Mitotic and Stress-induced Phosphorylation of HsPI3K-C2α Targets the Protein for Degradation
2003; Elsevier BV; Volume: 278; Issue: 28 Linguagem: Inglês
10.1074/jbc.m301657200
ISSN1083-351X
AutoresSvetlana A. Didichenko, Cristina M. Fragoso, Marcus Thelen,
Tópico(s)Cellular transport and secretion
ResumoActivation of the phosphoinositide 3-kinases (PI 3-kinases) has been implicated in multiple cellular responses such as proliferation and survival, membrane and cytoskeletal reorganization, and intracellular vesicular trafficking. The activities and subcellular localization of PI 3-kinases were shown to be regulated by phosphorylation. Previously we demonstrated that class II HsPIK3-C2α becomes phosphorylated upon inhibition of RNA pol II-dependent transcription (Didichenko, S. A., and Thelen, M. (2001) J. Biol. Chem. 276, 48135–48142). In this study we investigated cell cycle-dependent and genotoxic stress-induced phosphorylation of HsPIK3-C2α. We find that the kinase becomes phosphorylated upon exposure of cells to UV irradiation and in proliferating cells at the G2/M transition of the cell cycle. Stress-dependent and mitotic phosphorylation of HsPIK3-C2α occurs on the same serine residue (Ser259) within a recognition motif for proline-directed kinases. Mitotic phosphorylation of HsPIK3-C2α can be attributed to Cdc2 activity, and stress-induced phosphorylation of HsPIK3-C2α is mediated by JNK/SAPK. The protein level of HsPIK3-C2α is regulated by proteolysis in a cell cycle-dependent manner and in response of cells to stress. Phosphorylation appears to be a prerequisite for proteasome-dependent degradation of HsPIK3-C2α and may therefore contribute indirectly to the regulation of the activity of the kinase. Activation of the phosphoinositide 3-kinases (PI 3-kinases) has been implicated in multiple cellular responses such as proliferation and survival, membrane and cytoskeletal reorganization, and intracellular vesicular trafficking. The activities and subcellular localization of PI 3-kinases were shown to be regulated by phosphorylation. Previously we demonstrated that class II HsPIK3-C2α becomes phosphorylated upon inhibition of RNA pol II-dependent transcription (Didichenko, S. A., and Thelen, M. (2001) J. Biol. Chem. 276, 48135–48142). In this study we investigated cell cycle-dependent and genotoxic stress-induced phosphorylation of HsPIK3-C2α. We find that the kinase becomes phosphorylated upon exposure of cells to UV irradiation and in proliferating cells at the G2/M transition of the cell cycle. Stress-dependent and mitotic phosphorylation of HsPIK3-C2α occurs on the same serine residue (Ser259) within a recognition motif for proline-directed kinases. Mitotic phosphorylation of HsPIK3-C2α can be attributed to Cdc2 activity, and stress-induced phosphorylation of HsPIK3-C2α is mediated by JNK/SAPK. The protein level of HsPIK3-C2α is regulated by proteolysis in a cell cycle-dependent manner and in response of cells to stress. Phosphorylation appears to be a prerequisite for proteasome-dependent degradation of HsPIK3-C2α and may therefore contribute indirectly to the regulation of the activity of the kinase. Phosphoinositide 3-kinases (PI 3-kinases) 1The abbreviations used are: PI 3-kinases, phosphoinositide 3-kinases; Cdk, cyclin-dependent protein kinase; CMV, cytomegalovirus; DMEM, Dulbecco's modified Eagle's medium; EGFP, enhanced green fluorescent protein; ERK, extracellular signal regulated kinase; GFP, green fluorescent protein; GST, glutathione S-transferase; HA, hemagglutinin; HEK, human embryonic kidney; JNK, c-Jun NH2-terminal kinase; JNKK, JNK kinase; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; MAP, mitogen-activated protein; MAPKK, MAP kinase kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; MS/MS, tandem mass spectrometry; PI3K, PI 3-kinases; PtdIns, phosphatidylinositol; SAPK, stress-activated protein kinase. regulate diverse cellular processes, which include cell signaling, intracellular protein sorting, cell cycle progression, cell survival, and apoptosis (2Fruman D.A. Meyers R.E. Cantley L.C. Annu. Rev. Biochem. 1998; 67: 481-507Crossref PubMed Scopus (1323) Google Scholar). PI 3-kinases phosphorylate the D3 hydroxyl group on the inositol ring leading to 3-phosphoinositides which act as membrane-embedded second messengers mediating the activation of downstream effectors (3Toker A. Cantley L.C. Nature. 1997; 387: 673-676Crossref PubMed Scopus (1229) Google Scholar). Class I PI 3-kinases are heterodimeric enzymes consisting of a catalytic and a regulatory subunit. They are involved primarily in growth factor and chemotactic agonist-mediated signal transduction (4Carpenter C.L. Cantley L.C. Curr. Opin. Cell Biol. 1996; 8: 153-158Crossref PubMed Scopus (576) Google Scholar, 5Stephens L.R. Jackson T.R. Hawkins P.T. Biochim. Biophys. Acta Mol. Cell Res. 1993; 1179: 27-75Crossref PubMed Scopus (426) Google Scholar). In vitro, these PI 3-kinases are able to utilize phosphatidylinositol (PtdIns), PtdIns(4)P, and PtdIns(4,5)P2 as substrates, but most likely produce PtdIns(3,4,5)P3 in vivo (6Leevers S.J. Vanhaesebroeck B. Waterfield M.D. Curr. Opin. Cell Biol. 1999; 11: 219-225Crossref PubMed Scopus (574) Google Scholar). Following activation of resting cells, these kinases are recruited rapidly from the cytosol to the plasma membrane where they generate PtdIns(3,4,5)P3. Class II PI 3-kinases are monomeric proteins. Three human isozymes, HsPI3K-C2α, HsPI3K-C2β, and HsPI3K-C2γ (7Brown R.A. Ho L.K.F. Weber-Hall S.J. Shipley J.M. Fry M.J. Biochem. Biophys. Res. Commun. 1997; 233: 537-544Crossref PubMed Scopus (61) Google Scholar, 8Domin J. Pages F. Volinia S. Rittenhouse S.E. Zvelebil M.J. Stein R.C. Waterfield M.D. Biochem. J. 1997; 326: 139-147Crossref PubMed Scopus (219) Google Scholar, 9Misawa H. Ohtsubo M. Copeland N.G. Gilbert D.J. Jenkins N.A. Yoshimura A. Biochem. Biophys. Res. Commun. 1998; 244: 531-539Crossref PubMed Scopus (66) Google Scholar, 10Rozycka M. Lu Y.J. Brown R.A. Lau M.R. Shipley J.M. Fry M.J. Genomics. 1998; 54: 569-574Crossref PubMed Scopus (53) Google Scholar), and their homologs in rodents have been characterized (11Molz L. Chen Y.W. Hirano M. Williams L.T. J. Biol. Chem. 1996; 271: 13892-13899Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 12Ono F. Nakagawa T. Saito S. Owada Y. Sakagami H. Goto K. Suzuki M. Matsuno S. Kondo H. J. Biol. Chem. 1998; 273: 7731-7736Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 13Virbasius J.V. Guilherme A. Czech M.P. J. Biol. Chem. 1996; 271: 13304-13307Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). PI 3-kinases of this class have been found also in Drosophila melanogaster (14MacDougall L.K. Domin J. Waterfield M.D. Curr. Biol. 1995; 5: 1404-1415Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar) and in Caenorhabditis elegans (15The C. elegans Sequencing ConsortiumScience. 1998; 282: 2012-2018Crossref PubMed Scopus (3634) Google Scholar), but not in yeast. Members of class II are distinguished from other PI 3-kinases by the presence of two tandem domains at their carboxyl terminus, a phox homology domain and a C2 domain, a module that is known to confer Ca2+-dependent phospholipid binding (16Wymann M.P. Pirola L. Biochim. Biophys. Acta Lipids Lipid Metab. 1998; 1436: 127-150Crossref PubMed Scopus (580) Google Scholar). However, the C2 domains of class II PI 3-kinases lack a critical Asp residue in the calcium binding loop (17Newton A.C. Curr. Biol. 1995; 5: 973-976Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar), which is consistent with the finding that they do not bind to membranes in a calcium-dependent manner (14MacDougall L.K. Domin J. Waterfield M.D. Curr. Biol. 1995; 5: 1404-1415Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 18Arcaro A. Zvelebil M.J. Wallasch C. Ullrich A. Waterfield M.D. Domin J. Mol. Cell. Biol. 2000; 20: 3817-3830Crossref PubMed Scopus (138) Google Scholar). In vitro, all class II PI 3-kinases phosphorylate PtdIns and PtdIns(4)P, but their in vivo substrate remains to be determined (2Fruman D.A. Meyers R.E. Cantley L.C. Annu. Rev. Biochem. 1998; 67: 481-507Crossref PubMed Scopus (1323) Google Scholar). Both HsPI3K-C2α and HsPI3K-C2β are implicated in signaling downstream of epidermal growth factor and platelet-derived growth factor receptors (18Arcaro A. Zvelebil M.J. Wallasch C. Ullrich A. Waterfield M.D. Domin J. Mol. Cell. Biol. 2000; 20: 3817-3830Crossref PubMed Scopus (138) Google Scholar). HsPI3K-C2α was shown to concentrate in the trans-Golgi network and in clathrin-coated pits (19Domin J. Gaidarov I. Smith M.E. Keen J.H. Waterfield M.D. J. Biol. Chem. 2000; 275: 11943-11950Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar), whereas PI3K-C2β was found in the nuclei of rat liver cells (20Sindic A. Aleksandrova A. Fields A.P. Volinia S. Banfic H. J. Biol. Chem. 2001; 276: 17754-17761Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). In general, the role of class II PI 3-kinases in signal transduction and mode of activation is poorly understood, and specific downstream targets have not been characterized. Class I PI 3-kinases activities were shown to be regulated by phosphorylation (21Carpenter C.L. Auger K.R. Duckworth B.C. Hou W.-M. Schaffhausen B. Cantley L.C. Mol. Cell. Biol. 1993; 13: 1657-1665Crossref PubMed Scopus (194) Google Scholar, 22Dhand R. Hiles I. Panayotou G. Roche S. Fry M.J. Gout I. Totty N.F. Truong O. Vicendo P. Yonezawa K. Kasuga M. Courtneidge S.A. Waterfield M.D. EMBO J. 1994; 13: 522-533Crossref PubMed Scopus (415) Google Scholar, 23Vanhaesebroeck B. Welham M.J. Kotani K. Stein R. Warne P.H. Zvelebil M.J. Higashi K. Volinia S. Downward J. Waterfield M.D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4330-4335Crossref PubMed Scopus (374) Google Scholar). Phosphorylation of the regulatory subunit p85 by the catalytic subunit p110α of class I PI 3-kinase (p110/p85 heterodimer) down-regulates lipid kinase activity of the complex (21Carpenter C.L. Auger K.R. Duckworth B.C. Hou W.-M. Schaffhausen B. Cantley L.C. Mol. Cell. Biol. 1993; 13: 1657-1665Crossref PubMed Scopus (194) Google Scholar, 22Dhand R. Hiles I. Panayotou G. Roche S. Fry M.J. Gout I. Totty N.F. Truong O. Vicendo P. Yonezawa K. Kasuga M. Courtneidge S.A. Waterfield M.D. EMBO J. 1994; 13: 522-533Crossref PubMed Scopus (415) Google Scholar). Phosphorylation of class II PI 3-kinases was demonstrated; however, the physiological role of this phosphorylation remained unclear. Increased phosphorylation of class II PI 3-kinase C2α was found to correlate with a moderately elevated enzyme activity in insulin-stimulated cells (24Brown R.A. Domin J. Arcaro A. Waterfield M.D. Shepherd P.R. J. Biol. Chem. 1999; 274: 14529-14532Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). In contrast, our data demonstrated that the phosphorylation status neither changes the lipid kinase activity of PI3K-C2α nor affects the substrate specificity, but influences the intranuclear localization (1Didichenko S.A. Thelen M. J. Biol. Chem. 2001; 276: 48135-48142Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). In this study we investigated the phosphorylation of HsPI3K-C2α induced by genotoxic stress and during the cell cycle. We show that the kinase becomes phosphorylated upon exposure of cells to UV irradiation and in proliferating cells at the G2/M transition of cell cycle. Stress-dependent and mitotic phosphorylation of HsPI3K-C2α occurs on the same serine residue (Ser259) within a recognition motif (serine-proline sequence) for proline-directed kinases, such as mitogen-activated protein (MAP) kinases and cyclin-dependent protein kinases (Cdk). By using different selective inhibitors of MAP kinases and Cdks in in vitro and in vivo assays, we found that Cdc2 mediates mitotic phosphorylation, whereas JNK/SAPK is responsible for stress-induced phosphorylation of HsPI3K-C2α. In either case phosphorylation provides a signal for proteasome-dependent degradation of the protein. Antibodies—Antibodies against PI3K-C2α (AXIX and AXXIII) were described previously (1Didichenko S.A. Thelen M. J. Biol. Chem. 2001; 276: 48135-48142Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Anti-GFP rabbit polyclonal antibody was purchased from Clontech, anti-Cdc2 polyclonal rabbit antibody was from Oncogene, anti-human cyclin B1 antibody was from Pharmingen (BD), anti-phospho-Jun (pSer63) and anti-phospho-Jun (pSer73) rabbit immunoaffinity-purified IgG were from Upstate Biotechnology. Secondary horseradish peroxidase-conjugated goat anti-rabbit and goat anti-mouse antibodies were obtained from Bio-Rad. Plasmids—The cDNA encoding human HsPI3K-C2α (8Domin J. Pages F. Volinia S. Rittenhouse S.E. Zvelebil M.J. Stein R.C. Waterfield M.D. Biochem. J. 1997; 326: 139-147Crossref PubMed Scopus (219) Google Scholar) was a kindly provided by Dr. J. Domin (London). Various HsPI3K-C2α cDNAs were amplified by the PCR using gene-specific primers with incorporated restriction sites to facilitate their cloning into appropriate vectors. For expression in bacterial cells, GST-tagged fusion constructs were generated by cloning wild-type and mutant HsPI3K-C2α cDNAs into the BamHI site of pGEX-2T (Amersham Biosciences). For expression in mammalian cells the cDNAs were cloned into the eukaryotic expression vector pEGFP-C1 or pEGFP-N1 (Clontech). To generate pEGFP: ΔHsPI3K-C2α expressing GFP-ΔHsPI3K-C2α, a PCR product corresponding to the amino acids 240–275 of HsPI3K-C2α was inserted into XhoI-BamHI sites of pEGFP-C1. HsPI3K-C2α point mutations, S254A, S259A, S259D, S259E, S262A, and S264A were created by PCR amplification from pEGFP-C1:ΔHsPI3K-C2α, using mutant sequence oligonucleotides. pBK-CMV:myc-HsPI3K-C2α, which encodes full-length HsPI3K-C2α tagged at the NH2 terminus with myc epitope was constructed as follows. The SacI-BspEI fragment from pBK-CMV-HsPI3K-C2α (8Domin J. Pages F. Volinia S. Rittenhouse S.E. Zvelebil M.J. Stein R.C. Waterfield M.D. Biochem. J. 1997; 326: 139-147Crossref PubMed Scopus (219) Google Scholar) containing the 5′-untranslated region and the first 52 nucleotides of the HsPI3K-C2α coding sequence was replaced by the SacI-BspEI PCR fragment carrying a Kozak consensus sequence, an ATG start codon, and the sequence of myc tag joined in-frame to the HsPI3K-C2α coding sequence (4–52 bp). pBK-CMV:HA-HsPI3K-C2α, which encodes full-length HsPI3K-C2α tagged at the NH2 terminus with HA epitope, was constructed using a similar approach. To generate pEGFP-C1:GFP-HA-HsPI3K-C2α, which encodes complete HsPI3K-C2α double tagged at the NH2 terminus with GFP and HA epitope (GFP-HsPI3K-C2α), the SacI-EcoRI fragment from pBK-CMV:HA-HsPI3K-C2α was cloned into SacI-EcoRI sites of pEGFP-C1. To generate pEGFP-N1:myc-HsPI3K-C2α-GFP that encoded full-length HsPI3K-C2α tagged at the NH2 terminus with myc epitope and at the COOH terminus with GFP (HsPI3K-C2α-GFP), the SacI-AccI fragment from pBK-CMV:myc-HsPI3K-C2α containing the myc-tagged HsPI3K-C2α coding sequence up to 4884 bp was cloned into to pEGFP-N1:HsPI3K-C2α-ΔN, in which the COOH-terminal fragment of HsPI3K-C2α (4884–5055 bp) was joined in-frame to GFP. JNKK2 mammalian expression vector (25Wu Z. Wu J. Jacinto E. Karin M. Mol. Cell. Biol. 1997; 17: 7407-7416Crossref PubMed Scopus (95) Google Scholar) was a gift from G. Natoli (Bellinzona). Cell Culture, Synchronization, Transient and Stable Expressions— HeLa (ATCC), MCF7, COS-7, and HEK-293 cells were cultured at 37 °C in Dulbecco's modified Eagle's medium (DMEM, Invitrogen) supplemented with 10% fetal calf serum and antibiotics. The following protocols were used to obtain HeLa cells arrested at specific stages of the cell cycle. Cells, enriched in G1, were obtained by treatment with mimosine (Sigma) at 400 μm for 20 h. For early S phase arrest, subconfluent cultures were blocked by serum deprivation for 48 h followed by the addition of 5 μg/ml aphidicolin (Sigma) for 24 h (26Al Mohanna M.A. Al Khodairy F.M. Krezolek Z. Bertilsson P.A. Al Houssein K.A. Aboussekhra A. Carcinogenesis. 2001; 22: 573-578Crossref PubMed Scopus (27) Google Scholar). To obtain a cell population enriched in G2 phase, cells were presynchronized in S phase as described above and then released by transferring into fresh medium for 8 h. For M phase synchronization, cells were treated with nocodazole (Sigma) at 400 ng/ml for 16 h before collecting mitotic cells by shake off. Cell cycle distributions were confirmed by flow cytometry (fluorescence-activated cell sorter). For fluorescence-activated cell sorter analysis of DNA content, cells were washed twice in phosphate-buffered saline and fixed in 90% methanol at -20 °C for 15 min. After an additional wash in phosphate-buffered saline cells were resuspended in 4 mm sodium-citrate, 0.1% Triton X-100 and treated with 10 μg/ml RNase A in the presence of 50 μg/ml propidium iodide for 10 min at 37 °C. To inhibit proteasome activity, MG132 (Calbiochem) was added to cells at a concentration of 20 μm. Transient and stable transfections were carried out using PolyFect reagent (Qiagen) according to the manufacturer's instructions. For transient expression of GFP-HsPI3K-C2α and the mutants (S259A and S259D) COS-7 cells were transfected with the corresponding plasmids: pEGFP-C1:GFP-HA-HsPI3K-C2α, pEGFP-C1:GFP-HA-HsPI3K-C2α/S259A, and pEGFP-C1:HA-HsPI3K-C2α/S259D. For generation of stable HEK-293 lines expressing wild-type and mutant HsPI3K-C2α-GFP fusion proteins, HEK-293 cells were transfected with pEGFP-N1:myc-HsPI3K-C2α-GFP or pEGFP-N1:myc-HsPI3K-C2α/S259A-GFP. Two days after transfection, cells were replated in medium containing 1 mg/ml G418. G418-resistant colonies, selected at 2–3 weeks after transfection, were subcloned and analyzed for the expression of recombinant proteins by immunoblotting with anti-GFP antibody. UV and γ-Irradiation—Cells were exposed to genotoxic agents and analyzed 1.5 h later. An UV dose of 300 J/m2 was delivered in a single pulse using a Stratalinker (Stratagene). Prior to pulsing, the medium was removed, being replaced immediately after the treatment. 100 Gy of γ-irradiation was delivered using a Gammacell 1000 apparatus. Gel Electrophoresis, Immunoprecipitation, and Western Blot Analysis—Proteins were separated on 8 or 6% SDS-polyacrylamide gels prepared from the stock (33.5% acrylamide, 0.3% bisacrylamide) and blotted onto Immobilon-P (Millipore). Membranes were blocked with 5% non-fat milk in Tris-buffered saline containing 0.1% Triton X-100 and probed with specific antibodies. Immunoreactive bands were decorated with horseradish peroxidase-labeled secondary antibodies and visualized by enhanced chemiluminescence (Pierce). For immunoprecipitation cells were washed twice in phosphate-buffered saline and lysed in buffer (1% Nonidet P-40, 150 mm NaCl, 50 mm Tris-HCl (pH 7.5), 1.5 mm MgCl2, 1 mm EDTA), supplemented with phosphatase inhibitors (40 mm NaF, 0.5 mm sodium orthovanadate, 40 μm β-glycerophosphate, 5 mm sodium pyrophosphate) and protease inhibitors (Complete, Roche). Cell homogenates were centrifuged at 13,000 × g for 10 min, and supernatants were precleared with Gamma-Bind Plus-Sepharose (Amersham Biosciences) for 15 min. Immunoprecipitation of HsPI3K-C2α with antibody AXXIII was carried out at 4 °C for 1–2 h. Immune complexes were bound to GammaBind Plus-Sepharose for 30 min, collected by centrifugation, and washed twice in lysis buffer, once in 10 mm Tris-HCl (pH 8), 0.5 m NaCl, 0.5% Nonidet P-40, 0.5% deoxycholate, 0.05% SDS; then in 10 mm Tris-HCl (pH 8), once in 10 mm Tris-HCl (pH 8), 150 mm NaCl, 0.5% Nonidet P-40, 0.5% deoxycholate, 0.05% SDS, and finally in 10 mm Tris-HCl (pH 8), 0.05% SDS. For λ-phosphatase treatment immunoprecipitates were additionally washed twice in phosphatase buffer (50 mm Tris-HCl (pH 7.5), 2 mm MnCl2, 0.1 mm EDTA, 5 mm dithiothreitol, 0.01% Brij 35) and resuspended in 50 μl of the same buffer. After warming up at 30 °C for 3 min, 50 units of λ-phosphatase (New England Biolabs) was added, and samples were incubated at 30 °C for 40 min. Pulse-Chase Experiments—Subconfluent cultures of HeLa cells were labeled overnight with 50 μCi of [35S]methionine/cysteine (Amersham Biosciences)/ml in methionine-free DMEM (Invitrogen) supplemented with 10% dialyzed fetal calf serum. After labeling cells were washed in phosphate-buffered saline, replated (1:3 dilution), and chased with complete DMEM containing 10% fetal calf serum for 48 h. To obtain mitotic cells, 400 ng/ml nocodazole was added to the medium for the last 36 h of the chase. Labeled mitotic cells were collected by shake off, washed three times in prewarmed DMEM, and released into fresh complete medium for 3 h. For metabolic labeling of cells at M/G1 transition of cell cycle, nocodazole-treated mitotic HeLa cells were released into the labeling DMEM in the presence of 50 μCi of [35S]methionine/cysteine for 1 or 3 h. Cells were subsequently subjected to immunoprecipitation analysis with anti-HsPI3K-C2α antibody (AXXIII) as described above. Immunoprecipitated proteins were resolved by SDS-PAGE, transferred to polyvinylidene difluoride membranes, and visualized by autoradiography and immunoblotting. Kinase and Protease Inhibitor Treatments—Roscovitine (Calbiochem) was used to inhibit Cdk activity. HeLa cells at late S phase (6 h after release from aphidicolin block) were treated or not with 30 μm roscovitine for 2 h. 400 ng/ml nocodazole was added and treatment continued for 15 h. Nonadhering mitotic and adhering G2 cells were collected by mechanical shock and trypsin treatment, respectively. Nocodazole-arrested mitotic HeLa cells were treated with 75 μm roscovitine for 15, 45, and 90 min. For okadaic acid treatment nocodazole-arrested mitotic HeLa cells were treated with 0.5 μm okadaic acid for 30 min, then 75 μm roscovitine was added, and treatment continued for 30 min. SP600125 (Tocris) was used to inhibit JNK activation, and PD98059 and SB202190 (both from Alexis) were used to inhibit ERK and p38 activation, respectively. HeLa cells were preatreated with the inhibitors at concentrations indicated for 30 min before UV irradiation. Irradiated cells were cultured for 90 min in the presence of the inhibitors before harvesting. The specific protease inhibitors MG132, ALLM, and lactacystin were from Calbiochem. HEK-293 cells were UV irradiated as described above. After 2 h of recovery in fresh medium, cells were treated with protease inhibitors (20 μm MG132, 100 μm ALLM, or 50 μm lactacystin) for the indicated times prior to Western blot analysis. In Vitro Kinase Assay—HsPI3K-C2α was immunoprecipitated from mimosine-treated HeLa cells using affinity-purified anti-HsPI3K-C2α antibody AXIX (1Didichenko S.A. Thelen M. J. Biol. Chem. 2001; 276: 48135-48142Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). The antibody-antigen complexes were collected with GammaBind Plus-Sepharose and used as substrate for in vitro phosphorylation by cellular extracts. To obtain cellular extracts, pellets of interphase or mitotic HeLa cells were resuspended in 2 pellet volumes of ice-cold hypotonic buffer (50 mm Tris-HCl (pH 7.5), 1 mm dithiothreitol, 40 mm NaF, 0.5 mm sodium orthovanadate, 40 mm β-glycerophosphate, 5 mm sodium pyrophosphate, and a mixture of protease inhibitors (Complete, Roche)) and disrupted by brief sonication. The resulting homogenates were centrifuged at 400,000 × g for 15 min at 4 °C. Supernatants (∼10 mg of protein/ml) were supplemented with 150 mm NaCl and 10 mm MgCl2 and used as a source of kinases. HsPI3K-C2α immunoprecipitates were mixed with supernatants in a final volume of 100 μl, and phosphorylation assays were initiated by adding 1 mm ATP. Assays were carried out at 30 °C for 1 h and terminated by the addition of ice-cold Tris-buffered saline containing 0.1% of Triton X-100. HsPI3K-C2α immunoprecipitates were collected by centrifugation, washed twice with Tris-buffered saline, and analyzed by Western blotting as described above. For in vitro phosphorylation GST-ΔHsPI3K-C2α fusion proteins were expressed in Escherichia coli strain (BL21) and purified by absorption to glutathione-Sepharose beads (Amersham Biosciences). Fusion proteins were left attached to the beads, and phosphorylation reactions with cellular extracts were carried out as described above in the presence of 50 μCi of [γ-32P]ATP and 1 mm ATP. In vitro phosphorylation of GST-ΔHsPI3K-C2α and GFP-HsPI3K-C2α fusion proteins by 10 units of purified recombinant human Cdc2-cyclin B (Calbiochem) was performed in Cdc2-kinase buffer (50 mm Tris-HCl (pH 7.5), 2 mm dithiothreitol, 10 mm MgCl2, 1 mm EGTA) in the presence of 10 μCi of [γ-32P]ATP and 100 μm ATP. Soluble GST-ΔHsPI3K-C2α was used as substrate in phosphorylation assays with immunoprecipitated Cdc2. Cdc2 was immunoprecipitated from cytosols of HeLa cells as described above, immunocomplexes bound to GammaBind Plus-Sepharose beads were additionally washed twice in the kinase buffer, and the reaction was initiated by addition of purified GST-ΔHsPI3K-C2α and ATP (100 μm ATP, 10 μCi of [γ-32P] ATP). Immunofluorescence—Immunofluorescence experiments were performed as described previously (1Didichenko S.A. Thelen M. J. Biol. Chem. 2001; 276: 48135-48142Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar) using the methanol fixation protocol. UV-induced and Cell Cycle-dependent Phosphorylation of HsPI3K-C2α—Our previous immunofluorescence studies revealed that in interphase HeLa cells HsPI3K-C2α is localized to nuclear speckles together with the components of the splicing apparatus (1Didichenko S.A. Thelen M. J. Biol. Chem. 2001; 276: 48135-48142Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Inhibition of transcription by actinomycin D or α-amanitin causes subnuclear relocation of the kinase and is accompanied by phosphorylation of the protein, which can be measured as mobility shift of HsPI3K-C2α during SDS-PAGE. To investigate whether down-regulation of transcription caused by different types of genotoxic stress results in phosphorylation of HsPI3K-C2α, we exposed cells to DNA-damaging treatment such as UV light or ionizing radiation. Exposure of HeLa and MCF7 cells to UV irradiation induced a collapse of nuclear HsPI3K-C2α-positive speckles (Fig. 1A) similar to that observed in actinomycin D-treated cells: speckles lose their irregular shape, become round, and fuse into larger clusters (1Didichenko S.A. Thelen M. J. Biol. Chem. 2001; 276: 48135-48142Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). This effect was associated with the increased phosphorylation of HsPI3K-C2α, as measured by its mobility shift on SDS-polyacrylamide gels (Fig. 1B). When cell extracts were treated with λ-phosphatase the appearance of the slower migrating band was abolished (not shown). In contrast to UV-treated cells, exposure of cells to γ-irradiation neither changed subnuclear localization of HsPI3K-C2α (not shown) nor induced its phosphorylation (Fig. 1B). These results suggest that HsPI3K-C2α specifically participates in UV-induced damage response. The observation that phosphorylation of HsPI3K-C2α correlates with changes in its subnuclear localization let us to speculate that the phosphorylation status of the kinase may also be cell cycle-dependent, because in mitotic cells HsPI3K-C2α-positive speckles dissolve, and the kinase becomes equally distributed over the cytoplasm (Fig. 1A). We used HeLa cells to examine whether HsPI3K-C2α demonstrates different phosphorylation patterns during the cell cycle. Cells were synchronized in different stages of the cell cycle as follows: at late G1 with mimosine, at M with nocodazole, in early S phase by serum deprivation followed by an aphidicolin block, and cells enriched in G2 phase were obtained 8 h after release from aphidicolin block (26Al Mohanna M.A. Al Khodairy F.M. Krezolek Z. Bertilsson P.A. Al Houssein K.A. Aboussekhra A. Carcinogenesis. 2001; 22: 573-578Crossref PubMed Scopus (27) Google Scholar). Proteins from corresponding cell lysates were fractioned by SDS-PAGE, and HsPI3K-C2α was analyzed by immunoblotting (Fig. 2A). In mimosine-treated cells HsPI3K-C2α was detected as a single band, in cells blocked in S phase a second slower migrating band became visible. Two bands, a faster and a slower migrating, of equal intensity were apparent in cells enriched in G2 phase. A single slower migrating band was found in prometaphase-blocked mitotic cells. To confirm that altered gel mobility of HsPI3K-C2α was the result of phosphorylation, protein extracts from synchronized HeLa cells were treated with λ-phosphatase (Fig. 2). Phosphatase treatment resulted in the collapse of the slower migrating band of the kinase, indicating that indeed retarded mobility of HsPI3K-C2α during SDS-PAGE is a consequence of phosphorylation. These results demonstrate that HsPI3K-C2α undergoes a cell cycle-regulated phosphorylation that reaches its maximum in mitosis. HsPI3K-C2α Can Be Phosphorylated in Vitro by Kinases Present in HeLa Cell Extracts—The initial strategy to identify residues on which HsPI3K-C2α becomes phosphorylated upon UV irradiation and during cell cycle was to label HeLa cells metabolically in the presence of 32Pi. In several attempts we did not succeed to obtain sufficient amounts of in vivo 32P-labeled HsPI3K-C2α by immunoprecipitation to perform phosphopeptide mapping analysis. To overcome this problem, we developed an in vitro phosphorylation assay that allowed the identification of potential phosphorylation sites. As a substrate for phosphorylation we used HsPI3K-C2α immunoprecipitated from mimosine-treated HeLa cells. Concentrated high speed supernatants (S100) prepared from HeLa cells (asynchronously growing or mitotic) were used as sources of kinases. The supernatants are devoid of HsPI3K-C2α as shown previously (1Didichenko S.A. Thelen M. J. Biol. Chem. 2001; 276: 48135-48142Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar), which excluded any additional input of the a
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