibition of SAP kinase binding when using an unrelated peptide (LQHPYINVWYDP) as competitor (data not shown). Previous studies have demonstrated that IR also induces MAP (Erk1/2) and pp90rsk kinase activity(
      • Kharbanda S.
      • Saleem A.
      • Shafman T.
      • Emoto Y.
      • Weichselbaum R.
      • Kufe D.
      ). To assess specificity of SAP kinase binding to SH3 domains of Grb2, experiments were performed in which the IR-induced lysates were incubated with GST-Grb2 N-SH3 or GST-Grb2 C-SH3 and the adsorbed proteins assayed by immunoblotting with either anti-MAP or anti-pp90rsk antibodies. The results demonstrate that, unlike SAP kinase, there is no detectable association of MAP and pp90rsk with the Grb2 SH3 domains (Fig. 2C and data not shown). These findings indicate that the IR-induced binding of Grb2 and SAP kinase is mediated through the SH3 domains of Grb2 and the PPPKIP site in SAP kinase.
      The significance of Grb2 binding to SAP kinase in irradiated cells is presumably to link SAP kinase to an upstream or downstream effector in the IR response. We therefore studied anti-p54 immunoprecipitates to define other proteins that might associate with a Grb2-SAP kinase complex. Staining demonstrated increased binding of a protein at approximately 85 kDa (data not shown). Because the PI 3-kinase consists of 85 and 110 kDa subunits, we asked whether p85α of PI 3-kinase associates with SAP kinase. Analysis of anti-p54 immunoprecipitates with anti-PI 3-kinase (p85α) demonstrated increased reactivity in IR-treated as compared with control cells (Fig. 3A). Although these findings suggest that SAP kinase and PI 3-kinase associate in irradiated cells, the results do not distinguish between direct binding of these molecules or an indirect association through other proteins. One possible explanation for an association between SAP kinase and PI 3-kinase is through Grb2. In this regard, recent studies have demonstrated that PI 3-kinase associates with Grb2 during growth factor stimulation(
      • Saleem A.
      • Kharbanda S.
      • Yuan Z.-M.
      • Kufe D.
      ). To address this issue, we assayed Grb2 immunoprecipitates for the presence of PI 3-kinase p85α. The results demonstrate binding of p85α to Grb2 in control and irradiated cells (Fig. 3B, left panel). Analysis of anti-p85α immunoprecipitates confirmed binding to Grb2 (Fig. 3B, right panel), although there was no detectable anti-Grb2 reactivity when assaying precipitates with preimmune rabbit serum or anti-c-Abl (data not shown). In order to define the domain of Grb2 involved in binding to PI 3-kinase, lysates from control and IR-treated U-937 cells were subjected to affinity chromatography with GST-Grb2 (full-length), GST-Grb2 SH2, GST-Grb2 C-SH3, or GST-Grb2 N-SH3 domains. The adsorbed proteins were then immunoblotted with anti-p85α. The results demonstrate that association of p85α with the SH2 domain of Grb2 is increased by IR exposure (Fig. 3C). Moreover, the finding that tyrosine phosphorylation of p85α is increased in irradiated cells provides an explanation for the IR-stimulated interaction of this protein with the Grb2 SH2 domain (Fig. 3D).
      Figure thumbnail gr3
      Figure 3:Association of the p85α subunit of PI 3-kinase with p54 SAP kinase and Grb2. A, lysates from control and IR-treated cells were subjected to immunoprecipitation with anti-p54 antibody. Proteins were resolved by 7.5% SDS-PAGE and analyzed by immunoblotting with anti-p85α antibody. B, lysates from control and IR-treated cells were immunoprecipitated with anti-p85α and anti-Grb2 antibodies. The proteins were resolved by SDS-PAGE and immunoblotted with anti-Grb2 and anti-p85α. C, lysates from control and IR-treated cells were incubated with GST-Grb2 SH2 protein. Lysates from IR-treated cells were also incubated with GST and GST-Grb2 (full-length). The bound proteins were resolved by 7.5% SDS-PAGE and subjected to immunoblotting with anti-p85α antibody. D, lysates from control and IR-treated cells were subjected to immunoprecipitation with anti-Tyr(P) (anti-P-Tyr) antibody. The bound proteins were resolved by 7.5% SDS-PAGE and analyzed by immunoblotting with anti-P85α.
      Whereas these findings are in support of a Grb2-mediated association between SAP kinase (through the Grb2 SH3 domains) and PI 3-kinase p85α (through the Grb2 SH2), we also asked whether there is a potential functional relationship between these two proteins. In addressing this possibility, we pretreated cells with wortmannin, a potent PI 3-kinase inhibitor(
      • Yano H.
      • Nakanishi S.
      • Kimura K.
      • Hanai N.
      • Saitoh Y.
      • Fukui Y.
      • Nonomura Y.
      • Matsuda Y.
      ,
      • Acaro A.
      • Wymann M.P.
      ), to determine whether PI 3-kinase is coupled to IR-induced SAP kinase signaling. Wortmannin treatment was associated with stimulation of constitutive levels of SAP kinase activity (Fig. 4A). Moreover, activation of SAP kinase in cells treated with both wortmannin and IR was similar to that obtained with wortmannin alone (Fig. 4A). In contrast, rapamycin (inhibitor of pp70s6k)(
      • Chen R.H.
      • Sarnecki C.
      • Blenis J.
      ), H-7 (a nonspecific protein kinase C inhibitor)(
      • Hidaka H.
      • Inagaki M.
      • Kawamoto S.
      • Sasaki Y.
      ), and HA1004 (an inhibitor of cyclic nucleotide-dependent protein kinase) (
      • Asano T.
      • Hidaka H.
      ) had little effect on SAP kinase activity (Fig. 4B and data not shown). These results suggest a potential role for PI 3-kinase in the regulation of SAP kinase.
      Figure thumbnail gr4
      Figure 4:Activation of SAP kinase by wortmannin. A, cells were exposed to IR, harvested at 1 h, pretreated with 250 nM wortmannin for 30 min (WORT) or with wortmannin and then IR (WORT/IR). Lysates were immunoprecipitated with anti-p54 antibody, and the resulting immune complexes were incubated in kinase buffer containing 10 μg of GST-Jun and [γ-32P]ATP for 15 min at 30°C. The reactions were stopped by the addition of SDS-PAGE sample buffer. Phosphorylated proteins were separated by 10% SDS-PAGE, dried, and analyzed by autoradiography. B, cells were treated with 1 ng/ml rapamycin (RAPA), 250 nM wortmannin (WORT), 50 μM H-7, or 50 μM HA1004 (data not shown) for 30 min. Lysates were subjected to immunoprecipitation with anti-p54 antibody. GST-Jun phosphorylation assays were performed as described above.
      PI 3-kinase possesses lipid and serine kinase activities(
      • Carpenter C.L.
      • Auger K.R.
      • Duckworth B.C.
      • Hou W.M.
      • Schaffhausen B.
      • Cantley L.C.
      ,
      • Dhand R.
      • Hiles I.
      • Panayotou G.
      • Roche S.
      • Fry J.M.
      • Gout I.
      • Totty N.F.
      • Truong O.
      • Vicendo P.
      • Yonezawa K.
      • Kasuga M.
      • Courtneidge S.A.
      • Waterfield M.D.
      ). The 85-kDa subunit of PI 3-kinase binds to tyrosine-phosphorylated proteins through its SH2 domains, and the 110-kDa subunit exhibits catalytic activity. Although IR treatment is associated with increased tyrosine phosphorylation of p85α (Fig. 3D), the precise mechanism by which p85α interacts with the SH2 domain of Grb2 requires further study. The activation of PI 3-kinase results in the formation of PI-3,4-P2 and PI-3,4,5-P3. The finding that the Ca2+- and phorbol ester-insensitive ζ isoform of protein kinase C is activated by PI-3,4,5-P3 has suggested that this kinase may be a target for PI 3-kinase signaling(
      • Nakanishi H.
      • Brewer K.A.
      • Exton J.H.
      ). The serine kinase activity of PI 3-kinase contributes to the phosphorylation of p85 subunit(
      • Dhand R.
      • Hiles I.
      • Panayotou G.
      • Roche S.
      • Fry J.M.
      • Gout I.
      • Totty N.F.
      • Truong O.
      • Vicendo P.
      • Yonezawa K.
      • Kasuga M.
      • Courtneidge S.A.
      • Waterfield M.D.
      ). Other work has indicated that PI 3-kinase phosphorylates the insulin receptor substrate 1 on serine(
      • Lam K.
      • Carpenter C.L.
      • Ruderman N.B.
      • Friel J.C.
      • Kelly K.L.
      ). Wortmannin inhibits both the lipid and serine phosphorylation activities of PI 3-kinase(
      • Lam K.
      • Carpenter C.L.
      • Ruderman N.B.
      • Friel J.C.
      • Kelly K.L.
      ). Thus, the effects of wortmannin on PI 3-kinase signaling pathways may be complex. The present demonstration that wortmannin treatment is associated with activation of SAP kinase suggests that PI 3-kinase may be involved in the regulation of SAP kinase. The findings that PI 3-kinase and SAP kinase form a complex in irradiated cells also supports a possible functional interaction between these two molecules. However, although SAP kinase-activating kinase 1 has been shown to directly stimulate SAP kinase activity(
      • Sanchez I.
      • Hughes R.T.
      • Mayer B.J.
      • Yee K.
      • Woodgett J.R.
      • Avruch J.
      • Kyriakis J.M.
      • Zon L.I.
      ,
      • Yan M.
      • Dai T.
      • Deak J.C.
      • Kyriakis J.M.
      • Zon L.I.
      • Woodgett J.R.
      • Templeton D.J.
      ), further studies are needed to more precisely define a potential interaction between PI 3-kinase and SAP kinase.

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