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• W2028274300 abstract "Microtubule-interfering agents are widely used in cancer chemotherapy, and prognostic results vary significantly from tumor to tumor, depending on the p53 status. In preliminary experiments, we compared the expression and phosphorylation profiles of more than 100 protein kinases and protein phosphatases in human colorectal carcinoma cell line HCT116 between p53+/+ and p53−/− cells in response to short term nocodazole treatment through application of Kinetworks™ immunoblotting screens. Among the proteins tracked, the regulation of the phosphorylation of c-Jun N-terminal kinase (JNK)1/2 at Thr-183/Tyr-185 was the major difference between p53+/+ and p53−/− cells. With the loss of the p53 gene, the levels of phosphorylation of Ser-63 of c-Jun and Thr-183/Tyr-185 of JNK1/2 in p53−/− cells did not increase as markedly as in p53+/+ cells in response to a 1-h treatment with nocodazole or other microtubule-disrupting drugs such as vinblastine and colchicine. Similar observations were also made in MCF-7 and A549 tumor cells, which were rendered p53-deficient by E6 oncoprotein expression. However, arsenate-induced JNK activation in p53−/− cells was preserved. Inhibition of p53 expression by its antisense oligonucleotide also attenuated nocodazole-induced JNK activation in p53+/+ cells. Surprisingly, cotransfection of p53+/+ cells with dominant negative mutants of JNK isoforms and treatment of p53+/+ cells with the JNK inhibitor SP600125 actually further enhanced apoptosis in p53+/+ cells by up to 2-fold in response to nocodazole. These findings indicate that inhibition of p53-mediated JNK1/2 activity in certain tumor cells could serve to enhance the apoptosis-inducing actions of cancer chemotherapeutic agents that disrupt mitotic spindle function. Microtubule-interfering agents are widely used in cancer chemotherapy, and prognostic results vary significantly from tumor to tumor, depending on the p53 status. In preliminary experiments, we compared the expression and phosphorylation profiles of more than 100 protein kinases and protein phosphatases in human colorectal carcinoma cell line HCT116 between p53+/+ and p53−/− cells in response to short term nocodazole treatment through application of Kinetworks™ immunoblotting screens. Among the proteins tracked, the regulation of the phosphorylation of c-Jun N-terminal kinase (JNK)1/2 at Thr-183/Tyr-185 was the major difference between p53+/+ and p53−/− cells. With the loss of the p53 gene, the levels of phosphorylation of Ser-63 of c-Jun and Thr-183/Tyr-185 of JNK1/2 in p53−/− cells did not increase as markedly as in p53+/+ cells in response to a 1-h treatment with nocodazole or other microtubule-disrupting drugs such as vinblastine and colchicine. Similar observations were also made in MCF-7 and A549 tumor cells, which were rendered p53-deficient by E6 oncoprotein expression. However, arsenate-induced JNK activation in p53−/− cells was preserved. Inhibition of p53 expression by its antisense oligonucleotide also attenuated nocodazole-induced JNK activation in p53+/+ cells. Surprisingly, cotransfection of p53+/+ cells with dominant negative mutants of JNK isoforms and treatment of p53+/+ cells with the JNK inhibitor SP600125 actually further enhanced apoptosis in p53+/+ cells by up to 2-fold in response to nocodazole. These findings indicate that inhibition of p53-mediated JNK1/2 activity in certain tumor cells could serve to enhance the apoptosis-inducing actions of cancer chemotherapeutic agents that disrupt mitotic spindle function. Given the pivotal roles of microtubules in numerous biological processes such as mitotic spindle formation, treatment of cells with nocodazole and other microtubule-interfering agents evokes the activation of stress response pathways, cell cycle arrest, and the induction of apoptosis. This accounts for the extensive use of microtubule-interfering agents in tumor chemotherapy. In recent years, a great deal of effort has been devoted to elucidating the signaling pathways that mediate the biological activities of microtubule-interfering agents (1Gundersen G.G. Cook T.A. Curr. Opin. Cell Biol. 1999; 11: 81-94Crossref PubMed Scopus (361) Google Scholar). The p53 tumor suppressor protein is a short lived transcription factor that serves as a key player in the cellular response to a variety of extra- and intracellular insults, such as DNA damage, oncogenic activation, and microtubule disruption (2Meek D.W. Cell Signal. 1998; 10: 159-166Crossref PubMed Scopus (177) Google Scholar, 3Ryan K.M. Phillips A.C. Vousden K.H. Curr. Opin. Cell Biol. 2001; 13: 332-337Crossref PubMed Scopus (567) Google Scholar). It is known that p53 exerts its function mainly through transcriptional activation of target genes such as the CDK 1The abbreviations used are: CDK, cyclin-dependent kinase; CK, casein kinase; ERK, extracellular signal-regulated kinase; GST, glutathioneS-transferase; HA, hemagglutinin; JNK, c-Jun N-terminal kinase; MAP, mitogen-activated protein; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; MKP, MAP kinase phosphatase; PKA, protein kinase A; PKC, protein kinase C; PTP, protein-tyrosine kinase. For additional abbreviations, see TableI 1The abbreviations used are: CDK, cyclin-dependent kinase; CK, casein kinase; ERK, extracellular signal-regulated kinase; GST, glutathioneS-transferase; HA, hemagglutinin; JNK, c-Jun N-terminal kinase; MAP, mitogen-activated protein; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; MKP, MAP kinase phosphatase; PKA, protein kinase A; PKC, protein kinase C; PTP, protein-tyrosine kinase. For additional abbreviations, see TableIinhibitor, p21Waf1/Cip1, for arresting the cell cycle and the proapoptotic protein, Bax, for inducing apoptosis (4Miyashita T. Reed J.C. Cell. 1995; 80: 293-299Abstract Full Text PDF PubMed Scopus (301) Google Scholar, 5El Deiry W.S. Curr. Top. Microbiol. Immunol. 1998; 227: 121-137PubMed Google Scholar). Similar to other stresses, microtubule disruption results in an increase of p53 phosphorylation at multiple sites in a drug- and cell-specific manner, with resultant accumulation of transcriptionally active protein (6Sablina A.A. Chumakov P.M. Levine A.J. Kopnin B.P. Oncogene. 2001; 20: 899-909Crossref PubMed Scopus (65) Google Scholar, 7Stewart Z.A. Tang L.J. Pietenpol J.A. Oncogene. 2001; 20: 113-124Crossref PubMed Scopus (59) Google Scholar). Recently, we have demonstrated that nocodazole-induced phosphorylation of p53 at Ser-392, one of its key activating sites, is mediated through direct p38 MAP kinase stimulation of casein kinase 2 (CK2) in the HeLa cervical and HCT116 colon carcinoma cell lines (8Sayed M. Kim S.O. Salh B.S. Issinger O.G. Pelech S.L. J. Biol. Chem. 2000; 275: 16569-16573Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 9Sayed M. Pelech S.L. Wong C. Marotta A. Salh B. Oncogene. 2001; 20: 6994-7005Crossref PubMed Scopus (51) Google Scholar). To explore downstream p53-dependent regulation of signaling proteins in the early response of cells to nocodazole treatment, we applied three of our Kinetworks™ screens to track quantitatively the expressions of 75 protein kinases and 25 protein phosphatases and the states of 31 known phosphorylation sites in these and other phosphoproteins. This was accomplished by comparing the expression and phosphorylation profiles of these proteins in a human colon carcinoma cell line HCT116 p53+/+ and its derivative HCT116 p53−/−, where the p53 gene was disrupted through homologous recombination (10Waldman T. Kinzler K.W. Vogelstein B. Cancer Res. 1995; 55: 5187-5190PubMed Google Scholar, 11Bunz F. Dutriaux A. Lengauer C. Waldman T. Zhou S. Brown J.P. Sedivy J.M. Kinzler K.W. Vogelstein B. Science. 1998; 282: 1497-1501Crossref PubMed Scopus (2505) Google Scholar). Among the known phosphoproteins tracked, the p53-dependent increase in the phosphorylation and activation of the c-Jun N-terminal kinase (JNK) was the only significant difference between p53+/+ and p53−/− cells. Even though JNK is well known to play an important role in coordinating the cellular response to stress by phosphorylating the transcription factors c-Jun and p53 (12Hu M.C.T. Qiu W.R. Wang Y.P. Oncogene. 1997; 15: 2277-2287Crossref PubMed Scopus (106) Google Scholar, 13Buschmann T. Potapova O. Bar-Shira A. Ivanov V.N. Fuchs S.Y. Henderson S. Fried V.A. Minamoto T. Alarcon-Vargas D. Pincus M.R. Gaarde W.A. Holbrook N.J. Shiloh Y. Roni Z. Mol. Cell. Biol. 2001; 21: 2743-2754Crossref PubMed Scopus (248) Google Scholar), this report is the first time that a p53-mediated JNK activity has been unambiguously identified. In addition, we provide evidence for the possible involvement of the p53-mediated JNK activity in a protective response elicited by the stress of microtubule disruption. HCT116 p53 wild-type (p53+/+) and knockout derivative (p53−/−) (11Bunz F. Dutriaux A. Lengauer C. Waldman T. Zhou S. Brown J.P. Sedivy J.M. Kinzler K.W. Vogelstein B. Science. 1998; 282: 1497-1501Crossref PubMed Scopus (2505) Google Scholar) cells were kindly provided by Dr. Bert Vogelstein (Howard Hughes Medical Institute, Baltimore, MD). Human breast carcinoma MCF-7 and MCF-7 p53-deficient derivative, and human lung carcinoma A549 and A549 p53-deficient derivative were from Dr. Michel Roberge (University of British Columbia, Vancouver, BC, Canada). Both MCF-7 and A549 p53-deficient cell lines are derived from E6 oncogene overexpression. Cells were maintained in monolayer culture in a humidified 5% CO2atmosphere at 37 °C in Dulbecco's modified Eagle medium (Invitrogen) supplemented with 10% fetal bovine serum and penicillin/streptomycin. Anti-JNK1, p53, MEK4, MKP1, MKP2, β-actin, and horseradish peroxidase)-conjugated secondary antibodies were purchased from Santa Cruz Biotechnologies (Santa Cruz, CA). Anti-phospho-[Thr-183, Tyr-185]JNK polyclonal antibody was purchased from Promega (Madison, WI). The phospho-[Ser-63]c-Jun antibody was obtained from New England Biolabs (Beverly, MA). GST-c-Jun(1–79)-agarose conjugate was purchased from StressGen Biotechnologies (Victoria, BC, Canada). GST-JNK2 agarose conjugate was obtained from Upstate Biotechnology (Lake Placid, NY). Nocodazole, vinblastine, colchicine, taxol, and sodium arsenate were purchased from Sigma. The JNK inhibitor SP600125 was from Tocris Cookson Ltd. (Bristol, UK). Other reagents were all from commercial sources, unless otherwise stated. Fluorescein-labeled phosphorothioate p53 antisense (5′-CCCTGCTCCCCCCTGGCTCC-3′) and control nonsense (5′-CGGTGATCTCCAGAGTATGC-3′) oligonucleotides were synthesized by the NAPS unit of University of British Columbia on a 349 DNA/RNA synthesizer (Applied Biosystems, Foster City, CA). The antisense oligonucleotide is complementary to nucleotides 1071–1090 of exon 10 of the p53 gene, which is located in the C-terminal region that is required for oligomerization of p53 (14Hirota Y. Horiuchi T. Akahane K. Jpn. J. Cancer Res. 1996; 87: 735-742Crossref PubMed Scopus (21) Google Scholar). The oligonucleotides were purified twice by ethanol precipitation. Cells were transfected with oligonucleotides at the concentrations indicated using Lipofectin transfection reagent from Invitrogen according to the manufacturer's protocol. The pcDNA3-HA-MEK4(AL) dominant negative mutant plasmid was provided by Dr. Jim Woodgett (Ontario Cancer Institute, ON, Canada), and pLNCX vectors containing wild-type and dominant negative mutants (APF) of JNKs, HAp40JNK1α, β, HAp40JNK2α, β, were gifts from Dr. Lynn Heasley (University of Colorado, Denver) (15Butterfield L. Storey B. Maas L. Heasley L.E. J. Biol. Chem. 1997; 272: 10110-10116Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). Transfections of HCT116 cells with these constructs were performed using LipofectAMINE Plus reagent (Invitrogen). For flow cytometry analyses of DNA staining profile, transfected or nontransfected cells in 100-mm dishes at ∼60–80% confluence were treated with 200 ng/ml nocodazole. At various times as indicated under “Results,” the cells were harvested by trypsin treatment, combined with floating cells in the medium, washed once in phosphate-buffered saline, and fixed in methanol for 30 min at −20 °C. After three washes in phosphate-buffered saline, the cells were resuspended in phosphate-buffered saline containing 25 μg/ml RNase A and 25 μg/ml propidium iodine at 37 °C for 30 min. The DNA fluorescence was measured using a BD Biosciences FACScan; data acquisition and analysis were performed with the Cell Quest software. DNA fragmentation assays were performed as described by Huang et al. (16Huang C. Zhang Z. Ding M., Li, J., Ye, J. Leonard S. Shen H.-M. Butterworth L., Lu, Y. Costa M. Rojanasakul Y. Castranova V. Vallyathan V. Shi X. J. Biol. Chem. 2000; 275: 32516-32522Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). Total cell lysates were prepared as described previously (17Zhang H. Shi X. Hampong M. Blanis L. Pelech S. J. Biol. Chem. 2001; 276: 6905-6908Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). Briefly, cells were washed with ice-cold phosphate-buffered saline, scraped in lysis buffer (20 mmTris, 20 mm β-glycerophosphate, 150 mm NaCl, 3 mm EDTA, 3 mm EGTA, 1 mmNa3VO4, 0.5% Nonidet P-40, 1 mmdithiothreitol) supplemented with 1 mm phenylmethylsulfonyl fluoride, 2 μg/ml leupeptin, 4 μg/ml aprotinin, and 1 μg/ml pepstatin A, sonicated for 15 s. Cell debris was removed by centrifugation at 13,000 rpm for 15 min at 4 °C. Protein concentration was determined by the method of Bradford (18Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (211983) Google Scholar). Aliquots of cell lysates were resolved on SDS-PAGE (13% gel), transferred to nitrocellulose membranes, and incubated with various primary antibodies followed by relevant horseradish peroxidase-conjugated secondary antibodies. The blots were developed with ECL Plus reagent (AmershamBiosciences), and signals were then captured by Fluor-S MultiImager and quantified using Quantity One software (Bio-Rad). For the preparation of cytosolic and particulate fractions for Kinetworks™ analyses, cells were homogenized using a Dounce homogenizer in the above lysis buffer without NaCl and Nonidet P-40. After ultracentrifugation at 100,000 rpm for 30 min at 4 °C, the supernatant was collected as a cytosolic fraction. The pellet fraction was then rehomogenized in lysis buffer with NaCl and 0.5% Nonidet P-40. After ultracentrifugation, the detergent-solubilized supernatant was saved as a particulate fraction. Kinetworks™ analyses were performed on 300–600 μg of protein/sample. The Kinetworks™ analyses carried out included KPKS 1.0 for 75 protein kinases, KPPS 1.1 for 25 protein phosphatases, and KPSS 1.0 for 33 phosphoproteins. The immunoblotting analyses involved probing with mixes of in-house validated primary antibodies from commercial sources and the application of each mix into a separate lane of a 20-lane multiblotter (Immunetics). Detailed protocols for the Kinetworks™ analyses can be found at the Kinexus Bioinformatics website (www.kinexus.ca). For JNK kinase assay, endogenous JNK was immunoprecipitated from cell lysate using anti-JNK1 antibody. In vitro JNK kinase assay was performed as described (19Potapova O. Gorospe M. Dougherty R.H. Dean N.M. Gaarde W.A. Holbrook N.J. Mol. Cell. Biol. 2000; 20: 1713-1722Crossref PubMed Scopus (128) Google Scholar). The JNK activity was assayed by phosphorylation of GST-c-Jun(1–79), as revealed by Western blot analysis using anti-phospho-[Ser-63]c-Jun antibody. MEK4 kinase activity was assayed similarly except using GST-JNK2 as substrate, instead of GST-c-Jun, whose phosphorylation was detected by Western blotting with anti-phospho-[Thr-183,Tyr-185]JNK antibody. Previous studies have revealed a correlation between p53 status and sensitivity of tumor cells to chemotherapeutic drugs (20Wu G.S. El-Deiry W.S. Nat. Med. 1996; 2: 255-256Crossref PubMed Scopus (94) Google Scholar, 21O'Connor P.M. Jackman J. Bae I. Myers T.G. Fan S. Mutoh M. Scudiero D.A. Monks A. Sausville E.A. Weinstein J.N. Friend S. Fornace Jr., A.J. Kohn K.W. Cancer Res. 1997; 57: 4285-4300PubMed Google Scholar, 22Bunz F. Hwang P.M. Torrance C. Waldman T. Zhang Y. Dillehay L. Williams J. Lengauer C. Kinzler K.W. Vogelstein B. J. Clin. Invest. 1999; 104: 263-269Crossref PubMed Scopus (915) Google Scholar). The difference in drug response between tumors with wild-type p53 and those harboring p53 loss-of-function mutations can be explained in part by p53-mediated apoptosis (23Vousden K.H. Cell. 2000; 103: 691-694Abstract Full Text Full Text PDF PubMed Scopus (703) Google Scholar). Treatment of HCT116 cells with 200 ng/ml nocodazole induced a much stronger apoptotic response in p53+/+ cells than in p53−/− cells. As shown in Fig.1, endonucleolytic cleavage of genomic DNA, an indicator of apoptosis, was much more evident in p53+/+ than in p53−/− cells at 72 h (Fig. 1 A). Consistent with this, flow cytometry revealed that most cells from both cell lines were arrested at G2/M transition (4 n DNA) after a 72-h nocodazole treatment (Fig. 1, C and D). However, most of these cells were in G1 phase (2 n DNA) when cultured in the absence of this microtubule disrupter (Fig.1 B). About 30% of cells contained less than 2 n DNA content (sub-G1) characteristic of apoptotic cells in p53+/+ cells (Fig. 1 C) compared with less than 13% in p53−/− cells 72 h after nocodazole treatment (Fig.1 D). To examine the signaling pathways downstream of p53 which might account for the difference in apoptosis induction, we undertook an unbiased proteomics-based approach to focus on signaling proteins that may be important in mediating the actions of the tumor suppressor protein p53 in response to nocodazole. In preliminary studies, we applied three Kinetworks™ screens to analyze the expression profiles of up to 75 protein kinases and 25 protein phosphatases, as well as the phosphorylation states of 25 of these protein kinases (TableI) and 10 other known phosphoproteins (data not shown) from p53+/+ and p53−/− cells treated with 200 ng/ml nocodazole for 1 h. 48 distinct protein kinases isoforms were clearly evident in the HCT116 cell lines (listed in Table I), and weaker signals were recorded for 14 other protein kinases (calmodulin-dependent kinase 1, calmodulin-dependent kinase kinase, casein kinase 1ε, cGMP-dependent protein kinase, cyclin-dependent kinases 2, 6, and 9, death-associated kinase 1, JAK2, Lyn, MST1, protein kinase Cδ, RhoA kinase, and Syk; data not shown) of 75 kinases that can be detected by the Kinetworks™ KPKS 1.0 screen. Of these 48 kinases, 18 protein kinases revealed expression differences of 49% or greater between the untreated p53+/+ and p53−/− cell lines when either the cytosolic or particulate fractions of these cells were investigated separately. In particular, loss of p53 function was associated with increases in cytosolic CK2, ERK6, MEK6, MEK7, Pim1, and RafB; increases in particulate MEK1, MEK2, MEK6, Pim1, PKC-ζ, RafB, and S6 kinase; decreases in cytosolic CDK1, PKC-β1, Rsk1 and Yes; and reductions in particulate Hpk1, MosI and p40 JNK (SAPK-β). 2JNK1 and JNK2 have been reported previously to have at least four different isoforms, p46α, p46β, p54α, and p54β, because of alternative splicing (34Yujiri T. Sather S. Fanger G.R. Johnson G.L. Science. 1998; 282: 1911-1914Crossref PubMed Scopus (280) Google Scholar, 35Kallunki T., Su, B. Tsigelny I. Sluss H.K. Derijard B. Moore G. Davis R. Karin M. Genes Dev. 1994; 8: 2996-3007Crossref PubMed Scopus (591) Google Scholar, 36Kyriakis J.M. Woodgett J.R. Avruch J. Ann. N. Y. Acad. Sci. 1995; 766: 303-319Crossref PubMed Scopus (61) Google Scholar). From our study we believe that p54 JNK corresponds to p47 JNK, and p46 JNK corresponds to p40 JNK. The difference in the apparent molecular masses may result from different electrophoresis conditions used in the Kinetworks™ analysis. The 3.8-fold or greater increased productions of Pim1 and MEK6 in the p53−/− cells were the most striking. These findings indicated extensive alterations in protein kinase regulation as a function of p53 status. There were also marked differences in the responses of the p53+/+ and p53−/− cells to a 1-h exposure to nocodazole. In the p53+/+ cells, nocodazole led to 1.5–1.7-fold increases in cytosolic CK2 and Csk, and particulate MEK7, and it was associated with 53–74% reductions in the levels of cytosolic PKC-β1 and particulate ERK2, ERK3, and Yes (the Yes change is not likely to be significant because of low signal:noise ratio). In the p53−/− cells, the nocodazole treatment caused 1.5–3-fold increases in cytosolic Csk, CDK1, CDK7, GCK, PKA, PKC-β1, Rsk1, Yes, ZAP70, and ZIP kinase, and particulate ERK1, PKC-ε, and p40 JNK; and 57–100% reductions in cytosolic Pim1, and particulate Csk1 and Fyn. MosI expression was elevated 5-fold in the particulate fraction of nocodazole-treated p53−/− cells but unaffected in the p53+/+ cells. Of 25 different protein phosphatases that could be potentially detected with the Kinetworks™ KPPS 1.1 screen, 15 were clearly detected in the HCT116 cell lines (Table I), and 5 others were weakly detected (i.e. LAR, MKP1, MKP3, protein phosphatase 2A catalytic subunit and protein phosphatase X A′2 subunit; data not shown). In the p53−/− cells compared with the p53+/+ cells, there were 1.5–2-fold elevated protein levels of particulate PTP-1B and cytosolic protein phosphatase 1γ catalytic subunit, PTP-PEST, and PTP-1C, and 48–83% reductions in the expressions of particulate MKP2, P5/PPT, and protein phosphatase 1α catalytic subunit. The elevation of cytosolic PTP-1C and reduction of particulate PTP-1C in the p53−/− cells may reflect translocation of this protein-tyrosine phosphatase. Nocodazole caused 2.1–2.5-fold increases in the cytosolic levels of protein phosphatase V catalytic subunit in both the p53−/− and p53+/+ cells, and this might also arise from redistribution of this phosphatase away from the particulate fraction in at least the p53−/− cells. However, nocodazole treatment also had differential effects on other protein phosphatases in the two HCT116 cell lines. It selectively evoked a 2.7-fold increase in cytosolic KAP and 4-fold more particulate MKP2 in p53−/− cells, and 1.8–2.5-fold increased expressions of particulate KAP, protein phosphatase 1Cγ and PTP-1B in p53+/+ cells. The extensive p53-dependent changes in the levels of the protein kinases and protein phosphatases in the HCT116 cells were also accompanied by many altered states of phosphorylation of protein kinases as detected with phosphorylation site-specific antibodies employed in the Kinetworks™ KPSS 1.1 screen (Fig.2 and Table I). 19 distinct phosphorylation sites in 17 different protein kinases were observed to undergo phosphorylation, and 12 of these kinases demonstrated at least 50% increases or decreases in their phosphorylation signals between the p53+/+ and p53−/− cell lines in the absence of nocodazole treatment. After exposure to nocodazole, there were also p53-dependent changes in phosphorylation states of 11 protein kinases which exceeded 50%. In several cases, the altered states of phosphorylation reflected at least in part changes in the protein levels of the various protein kinases. This was observed for the CDK1 inhibitory Tyr-14, ERK2-activating Thr-185/Tyr-187, GSK3β-activating Tyr-216, PKB-α- activating Ser-473, Raf1 p72 Ser-259, Rsk1-activating Thr-360/Ser-364, and Src inhibitory Tyr-529 phosphorylation changes. The “specific phosphorylation” of a protein takes into account the magnitude of the phosphorylation signal relative to the amount of that protein in a sample. Therefore, the specific phosphorylations of the aforementioned protein kinases were not affected markedly by the p53 status or by nocodazole treatment. However, there were cases where loss of p53 function was associated with increased specific phosphorylation of protein kinases, and although the specific phosphorylation of these kinases could be stimulated further by nocodazole in the p53+/+ cells, this was not evident in the p53−/− cells. For example, the specific phosphorylation of cytosolic MEK1/MEK2-activating Ser-217/Ser221 was enhanced in untreated p53−/− cells compared with p53 +/+ cells (percent change in phosphorylation state versus percent change in protein level: 384/79 = 4.9-fold), 3The -fold change in specific phosphorylation of each protein = (100 + % change in phosphorylation state)/(100 + % change in total protein level). and after nocodazole exposure it was increased (265/103 = 2.6-fold) in p53+/+ cells, but not further (122/138 = 0.9-fold) in p53−/− cells. Likewise, the specific phosphorylation of cytosolic PKC-β1 Thr-638/Ser-657 (128/38 = 3.4-fold) and Thr-641 (149/38 = 3.9-fold) were increased in untreated p53−/− cells compared with p53+/+ cells. After nocodazole treatment, these specific phosphorylations of cytosolic PKC β-1 were reduced in p53−/− cells (Thr-638/Ser-657 (66/188 = 0.35-fold) and Thr-641 (69/188 = 0.37-fold)) but were enhanced in p53+/+ cells (Thr-638/Ser-657 (111/47 = 2.4-fold) and Thr-641 (91/47 = 1.9-fold)). By contrast, there was a complete loss of detectable Ser-259 specific phosphorylation of both cytosolic and particulate p62 Raf1 in untreated p53−/− cells compared with p53+/+ cells. Of greatest interest from the Kinetworks™ analyses was the p53-dependent differential regulation of p40 JNK and p47 JNK phosphorylation at their activation sites. In the untreated p53−/− cells compared with p53+/+ cells, there were marked reductions of the specific phosphorylations of cytosolic p40 JNK (56/111 = 0.5-fold), particulate p40 JNK (1/51 = 0.02-fold), and cytosolic p47 JNK phosphorylation (1/109 = 0.01-fold). Although nocodazole treatment evoked clear stimulations of the specific phosphorylations of cytosolic (247/116 = 2.1-fold) and particulate (336/90 = 3.7-fold) p40 JNK, and cytosolic (258/100 = 2.6-fold) and particulate (134/100 = 1.3-fold) p47 JNK in p53+/+ cells, no increases in JNK specific phosphorylation occurred after exposure of the p53−/− cells to nocodazole. These p53 loss of function-associated reductions in basal phosphorylation of JNK along with the abrogation of nocodazole-induced phosphorylation at these activating sites, revealed that JNK acts downstream of p53 in a signaling cascade in the HCT116 p53+/+ cells. The remainder of this study focuses on confirming this finding and establishing its physiological significance. To confirm the results of the Kinetworks™ analysis with respect to JNK regulation in the two HCT116 cell lines in response to nocodazole, we monitored p40 JNK protein and phosphorylation levels by Western blot analysis. Maximum phosphorylation of p40 JNK was induced with 200 ng/ml nocodazole in the p53+/+ cells in 1 h (Fig. 3,A and C). By contrast, there was a slightly higher level of expression of p40 JNK in the p53−/− cells even before nocodazole treatment. However, the protein level of p40 JNK remained relatively constant during the course of treatment (Fig.3 B). These findings further indicate a marked loss in the ability of this microtubule-interfering agent to activate p40 JNK in p53−/− cells. In correlation with the higher levels of immunoreactive phosphorylated JNK, the JNK immunoprecipitated from p53+/+ cells treated with nocodazole exhibited much higher phosphotransferase activity toward GST-c-Jun when assayed in vitro compared with p53−/− counterparts (Fig. 3 D). Therefore, the difference observed after nocodazole treatment in these two cell lines in p40 JNK phosphorylation was caused by the differential activation of the kinase. To examine further the dependence of nocodazole-induced JNK activation on p53 status, we investigated the effect of nocodazole on JNK activity in p53-deficient derivatives of two other cell lines, MCF-7 and A549, in which p53 function was abrogated through expression of viral E6 oncoprotein. Although MCF-7 p53-deficient cells displayed a much higher level of p40 JNK protein expression, MCF-7 parental cells exhibited a marked increase of phosphorylated p40 JNK compared with their p53-deficient counterparts in response to nocodazole treatment (Fig. 3, E andF). Similarly, a higher degree of JNK phosphorylation was observed in A549 parental cells upon nocodazole treatment than in A549 p53-deficient cells, although they exhibited similar JNK protein expression levels (Fig. 3, G and H). Therefore, it is evident that the p53-dependent JNK activation upon nocodazole treatment is not limited to HCT116 cells. However, because of the possible complication of other activities of E6 oncoprotein, we chose to focus subsequent work on the HCT116 cells. Furthermore, to ascertain whether the p53-dependent JNK activation was a general response to microtubule interference, we examined the effects of other microtubule-interfering agents including vinblastine, colchicine, and taxol on JNK activity in HCT116 cells. The higher degree of JNK phosphorylation in HCT116 p53+/+ cells was observed after treatment with microtubule-depolymerizing drugs, vinblastine and colchicine (Fig. 4,A–D), but not with microtubule-stabilizing drug, taxol (data not shown), indicating that the p53-dependent JNK activation might be related to the microtubule-depolymerizing activity of nocodazole, vinblastine, and colchicine. It was conceivable that the lack of significant activation of p40 JNK in p53−/− cells upon nocodazole treatment may result from defects in the JNK signaling pathway in p53−/− cells. To examine this possibility, we used sodium arsenate, a potent activator of stress response pathways involving p38 MAP kinase and JNK, to treat HCT116 cells. Treatment with 0.5 mm sodium arsenate for 0.5 h increased the phospho- p40 JNK levels in p53−/− cells, equivalent to, if not better than tha" @default.
• W2028274300 created "2016-06-24" @default.
• W2028274300 creator A5001273370 @default.
• W2028274300 creator A5015314431 @default.
• W2028274300 creator A5027924631 @default.
• W2028274300 creator A5067092208 @default.
• W2028274300 creator A5069849278 @default.
• W2028274300 creator A5071582287 @default.
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• W2028274300 date "2002-11-01" @default.
• W2028274300 modified "2023-10-15" @default.
• W2028274300 title "Nocodazole-induced p53-dependent c-Jun N-terminal Kinase Activation Reduces Apoptosis in Human Colon Carcinoma HCT116 Cells" @default.
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