Cancer Research Infection and Cancer: Biology, Therapeutics, and Prevention Translational Medicine Conference in Israel
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Yang, C.-P. H.
Right arrow Articles by Horwitz, S. B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Yang, C.-P. H.
Right arrow Articles by Horwitz, S. B.
[Cancer Research 60, 5171-5178, September 15, 2000]
© 2000 American Association for Cancer Research

Experimental Therapeutics

Taxol Mediates Serine Phosphorylation of the 66-kDa Shc Isoform1

Chia-Ping Huang Yang and Susan Band Horwitz2

Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In the human lung carcinoma cell line A549, Taxol (20 nM) causes a decreased electrophoretic mobility of the 66-kDa Shc isoform (p66shc), beginning 4 h after drug exposure, and reaching a maximum at 9–18 h. No shift was observed for the 52- and 46-kDa isoforms of Shc. The electrophoretic mobility shift of p66shc caused by Taxol is not the result of tyrosine phosphorylation, and there is no indication of a Shc/Grb2 complex in Taxol-treated A549 cells. This modification is blocked by the serine/threonine protein phosphatase 2A. In vivo 32P-labeling and subsequent phosphoamino acid analysis of p66shc indicated that both the original and the shifted p66shc were predominantly serine phosphorylated. Cyanogen bromide digestion of p66shc produced a phosphorylated fragment with an apparent molecular weight of ~7.9 kDa from the untreated cells and two phosphorylated fragments, of ~7.9 and ~9.6 kDa, from the Taxol-treated cells. The domain of Taxol-induced serine phosphorylation is thought to be in the cyanogen bromide fragment containing residues 2–65. The Taxol-induced electrophoretic mobility shift of p66shc was inhibited by the protein synthesis inhibitor, cycloheximide, but not by the mitogen-activated and extracellular signal-regulated protein kinase kinase (MEK) inhibitor, PD98059. This mobility shift did not occur in Taxol-resistant A549-T12 cells treated with 20 nM Taxol. In addition to Taxol, other microtubule-interacting drugs caused a decreased electrophoretic mobility of p66shc. This Taxol-mediated serine phosphorylation seen in p66shc may result from a MEK-independent signaling pathway that is activated in cells that have a prolonged or abnormal mitotic phase of the cell cycle and may play a role in signaling events that lead to cell death.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Taxol, a natural product with significant antitumor activity, has been approved for the treatment of breast, ovarian, and lung carcinomas (1) . The major cellular target for Taxol is the tubulin/microtubule system (2) . Taxol has a specific binding site on the microtubule (3 , 4) , and incubation of cells with Taxol causes the formation of stable bundles of microtubules that disrupt the normal polymerization/depolymerization cycle of microtubules (5) and suppresses microtubule dynamics (6) . Treatment of cells with Taxol results in the arrest of cells in the G2-M phase of the cell cycle (5) , and prolonged exposure induces apoptotic death (7, 8, 9) .

In addition to its effects on microtubules, Taxol, particularly in mouse macrophages, induces the production of cytokines, such as tumor necrosis factor {alpha} and interleukin 1, and increases tyrosine phosphorylation of proteins, including MAP3 kinase (10, 11, 12, 13, 14) . It has been reported that Taxol- and LPS-induced activation of mouse macrophages is mediated by heat shock protein 90 (15) . CD18 also has been shown to be a cellular target for Taxol in murine macrophages (16) . In other studies using phage-displayed peptides, Bcl-2 has been identified as a Taxol binding protein (17) .

Several lines of evidence indicate that Taxol can activate a variety of signal transduction pathways, e.g.: (a) Taxol activates c-Jun NH2-terminal kinase through both Ras and apoptosis signal-regulating kinase 1 pathways (18 , 19) . c-Jun NH2-terminal kinase activation mediates both Taxol-induced gene expression and cell death (20) ; (b) Taxol induces activation of Raf-1 and ERK (11 , 19 , 21, 22, 23, 24) ; and (c) Taxol causes tyrosine phosphorylation of p38 in cells of monocytic origin (25) and induces activation of p38 in human breast cancer lines (26) . In addition, tyrosine phosphorylation of Shc and formation of a Shc/Grb2 complex have been demonstrated in Taxol-treated murine macrophage RAW 264.7 cells (23) .

The SH2-containing Shc proteins (27) are substrates of activated tyrosine kinases and have been implicated in a signaling cascade leading to Ras activation (28) . Shc proteins are phosphorylated by receptor tyrosine kinases, such as EGFR (27) , insulin receptor (29) , and erbB-2 (30) , and by cytoplasmic tyrosine kinases, such as activated Src, Fps, and Lck (31 , 32) . Ligand binding induces autophosphorylation of receptors, and the activated receptors associate with and phosphorylate Shc through Shc SH2 and/or phosphotyrosine binding domains (33) . Phosphorylation of Shc proteins generates a docking site for the SH2 domain of Grb2 (34) that in turn interacts with SOS, the guanine nucleotide exchange factor for Ras, through its SH3 domains. The formation of a Shc/Grb2/SOS complex results in the membrane relocalization of SOS and activation of Ras (35) .

Shc is expressed as three proteins of 46, 52, and 66 kDa (27) . The 46- and 52-kDa isoforms result from use of distinct initiation sites in the same transcript, whereas the 66-kDa isoform is believed to arise from an alternatively spliced message (27) . All three isoforms have been structurally characterized (27 , 36 , 37) . The 52- and the 46-kDa isoforms share a COOH-terminal SH2 domain, a collagen-homologous, glycine/proline-rich region (CH1) and an NH2-terminal phosphotyrosine binding domain. The predicted amino acid sequence of the 66-kDa isoform overlaps with that of the 52-kDa isoform and contains a unique NH2-terminal region, CH2, which is also rich in glycines and prolines (36 , 37) . The 66-kDa Shc, unlike the 52- and 46-kDa Shc isoforms, is unable to transform mouse fibroblasts when expressed in vitro (36) . In addition to tyrosine phosphorylation of the Shc isoforms, it has been reported that insulin and EGF cause an increase in serine/threonine phosphorylation of the 66-kDa Shc protein (37 , 38) . Unlike the 52- and 46-kDa Shc, the 66-kDa Shc is a negative regulator of the EGF-stimulated MAP kinase-fos signaling pathway, and its CH2 domain mediates this inhibitory effect (36) . Recently, it has been reported that p66shc controls the oxidative stress response and that a targeted mutation at the serine residue in the CH2 domain of the mouse p66shc gene induces stress resistance and prolongs life span (39) .

We have demonstrated previously that Taxol induces tyrosine phosphorylation of Shc, particularly the 52-kDa isoform, and formation of a Shc/Grb2 complex in the murine macrophage cell line RAW 264.7 (23) . The aim of this study was to determine the involvement of Shc in Taxol-treated, non-macrophage-like human cell lines. We found that Taxol caused a decreased electrophoretic mobility of p66shc in the human lung carcinoma cell line A549. This shift was not the result of tyrosine phosphorylation; rather, Taxol mediated an altered serine phosphorylation of p66shc. There was no indication of a Shc/Grb2 complex in these Taxol-treated cells. These results suggest that Taxol, at nanomolar concentrations and over a 9–18-h period, induces a signaling pathway that is distinct from one that involves immediate activation of tyrosine kinases by micromolar concentrations of Taxol, such as was reported in mouse macrophages (23) .


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials and Cells.
Monoclonal and polyclonal anti-Shc, monoclonal anti-Grb2, anti-phosphotyrosine (PY20), and anti-PARP antibodies were purchased from Transduction Laboratories/PharMingen. Anti-phospho-p44/42 MAP kinase monoclonal E10 antibody was from New England Biolabs. Anti-EGFR monoclonal antibody was from Neomarkers, Lab Vision Corp. Anti-PARP p85 fragment (apparent molecular weight) polyclonal antibody was from Promega. Protein phosphatase 2A1 was purchased from Upstate Biotechnology. MEK inhibitors, PD98059 and UO126, were from Calbiochem and Promega, respectively. Taxol was obtained from the Drug Development Branch, National Cancer Institute (Bethesda, MD). Human lung carcinoma cells, A549, and their Taxol-resistant derivative, A549-T12, were grown in RPMI 1640 containing 10% fetal bovine serum. A549-T12 cells do not express P-glycoprotein and are maintained in a final concentration of 12 nM Taxol, as described (40) .

Preparation of Cell Lysates.
Cells were washed three times with PBS, lysed with boiling buffer containing 1% SDS, 10 mM Tris-HCl (pH 7.4), boiled for an additional 5 min, passed six times through a 26-gauge needle, and centrifuged for 5 min in a microcentrifuge. Such denatured lysates were mixed with SDS-sample buffer and analyzed by SDS-PAGE. Nondenatured lysates were prepared by incubating cells in buffer A containing 1% Triton X-100, 150 mM NaCl, 10 mM Tris-HCl (pH 7.4), 1 mM EDTA, 1 mM EGTA, 0.2 mM sodium vanadate, 0.2 mM phenylmethylsulfonyl fluoride, and 0.5% NP40 at 4°C for 30 min. In some experiments, 1 mM NaF was included in buffer A. Aggregates were dispersed by passing through a 21-gauge needle five times, and insoluble material was removed by centrifugation for 15 min at 3000 rpm at 4°C.

Immunoprecipitation.
Equivalent amounts of protein (200–400 µg) of nondenatured lysates were precleared by incubating with 20 µl of protein A-agarose beads for 1 h at 4°C, followed by centrifugation at 13,000 rpm. The supernatants were incubated with specific primary antibodies in buffer A overnight at 4°C. Protein A-agarose beads were added and incubated for an additional 1 h at 4°C. Immunocomplexes were collected by centrifugation, washed five times with buffer A, eluted with boiling SDS-sample buffer, and analyzed by SDS-PAGE.

Western Blot Analysis.
Total cell lysates or immunoprecipitates were resolved by SDS-PAGE and transferred to nitrocellulose. Western blot analysis was performed as described (23) with ECL reagent. In some experiments, blots were stripped in 100 mM ß-mercaptoethanol, 2% SDS, and 62.5 mM Tris-HCl (pH 6.7) for 30 min at 55°C, reblocked, and reprobed.

Two-Dimensional Gel Electrophoresis.
Approximately 40 µg of nondenatured lysate was resolved by two-dimensional gel electrophoresis as described by O’Farrell (41) . Proteins were transferred to nitrocellulose, and Western blot analysis was done with monoclonal anti-Shc antibody.

Protein Phosphatase Treatment.
Cell lysates were immunoprecipitated with polyclonal anti-Shc antibody, and the immunoprecipitate/protein A agarose was incubated with 0.25 µg of protein phosphatase 2A1 in a buffer containing 25 mM Tris-HCl (pH 7.0), 0.2 mM MnCl2, 1 mM DTT, and 0.1 mg/ml BSA for 1 h at 30°C. The reaction was stopped by heating at 100°C in sample buffer prior to analysis by SDS-PAGE.

32P-Labeling of Intact Cells and Immunoprecipitation of Shc Proteins.
Cells were grown in complete medium for 12 h in the presence and absence of Taxol. Medium was aspirated and replaced with phosphate-free RPMI 1640 and fresh Taxol. After 1 h, [32P]Pi was added (0.4 mCi/ml) and incubated for an additional 4 h. Cells were washed twice with cold saline (0.9% NaCl) and lysed with 1% SDS, 10 mM Tris-HCl (pH 7.5). Lysates were diluted with water and 2x nondenaturing lysis buffer (final SDS concentration, <0.5%) and immunoprecipitated with polyclonal anti-Shc antibody. The immunoprecipitates were analyzed by SDS-PAGE.

Cyanogen Bromide Digestion of p66shc.
32P-Labeling of intact A549 cells and immunoprecipitation of lysates with anti-Shc antibody were performed as described above. Immunoprecipitates were resolved by SDS-PAGE and transferred to nitrocellulose. 32P-Labeled p66shc was visualized by autoradiography, and the appropriate area of the blot was carefully excised. The nitrocellulose strips were incubated with 100 mg/ml CNBr in 70% formic acid for 3 h at room temperature. The supernatant was taken to dryness in a Speed-vac, and the residue was dissolved and re-evaporated three times with water. Phosphopeptides were resolved on a 15% Tricine gel with 0.1 M Tris, 0.1 M Tricine, and 0.1% SDS as the cathode buffer (42) , transferred to nitrocellulose, and subjected to autoradiography.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Taxol Treatment Results in Alterations in the Electrophoretic Mobility of the 66-kDa Shc Isoform (p66shc).
A549 cells were treated with 5–200 nM Taxol for 16 h. A decreased electrophoretic mobility of p66shc was seen in those cells that were incubated with 20 nM or higher concentrations of the drug (Fig. 1A)Citation . No mobility shift was seen for either p52shc or p46shc. Similar results were obtained with HeLa and SKOV3 cells treated with Taxol (data not shown).



View larger version (44K):
[in this window]
[in a new window]
  		Fig. 1. Taxol alters the electrophoretic mobility of p66shc. A549 cells were treated with 5–200 nM Taxol for 16 h. Lysates were prepared, and Western blot analyses were performed as described in "Materials and Methods" with polyclonal anti-Shc (c-20; Santa Cruz Biotechnology, used only in A), monoclonal anti-Raf-1 (B), monoclonal anti-phospho-p44/42 MAP kinase (C), monoclonal anti-PARP (D), or polyclonal anti-PARP p85 fragment (E) antibodies. Anti-PARP antibody recognizes the NH2-terminal 24-kDa cleavage product of PARP, whereas the anti-PARP p85 fragment antibody only recognizes the COOH-terminal 89-kDa cleavage product. C, control cells.

 
A reduction in electrophoretic mobility of Raf-1 was observed in A549 cells at 20 nM, or higher concentrations, of Taxol (Fig. 1B)Citation . It has been demonstrated that reduction in electrophoretic mobility of Raf-1 after Taxol treatment correlated with the activation of Raf-1 kinase activity in A549 cells (24) . Taxol also caused the phosphorylation of ERK1/2 in a dose-dependent manner, as detected by an antibody specific for the phosphorylated form of ERK1/2 (Fig. 1C)Citation . In addition, Taxol, >10 nM, induced cleavage of PARP to its NH2-terminal 24-kDa and COOH-terminal 89-kDa fragments (Fig. 1, D and E)Citation . PARP is one of the earliest proteins targeted for specific cleavage during apoptosis (43) . Therefore, the Taxol-induced electrophoretic mobility shift of p66shc coincided with phosphorylation of Raf-1 and ERK1/2, as well as with cleavage of PARP. Previous studies with A549 cells indicated that apoptosis occurred in ~25% of the cells after an 18-h exposure to 12 nM Taxol (24) .

Although phosphorylation of ERK1/2 coincided with the cleavage of PARP, it was not clear whether ERK1/2 activation mediated apoptosis. The MEK inhibitor PD98059, which blocks the activation of MEK, and UO126, which inhibits both active and inactive MEK, clearly inhibited Taxol-induced phosphorylation of ERK1/2 but did not prevent Taxol-mediated PARP cleavage (Fig. 2)Citation . These results indicated that Taxol-induced PARP cleavage was not mediated by activated ERK1/2. It has been reported in leukemia cells that microtubule-active drugs induced apoptosis, which was associated with Raf-1 and Bcl-2 phosphorylation but independent of the MAP kinase pathway (44) . Similar results were found in MCF-7 cells (45) .



View larger version (39K):
[in this window]
[in a new window]
  		Fig. 2. Activation of ERK1/2 does not mediate the release of the 24-kDa PARP cleavage product. A549 cells were incubated with 10 or 50 µM PD98059 (A) or 5 µM UO126 (B) for 30 min, followed by treatment with 100 nM Taxol for 16 h in the presence of inhibitor. Cell lysates were prepared, and Western blot analyses were done with monoclonal anti-phospho-p44/42 MAP kinase or with monoclonal anti-PARP antibodies. C, control cells; T, cells treated with 100 nM Taxol.

 
The effect of Taxol on p66shc mobility was monitored over a period of 40 h. The shift was observed first at ~4 h after the sensitive A549 cells were treated with 20 nM Taxol, reaching a maximum at approximately 9–18 h and starting to decline at ~21 h after drug treatment (Fig. 3Citation , upper panel). An essentially identical time course was obtained for Taxol-induced phosphorylation of Raf-1 (Fig. 3Citation , lower panel).



View larger version (72K):
[in this window]
[in a new window]
  		Fig. 3. The Taxol-mediated electrophoretic mobility shift of p66shc and Raf-1 is transient. A549 cells were treated with 20 nM Taxol for 2–40 h. Lysates were prepared, and Western blot analyses with monoclonal anti-Shc or anti-Raf-1 antibodies were performed as described in "Materials and Methods."

 
Taxol Does Not Induce Tyrosine Phosphorylation of Shc.
It has been reported that incubation of Taxol with murine macrophage RAW 264.7 cells induced tyrosine phosphorylation of a number of proteins (10 , 23) . One of these proteins was Shc, particularly the p52shc. Taxol also caused formation of a Shc/Grb2 complex in these cells (23) . However, no increase in tyrosine phosphorylation of proteins was observed in A549 cells treated with Taxol (data not shown). Neither tyrosine phosphorylation of Shc proteins nor Shc/Grb2 complex formation was detected after Taxol treatment in A549 cells (Fig. 4)Citation . These results were determined by immunoprecipitation of cell lysates prepared from control and Taxol-treated A549 cells with polyclonal anti-Shc antibodies, followed by Western blot analysis with monoclonal anti-phosphotyrosine (Fig. 4ACitation , right panel) or anti-Grb2 antibody (Fig. 4BCitation , right panel). Equal levels of Grb2 were found in the cell lysates prepared from control and treated cells (data not shown). The blots were stripped, reblocked, and reprobed with anti-Shc monoclonal antibody to ensure equal loading of immunoprecipitated protein (Fig. 4C)Citation . The reciprocal immunoprecipitation using recombinant anti-phosphotyrosine antibody RC20, followed by Western blot analysis with monoclonal anti-Shc antibody, also demonstrated that Shc proteins were not tyrosine phosphorylated (data not shown). As positive controls, serum-starved A549 cells were treated with EGF for 5–15 min, and the lysates were immunoprecipitated with anti-Shc antibody. Western blot analysis using anti-phosphotyrosine and anti-Grb2 antibodies clearly demonstrated that Shc proteins were tyrosine phosphorylated (Fig. 4ACitation , left panel), and the Shc/Grb2 complex was formed upon EGF treatment (Fig. 4BCitation , left panel). As expected, EGF also caused tyrosine phosphorylation of the EGFR (Fig. 4D)Citation and EGFR/Shc complex formation (Fig. 4ECitation , left panel), results not obtained after Taxol treatment (Fig. 4ECitation , right panel).



View larger version (32K):
[in this window]
[in a new window]
  		Fig. 4. Taxol does not induce tyrosine phosphorylation of Shc proteins in A549 cells. Left panels, A549 cells were incubated in serum-free medium for 24 h and then treated with 50 ng/ml EGF for 5–15 min. Right panels, A549 cells were treated with 20–50 nM Taxol for 16 h in complete medium. Nondenatured cell lysates were prepared and immunoprecipitated with polyclonal anti-Shc (A, B, C, and E) or monoclonal anti-EGFR (D) antibodies, followed by Western blotting (WB) with monoclonal anti-phosphotyrosine (PY20; A and D), monoclonal anti-Grb2 (B), or monoclonal anti-EGFR (E) antibodies. Western blot analyses with monoclonal anti-Shc antibody were done to ensure equal loading of the Shc proteins after immunoprecipitation (C). The bands in the upper region of Fig. 4Citation A (left panel) most likely represent the tyrosine phosphorylated EGFR after 5 or 10 min EGF treatment. IP, immunoprecipitation.

 
The Taxol-induced Decreased Mobility of p66shc Results from Serine Phosphorylation, and the Phosphorylation Domain Is Contained within a 7.9-kDa CNBr Fragment.
Taxol did not cause tyrosine phosphorylation of Shc nor the formation of a Shc/Grb2 complex in A549 cells, events observed in Taxol-treated RAW 264.7 cells (23) and in EGF-treated A549 cells (see Fig. 4Citation ). To determine the nature of the p66shc modification, the Taxol-induced p66shc electrophoretic mobility shift was analyzed initially by two-dimensional gel electrophoresis. Nondenatured lysates prepared from control and Taxol-treated A549 cells were separated by isoelectric focusing followed by SDS-PAGE (41) . The resolved proteins were analyzed by immunoblotting using monoclonal anti-Shc antibody (Fig. 5A)Citation . No major differences were observed for p52shc and p46shc between control and Taxol-treated cells (data not shown). p66shc was visible as a poorly resolved charge train in control cells. Treatment with Taxol (50 or 100 nM) resulted in an altered charge train that was more negatively charged (Fig. 5A)Citation . In addition, serine/threonine PP2A1 inhibited the electrophoretic mobility shift of p66shc (Fig. 5B)Citation . It is known that PP2A is associated with microtubules and is regulated during the cell cycle (46 , 47) . Protein phosphatases 1 and 2B had no effect on the shifted form of p66shc at the concentrations tested (data not shown).



View larger version (41K):
[in this window]
[in a new window]
  		Fig. 5. Taxol-induced electrophoretic mobility shift of p66shc results from serine phosphorylation. A, two-dimensional gel electrophoresis analysis of p66shc after Taxol treatment in A549 cells. Nondenatured lysates were prepared from A549 cells treated with 50 or 100 nM Taxol for 16 h, followed by immunoprecipitation with polyclonal anti-Shc antibodies. Immunoprecipitates were resolved by two-dimensional gel electrophoresis and transferred to nitrocellulose, and Western blotting was carried out with monoclonal anti-Shc antibody. B, incubation of p66shc with protein phosphatase 2A1. Lysates prepared from control and Taxol-treated cells were immunoprecipitated with polyclonal anti-Shc antibodies, and the immunoprecipitates were treated with PP2A1 as described in "Materials and Methods," followed by Western blot analysis with monoclonal anti-Shc antibody. C, in vivo 32P-labeling of p66shc. Intact A549 cells were treated with Taxol for 16 h and labeled with [32P]Pi during the last 4 h. Cell lysates were prepared and immunoprecipitated with anti-Shc antibody. The immunoprecipitates were analyzed by SDS-PAGE and autoradiography. D, cyanogen bromide digestion of 32P-labeled p66shc. 32P-Labeled p66shc was transferred to nitrocellulose, treated with cyanogen bromide, and analyzed by SDS-PAGE on a 15% Tricine gel as described in "Materials and Methods." C, control cells; T, 100 nM Taxol-treated cells.

 
To confirm that Taxol induced phosphorylation of p66shc, 32P-labeled cell lysates prepared from control and Taxol-treated A549 cells were immunoprecipitated with anti-Shc antibody, and the immunoprecipitates were analyzed by SDS-PAGE (Fig. 5C)Citation . p66shc was phosphorylated in both control and Taxol-treated cells, but the Taxol treatment resulted in a decreased electrophoretic mobility of the phosphorylated p66shc. The levels of p66shc phosphorylation were approximately 30–70% higher in the Taxol-treated cells than in the control cells. Both the original and the shifted p66shc were predominantly serine phosphorylated, as determined by phosphoamino acid analysis (data not shown).

To localize the domain of the Taxol-induced serine phosphorylation, the 32P-labeled Shc proteins obtained by immunoprecipitation with anti-Shc antibody were resolved by SDS-PAGE and transferred to nitrocellulose, and the region of the blot containing p66shc was digested with CNBr in 70% formic acid. Approximately 70–90% of the 32P-labeled p66shc was released from the nitrocellulose after CNBr treatment. SDS-PAGE/autoradiography of the CNBr-released peptides from p66shc of control cells revealed the presence of a 32P-labeled fragment with an apparent molecular weight of ~7.9 kDa (Fig. 5D)Citation . An additional 32P-labeled band at ~9.6 kDa, which most likely represents a further phosphorylated form of the ~7.9 kDa fragment, was seen in the Taxol-treated samples. Minor labeling of 14–16-kDa peptides also was observed that may represent incomplete digested products of the CNBr reaction.

MEK Inhibitor PD98059 Does Not Influence the Taxol-induced Electrophoretic Mobility Shift of p66shc and Raf-1.
It has been demonstrated in CHO cells expressing the human EGFR that EGF induced serine/threonine phosphorylation of ~50% of the 66-kDa Shc proteins. Similar results were obtained with insulin. Both of these effects were blocked by PD98059, a MEK-specific inhibitor, probably through a feedback mechanism (37 , 38) . In A549 cells, as expected, PD98059 inhibited the EGF-stimulated electrophoretic mobility shift of both p66shc and Raf-1 (Fig. 6Citation , left panels). However, PD98059 did not inhibit the Taxol-induced mobility shift of either p66shc or Raf-1 (Fig. 6Citation , right panel), suggesting that posttranslational modifications of p66shc and Raf-1 mediated by Taxol do not involve activation of MEK.



View larger version (52K):
[in this window]
[in a new window]
  		Fig. 6. MEK inhibitor, PD98059, does not inhibit the Taxol-induced electrophoretic mobility shift of p66shc and Raf-1 in A549 cells. Left panels, A549 cells were incubated for 24 h in serum-free medium before preincubation with or without 100 µM PD98059 for 1 h and then treated with 50 ng/ml EGF for 5–15 min. Right panels, A549 cells were incubated with 50 µM PD98059 for 30 min, followed by treatment with 20–100 nM Taxol for 16 h in the presence of the inhibitor. Cell lysates were prepared, and Western blot analyses were done with monoclonal anti-Shc and anti-Raf-1 antibodies as described in Figs. 1Citation and 2Citation .

 
Taxol-induced Alteration in Electrophoretic Mobility of p66shc and Raf-1 Requires Protein Synthesis.
Although Taxol caused an electrophoretic mobility shift of p66shc and activation of Raf-1 and ERK1/2 in A549 cells, these effects were not observed for a minimum of 4 h, unlike the effects elicited by mitogens, such as EGF, that can be detected within 1 min (37) . Higher concentrations of Taxol (e.g., 20 µM) did not cause the shift to occur earlier (data not shown). The protein synthesis inhibitor, cycloheximide, which also inhibits cell cycle progression, blocks the electrophoretic mobility shift of both p66shc and Raf-1 (Fig. 7)Citation .



View larger version (73K):
[in this window]
[in a new window]
  		Fig. 7. Cycloheximide inhibits the electrophoretic mobility of p66shc and Raf-1. A549 cells were treated with 20–100 nM Taxol in the presence and absence of 3 x 10-5 M cycloheximide (CHX) for 16 h. Lysates were prepared and immunoblotted with monoclonal antibodies against Shc and Raf-1 as described in Figs. 1Citation and 2Citation .

 
An Electrophoretic Mobility Shift of p66shc Does Not Occur in Taxol-resistant Cells.
When Taxol-resistant A549-T12 cells were treated with 20 nM Taxol, there was no electrophoretic mobility shift of either p66shc or Raf-1 (Fig. 8)Citation . A Taxol-induced shift was observed in A549-T12 cells only at a significantly higher concentration of drug, such as 200-1000 nM, although the extent of shift even at these concentrations was diminished, compared with the sensitive cell line.



View larger version (76K):
[in this window]
[in a new window]
  		Fig. 8. Taxol-induced electrophoretic mobility shift of p66shc and Raf-1 is inhibited in Taxol-resistant A549 cells. Taxol-sensitive and -resistant A549 cells were treated with 20 nM Taxol for 6, 12, 18, and 24 h. Lysates were prepared, and Western blot analyses with monoclonal anti-Shc or anti-Raf-1 antibodies were performed as described in Figs. 1Citation and 2Citation . S, drug-sensitive A549 cells; R, Taxol-resistant A549-T12 cells.

 
Microtubule-interacting Agents Induce the p66shc Mobility Shift in A549 Cells.
A variety of microtubule-interacting agents were used to compare their effects on the electrophoretic mobility shift of p66shc with that of Taxol. These compounds included Taxotere, epothilones A and B, eleutherobin, all of which promote polymerization of stable microtubules, and colchicine and vinblastine, both of which depolymerize microtubules (48) . As shown in Fig. 9Citation , incubation of A549 cells with these drugs (16 h, 50 nM) led to a p66shc mobility shift and phosphorylation of Raf-1. Baccatin, an inactive Taxol analogue that, at 50 nM, does not stabilize microtubules or block cells in mitosis, did not induce these effects. The same results as those seen with baccatin were observed with Adriamycin (0.3 µM), methrotrexate (1 µM), etoposide (5 µM), and 5-fluorouracil (10 µM), all cytotoxic compounds that do not interact with the tubulin/microtubule system and are not antimitotic agents.



View larger version (75K):
[in this window]
[in a new window]
  		Fig. 9. Microtubule-interacting agents alter the electrophoretic mobility of p66shc and Raf-1. A549 cells were treated with 50 nM of a variety of microtubule interacting agents for 16 h. Cell lysates were prepared and immunoblotted with monoclonal antibodies against Shc proteins and Raf-1 as described in Figs. 1Citation and 2Citation .

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this report, we have demonstrated in A549 cells that: (a) Taxol as well as a variety of microtubule-interacting agents caused a decreased electrophoretic mobility of the 66-kDa Shc isoform. This alteration coincided with the phosphorylation of Raf-1 and ERK1/2, and with the release of both 24-kDa and 89-kDa PARP cleavage fragments. However, Taxol-induced PARP cleavage was not inhibited by MEK inhibitors; (b) the modification of p66shc caused by Taxol was not the result of tyrosine phosphorylation but rather of an altered serine phosphorylation. Shc/Grb2 complex formation was absent in Taxol-treated cells. The domain of Taxol-induced serine phosphorylation of p66shc was contained within a 7.9-kDa CNBr fragment; (c) the decreased electrophoretic mobility of p66shc and Raf-1 induced by Taxol was not inhibited by the MEK inhibitor PD98059. Time course and inhibitor studies suggested that protein synthesis was required; and (d) the Taxol-induced shift of p66shc and Raf-1 was inhibited in Taxol-resistant cells.

Inspection of the deduced amino acid sequence of human p66shc (36) revealed that there were only three predicted CNBr fragments with masses in the 6–8-kDa range. These correspond to residues 2–65 (6683 Da), 177–230 (5841 Da), and 258–329 (7957 Da). On the basis of the following premises, it is predicted that the ~7.9-kDa CNBr fragment represents residues 2–65: (a) p52shc does not shift upon Taxol treatment. Residues 177–230 and 258–329 are shared by both p52shc and p66shc, whereas residues 2–65 are in the unique CH2 domain of p66shc. Highly charged peptides may run anomalously on SDS-PAGE, and therefore the fragment with a 6683-Da mass may migrate in the 7.9-kDa mass range; (b) a recent report indicated that, in mouse embryo fibroblast cells transfected with p66shc, alanine substitution of serine 36, in the CH2 domain, resulted in the loss of the H2O2-induced gel mobility shift of p66shc and a significant reduction of serine phosphorylation upon H2O2 treatment (39) ; and (c) residues 2–65 contain 11 serines. Because a gradual shift of p66shc was observed as the concentration of Taxol increased, the ~9.6-kDa band is most probably derived from the ~7.9 kDa following phosphorylation after Taxol treatment. It is interesting to note that residues 2–65 are a proline-rich region and suggest that a proline-directed kinase may be responsible for the phosphorylation of serine in this domain of p66shc.

Several lines of evidence indicate that the effects of Taxol on Shc in non-macrophage cells are clearly distinguishable from those effects elicited by mitogens, such as EGF: (a) Taxol does not cause tyrosine phosphorylation of Shc and does not induce Shc/Grb2 complex formation, suggesting that Ras activation is not involved; (b) the time required to induce the electrophoretic shift of p66shc by Taxol is much longer than that for EGF; and (c) the MEK inhibitor PD98059 does not inhibit the Taxol-induced p66shc mobility shift. Our results indicate that this Taxol-induced signal transduction pathway differs from classical mitogen-activated signal transduction cascades. Consistent with these findings, it has been reported that domains unique to Shc isoforms, CH1 and CH2, may be involved in a network of protein-protein interactions, and Shc may have other roles in addition to its involvement in Ras activation (28) . Because p66shc contains an extra CH2 region that is not present in p52shc, it may have unique functions compared with other Shc isoforms. In fact, it has been shown that insulin stimulates the phosphorylation of the 66- and 52-kDa Shc isoforms by distinct pathways (38) . Opposite effects of these two isoforms on the EGFR-MAP kinase-fos signaling pathway also has been reported (36) . Although EGF also caused serine/threonine phosphorylation of 50% of p66shc in CHO cells expressing human EGF receptor, unlike Taxol-induced serine phosphorylation of p66shc, this phosphorylation event occurred subsequent to tyrosine phosphorylation and was a fast response (1–3 min) prevented by pretreatment of cells with PD98059 (37) . A recent report described the role of phosphorylation at serine 36 in the CH2 domain of p66shc in the regulation of stress apoptotic response (39) .

The effect of Taxol on signaling molecules in lung carcinoma A549 cells also differs from those effects observed in murine macrophages. In mouse macrophages, high concentrations of Taxol (e.g., 10 µM) were shown to mimic LPS activities in stimulating signaling pathways and gene expression. In contrast, other microtubule-interacting agents, such as Taxotere and epothilones that promote polymerization of stable microtubules, were not able to elicit LPS-mimetic activity in murine macrophages (14 , 49 , 50) . Our results indicating that tyrosine phosphorylation is not involved in Taxol-induced signaling in A549 cells are consistent with findings from other studies in which Taxol-mediated Raf-1 and Bcl-2 hyperphosphorylation was Lck-independent in a Lck-deficient clone from a T-cell leukemia cell line, J.CAM1.6 (44) . Lck is a tyrosine kinase that is important for T-cell signaling from the plasma membrane to Raf-1 kinase (51) .

Taxol can cause mitotic arrest and apoptotic cell death. However, it is not clear whether Taxol-induced apoptosis is a consequence of mitotic arrest through its interactions with the tubulin/microtubule system or is an independent event occurring via mechanisms unrelated to cell cycle arrest. Possible mechanisms of Taxol-induced apoptosis have been proposed (52) . In A549 cells, it has been proposed that Taxol-mediated cell death may result from two different mechanisms; at low Taxol concentrations (<9 nM), cell death may occur without a G2-M block, whereas at higher concentrations (>9 nM), cell death may result from terminal mitotic arrest (24) . In this study, we demonstrated that Taxol, at concentrations >10–20 nM, induced p66shc serine phosphorylation that coincided with Taxol-induced activation of Raf-1 and ERK1/2 and with Taxol-induced release of PARP cleavage products, an early event in apoptosis. Two lines of evidence suggest that Taxol-mediated serine phosphorylation of p66shc is related to the blockade of cells in mitosis: (a) the gel mobility shift of p66shc is induced not only by Taxol but also by other microtubule-interacting agents, all of which are antimitotic drugs; and (b) the maximum effect of the electrophoretic mobility shift of p66shc was seen at 9–18 h after Taxol treatment, a time at which a high proportion of the cells would be in mitosis (see Fig. 3Citation ). The recent finding that disruption of p66shc function, by mutating the serine phosphorylation site, enhances cellular resistance to apoptosis (39) suggests that the phosphorylation of p66shc reported here may modulate Taxol-induced apoptosis.

In a classical Ras-dependent signal transduction pathway that leads to the activation of ERK1/2, tyrosine phosphorylation of Shc results in Shc/Grb2/SOS ternary complex formation and the subsequent activation of Ras and Raf-1. In Taxol-treated A549 cells, although a Taxol-induced Raf-1 mobility shift coincides with phosphorylation of p66shc, Raf-1 phosphorylation may not be the downstream event of p66shc phosphorylation. Rather, both of these events may be the consequence of mitotic arrest resulting from the action of antimitotic agents.

The emergence of drug resistance is a significant clinical problem that develops during the treatment of most human malignancies with antitumor agents, including Taxol. Potential mechanisms involved in Taxol resistance include overexpression of P-glycoprotein (53) , mutations in tubulin that may influence its interaction with Taxol (54) , and overexpression of c-erbB-2/neu in breast cancer cells (55) . In Taxol-resistant A549-T12 cells that do not express P-glycoprotein, both the phosphorylation of p66shc and Raf-1 induced by Taxol were diminished, compared with drug-sensitive cells. The report that p66shc knockout mice have an increased resistance to oxidative stress and a longer life span (39) agrees with the results obtained with A549-T12 cells that are resistant to Taxol and whose p66shc is not activated by 20 nM Taxol.

In summary, we have identified a Taxol-mediated signaling event that is distinct from those effects elicited by micromolar concentrations of Taxol in mouse macrophages. It is also different from mitogen-activated processes, in which both tyrosine and serine/threonine phosphorylation of Shc proteins occurs rapidly. It is known that serine phosphorylation occurs during mitosis (56) , and it is probable that serine kinases are up-regulated or activated at this phase of the cell cycle. The altered serine phosphorylation of p66shc that occurs in the presence of a variety of molecules that interact with the tubulin/microtubule system may relate to the dramatically prolonged and abnormal time that such cells are in the mitotic phase of the cell cycle.


   ACKNOWLEDGMENTS
 
We thank Drs. George Orr and Hayley McDaid for many helpful discussions.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This research was supported in part by USPHS Grant CA83185. Back

2 To whom requests for reprints should be addressed, at Department of Molecular Pharmacology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461. Phone: (718) 430-2163; Fax: (718) 430-8922; E-mail: shorwitz{at}aecom.yu.edu Back

3 The abbreviations used are: MAP, mitogen-activated protein; LPS, lipopolysaccharide; ERK, extracellular signal-regulated kinase; MEK, mitogen activated and extracellular signal-regulated protein kinase kinase; PARP, poly(ADP-ribose) polymerase; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; SOS, son of sevenless; SH2, Src homology 2; CH, collagen homology; PP2A, protein phosphatase 2A. Back

Received 2/18/00. Accepted 7/19/00.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Rowinsky E. K., Donehower R. C. Paclitaxel (Taxol) [published erratum appears in N. Engl. J. Med., 333: 75, 1995]. N. Engl. J. Med., 332: 1004-1014, 1995.[Free Full Text]
  2. Manfredi J. J., Parness J., Horwitz S. B. Taxol binds to cellular microtubules. J. Cell Biol., 94: 688-696, 1982.[Abstract/Free Full Text]
  3. Schiff P. B., Fant J., Horwitz S. B. Promotion of microtubule assembly in vitro by Taxol. Nature (Lond.), 277: 665-667, 1979.[Medline]
  4. Rao S., He L., Chakravarty S., Ojima I., Orr G. A., Horwitz S. B. Characterization of the Taxol binding site on the microtubule. Identification of arg(282) in ß-tubulin as the site of photoincorporation of a 7-benzophenone analogue of Taxol. J. Biol. Chem., 274: 37990-37994, 1999.[Abstract/Free Full Text]
  5. Schiff P. B., Horwitz S. B. Taxol stabilizes microtubules in mouse fibroblast cells. Proc. Natl. Acad. Sci. USA, 77: 1561-1565, 1980.[Abstract/Free Full Text]
  6. Yvon A. M., Wadsworth P., Jordan M. A. Taxol suppresses dynamics of individual microtubules in living human tumor cells. Mol. Biol. Cell, 10: 947-959, 1999.[Abstract/Free Full Text]
  7. Bhalla K., Ibrado A. M., Tourkina E., Tang C., Mahoney M. E., Huang Y. Taxol induces internucleosomal DNA fragmentation associated with programmed cell death in human myeloid leukemia cells. Leukemia (Baltimore), 7: 563-568, 1993.[Medline]
  8. Wang T. H., Popp D. M., Wang H. S., Saitoh M., Mural J. G., Henley D. C., Ichijo H., Wimalasena J. Microtubule dysfunction induced by paclitaxel initiates apoptosis through both c-Jun N-terminal kinase (JNK)-dependent and -independent pathways in ovarian cancer cells. J. Biol. Chem., 274: 8208-8216, 1999.[Abstract/Free Full Text]
  9. Kottke T. J., Blajeski A. L., Martins L. M., Mesner P. W., Jr., Davidson N. E., Earnshaw W. C., Armstrong D. K., Kaufmann S. H. Comparison of paclitaxel-, 5-fluoro-2'-deoxyuridine-, and epidermal growth factor (EGF)-induced apoptosis. Evidence for EGF-induced anoikis. J. Biol. Chem., 274: 15927-15936, 1999.[Abstract/Free Full Text]
  10. Carboni J. M., Singh C., Tepper M. A. Taxol and lipopolysaccharide activation of a murine macrophage cell line and induction of similar tyrosine phosphoproteins. J. Natl. Cancer Inst. Monogr., 15: 95-101, 1993.
  11. Ding A., Sanchez E., Nathan C. F. Taxol shares the ability of bacterial lipopolysaccharide to induce tyrosine phosphorylation of microtubule-associated protein kinase. J. Immunol., 151: 5596-5602, 1993.[Abstract]
  12. Liu Y., Bhalla K., Hill C., Priest D. G. Evidence for involvement of tyrosine phosphorylation in Taxol-induced apoptosis in a human ovarian tumor cell line. Biochem. Pharmacol., 48: 1265-1272, 1994.[Medline]
  13. Manthey C. L., Perera P. Y., Salkowski C. A., Vogel S. N. Taxol provides a second signal for murine macrophage tumoricidal activity. J. Immunol., 152: 825-831, 1994.[Abstract]
  14. Burkhart C. A., Berman J. W., Swindell C. S., Horwitz S. B. Relationship between the structure of Taxol and other taxanes on induction of tumor necrosis factor-{alpha} gene expression and cytotoxicity. Cancer Res., 54: 5779-5782, 1994.[Abstract/Free Full Text]
  15. Byrd C. A., Bornmann W., Erdjument-Bromage H., Tempst P., Pavletich N., Rosen N., Nathan C. F., Ding A. Heat shock protein 90 mediates macrophage activation by Taxol and bacterial lipopolysaccharide. Proc. Natl. Acad. Sci. USA, 96: 5645-5650, 1999.[Abstract/Free Full Text]
  16. Bhat N., Perera P. Y., Carboni J. M., Blanco J., Golenbock D. T., Mayadas T. N., Vogel S. N. Use of a photoactivatable Taxol analogue to identify unique cellular targets in murine macrophages: identification of murine CD18 as a major Taxol-binding protein and a role for Mac-1 in Taxol-induced gene expression. J. Immunol., 162: 7335-7342, 1999.[Abstract/Free Full Text]
  17. Rodi D. J., Janes R. W., Sanganee H. J., Holton R. A., Wallace B. A., Makowski L. Screening of a library of phage-displayed peptides identifies human bcl-2 as a Taxol-binding protein. J. Mol. Biol., 285: 197-203, 1999.[Medline]
  18. Ichijo H., Nishida E., Irie K., ten Dijke P., Saitoh M., Moriguchi T., Takagi M., Matsumoto K., Miyazono K., Gotoh Y. Induction of apoptosis by ASK1, a mammalian MAPKKK that activates SAPK/JNK and p38 signaling pathways. Science (Washington DC), 275: 90-94, 1997.[Abstract/Free Full Text]
  19. Wang T. H., Wang H. S., Ichijo H., Giannakakou P., Foster J. S., Fojo T., Wimalasena J. Microtubule-interfering agents activate c-Jun N-terminal kinase/stress-activated protein kinase through both Ras and apoptosis signal-regulating kinase pathways. J. Biol. Chem., 273: 4928-4936, 1998.[Abstract/Free Full Text]
  20. Lee L. F., Li G., Templeton D. J., Ting J. P. Paclitaxel (Taxol)-induced gene expression and cell death are both mediated by the activation of c-Jun NH2-terminal kinase (JNK/SAPK). J. Biol. Chem., 273: 28253-28260, 1998.[Abstract/Free Full Text]
  21. Blagosklonny M. V., Schulte T. W., Nguyen P., Mimnaugh E. G., Trepel J., Neckers L. Taxol induction of p21WAF1 and p53 requires c-raf-1. Cancer Res., 55: 4623-4626, 1995.[Abstract/Free Full Text]
  22. Blagosklonny M. V., Giannakakou P., El-Deiry W. S., Kingston D. G. I., Higgs P. I., Neckers L., Fojo T. Raf-1/bcl-2 phosphorylation: a step from microtubule damage to cell death. Cancer Res., 57: 130-135, 1997.[Abstract/Free Full Text]
  23. Wolfson M., Yang C. P., Horwitz S. B. Taxol induces tyrosine phosphorylation of Shc and its association with Grb2 in murine RAW 264.7 cells. Int. J. Cancer, 70: 248-252, 1997.[Medline]
  24. Torres K., Horwitz S. B. Mechanisms of Taxol-induced cell death are concentration dependent. Cancer Res., 58: 3620-3626, 1998.[Abstract/Free Full Text]
  25. Han J., Lee J. D., Bibbs L., Ulevitch R. J. A MAP kinase targeted by endotoxin and hyperosmolarity in mammalian cells. Science (Washington DC), 265: 808-811, 1994.[Abstract/Free Full Text]
  26. Shtil A. A., Mandlekar S., Yu R., Walter R. J., Hagen K., Tan T. H., Roninson I. B., Kong A. N. Differential regulation of mitogen-activated protein kinases by microtubule-binding agents in human breast cancer cells. Oncogene, 18: 377-384, 1999.[Medline]
  27. Pelicci G., Lanfrancone L., Grignani F., McGlade J., Cavallo F., Forni G., Nicoletti I., Grignani F., Pawson T., Pelicci P. G. A novel transforming protein (SHC) with an SH2 domain is implicated in mitogenic signal transduction. Cell, 70: 93-104, 1992.[Medline]
  28. Bonfini L., Migliaccio E., Pelicci G., Lanfrancone L., Pelicci P. G. Not all Shc’s roads lead to Ras. Trends Biochem. Sci., 21: 257-261, 1996.[Medline]
  29. Pronk G. J., McGlade J., Pelicci G., Pawson T., Bos J. L. Insulin-induced phosphorylation of the 46- and 52-kDa Shc proteins. J. Biol. Chem., 268: 5748-5753, 1993.[Abstract/Free Full Text]
  30. Segatto O., Pelicci G., Giuli S., Digiesi G., Di Fiore P. P., McGlade J., Pawson T., Pelicci P. G. Shc products are substrates of erbB-2 kinase. Oncogene, 8: 2105-2112, 1993.[Medline]
  31. McGlade J., Cheng A., Pelicci G., Pelicci P. G., Pawson T. Shc proteins are phosphorylated and regulated by the v-Src and v-Fps protein-tyrosine kinases. Proc. Natl. Acad. Sci. USA, 89: 8869-8873, 1992.[Abstract/Free Full Text]
  32. Baldari C. T., Pelicci G., Di Somma M. M., Milia E., Giuli S., Pelicci P. G., Telford J. L. Inhibition of CD4/p56lck signaling by a dominant negative mutant of the Shc adaptor protein [published erratum appears in Oncogene 11: 2451, 1995]. Oncogene, 10: 1141-1147, 1995.[Medline]
  33. Kavanaugh W. M., Williams L. T. An alternative to SH2 domains for binding tyrosine-phosphorylated proteins. Science (Washington DC), 266: 1862-1865, 1994.[Abstract/Free Full Text]
  34. Rozakis-Adcock M., McGlade J., Mbamalu G., Pelicci G., Daly R., Li W., Batzer A., Thomas S., Brugge J., Pelicci P. G., et al Association of the Shc and Grb2/Sem5 SH2-containing proteins is implicated in activation of the Ras pathway by tyrosine kinases. Nature (Lond.), 360: 689-692, 1992.[Medline]
  35. Aronheim A., Engelberg D., Li N., al-Alawi N., Schlessinger J., Karin M. Membrane targeting of the nucleotide exchange factor Sos is sufficient for activating the Ras signaling pathway. Cell, 78: 949-961, 1994.[Medline]
  36. Migliaccio E., Mele S., Salcini A. E., Pelicci G., Lai K. M., Superti-Furga G., Pawson T., Di Fiore P. P., Lanfrancone L., Pelicci P. G. Opposite effects of the p52shc/p46shc and p66shc splicing isoforms on the EGF receptor-MAP kinase-fos signalling pathway. EMBO J., 16: 706-716, 1997.[Medline]
  37. Okada S., Kao A. W., Ceresa B. P., Blaikie P., Margolis B., Pessin J. E. The 66-kDa Shc isoform is a negative regulator of the epidermal growth factor-stimulated mitogen-activated protein kinase pathway. J. Biol. Chem., 272: 28042-28049, 1997.[Abstract/Free Full Text]
  38. Kao A. W., Waters S. B., Okada S., Pessin J. E. Insulin stimulates the phosphorylation of the 66- and 52-kilodalton Shc isoforms by distinct pathways. Endocrinology, 138: 2474-2480, 1997.[Abstract/Free Full Text]
  39. Migliaccio E., Giorgio M., Mele S., Pelicci G., Reboldi P., Pandolfi P. P., Lanfrancone L., Pelicci P. G. The p66shc adaptor protein controls oxidative stress response and life span in mammals. Nature (Lond.), 402: 309-313, 1999.[Medline]
  40. Kavallaris M., Kuo D. Y. S., Burkhart C. A., Regl D. L., Norris M. D., Haber M., Horwitz S. B. Taxol-resistant epithelial ovarian tumors are associated with altered expression of specific ß-tubulin isotypes. J. Clin. Investig., 100: 1282-1293, 1997.[Medline]
  41. O’Farrell P. H. High resolution two-dimensional electrophoresis of proteins. J. Biol. Chem., 250: 4007-4021, 1975.[Abstract/Free Full Text]
  42. Schagger H., von Jagow G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal. Biochem., 166: 368-379, 1987.[Medline]
  43. Duriez P. J., Shah G. M. Cleavage of poly(ADP-ribose) polymerase: a sensitive parameter to study cell death. Biochem. Cell Biol., 75: 337-349, 1997.[Medline]
  44. Blagosklonny M. V., Chuman Y., Bergan R. C., Fojo T. Mitogen-activated protein kinase pathway is dispensable for microtubule-active drug-induced Raf-1/Bcl-2 phosphorylation and apoptosis in leukemia cells. Leukemia (Baltimore), 13: 1028-1036, 1999.[Medline]
  45. Huang Y., Sheikh M. S., Fornace A. J., Jr., Holbrook N. J. Serine protease inhibitor TPCK prevents Taxol-induced cell death and blocks c-Raf-1 and Bcl-2 phosphorylation in human breast carcinoma cells. Oncogene, 18: 3431-3439, 1999.[Medline]
  46. Sontag E., Nunbhakdi-Craig V., Bloom G. S., Mumby M. C. A novel pool of protein phosphatase 2A is associated with microtubules and is regulated during the cell cycle. J. Cell Biol., 128: 1131-1144, 1995.[Abstract/Free Full Text]
  47. Tournebize R., Andersen S. S., Verde F., Doree M., Karsenti E., Hyman A. A. Distinct roles of PP1 and PP2A-like phosphatases in control of microtubule dynamics during mitosis. EMBO J., 16: 5537-5549, 1997.[Medline]
  48. Martello L. A., McDaid H. M., Regl D. L., Yang C-P., Meng D., Pettus T. R., Kaufman M. D., Arimoto H., Danishefsky S. J., Smith, III A. B., Horwitz S. B. Taxol and discodermolide represent a synergistic drug combination in human carcinoma cell lines. Clin. Cancer Res., 6: 1978-1987, 2000.[Abstract/Free Full Text]
  49. Muhlradt P. F., Sasse F. Epothilone B stabilizes microtubuli of macrophages like Taxol without showing Taxol-like endotoxin activity. Cancer Res., 57: 3344-3346, 1997.[Abstract/Free Full Text]
  50. Moos P. J., Fitzpatrick F. A. Taxane-mediated gene induction is independent of microtubule stabilization: induction of transcription regulators and enzymes that modulate inflammation and apoptosis. Proc. Natl. Acad. Sci. USA, 95: 3896-3901, 1998.[Abstract/Free Full Text]
  51. Li W., Whaley C. D., Mondino A., Mueller D. L. Blocked signal transduction to the ERK and JNK protein kinases in anergic CD4+ T cells. Science (Washington DC), 271: 1272-1276, 1996.[Abstract]
  52. Fan W. Possible mechanisms of paclitaxel-induced apoptosis. Biochem. Pharmacol., 57: 1215-1221, 1999.[Medline]
  53. Horwitz S. B., Cohen D., Rao S., Ringel I., Shen H. J., Yang C. P. Taxol: mechanisms of action and resistance. J. Natl. Cancer Inst. Monogr., 15: 55-61, 1993.
  54. Gonzalez-Garay M. L., Chang L., Blade K., Menick D. R., Cabral F. A ß-tubulin leucine cluster involved in microtubule assembly and paclitaxel resistance. J. Biol. Chem., 274: 23875-23882, 1999.[Abstract/Free Full Text]
  55. Yu D., Liu B., Tan M., Li J., Wang S. S., Hung M. C. Overexpression of c-erbB-2/neu in breast cancer cells confers increased resistance to Taxol via mdr-1-independent mechanisms. Oncogene, 13: 1359-1365, 1996.[Medline]
  56. Blagosklonny M. V., Fojo T. Molecular effects of paclitaxel: myths and reality (a critical review). Int. J. Cancer, 83: 151-156, 1999.[Medline]



This article has been cited by other articles:


Home page
 Mol. Biol. CellHome page
Y. Hu, X. Wang, L. Zeng, D.-Y. Cai, K. Sabapathy, S. P. Goff, E. J. Firpo, and B. Li
ERK Phosphorylates p66shcA on Ser36 and Subsequently Regulates p27kip1 Expression via the Akt-FOXO3a Pathway: Implication of p27kip1 in Cell Response to Oxidative Stress
Mol. Biol. Cell, August 1, 2005; 16(8): 3705 - 3718.
[Abstract] [Full Text] [PDF]

Home page
 J. Cell Biol.Home page
W. W. Smith, D. D. Norton, M. Gorospe, H. Jiang, S. Nemoto, N. J. Holbrook, T. Finkel, and J. W. Kusiak
Phosphorylation of p66Shc and forkhead proteins mediates A{beta} toxicity
J. Cell Biol., April 25, 2005; 169(2): 331 - 339.
[Abstract] [Full Text] [PDF]

Home page
 J. Clin. Endocrinol. Metab.Home page
E. Pagnin, G. Fadini, R. de Toni, A. Tiengo, L. Calo, and A. Avogaro
Diabetes Induces p66shc Gene Expression in Human Peripheral Blood Mononuclear Cells: Relationship to Oxidative Stress
J. Clin. Endocrinol. Metab., February 1, 2005; 90(2): 1130 - 1136.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
T. L. Tinley, D. A. Randall-Hlubek, R. M. Leal, E. M. Jackson, J. W. Cessac, J. C. Quada Jr., T. K. Hemscheidt, and S. L. Mooberry
Taccalonolides E and A: Plant-derived Steroids with Microtubule-stabilizing Activity
Cancer Res., June 15, 2003; 63(12): 3211 - 3220.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
S. Lee, W. Yang, K.-H. Lan, S. Sellappan, K. Klos, G. Hortobagyi, M.-C. Hung, and D. Yu
Enhanced Sensitization to Taxol-induced Apoptosis by Herceptin Pretreatment in ErbB2-overexpressing Breast Cancer Cells
Cancer Res., October 15, 2002; 62(20): 5703 - 5710.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
A. Faisal, M. El-Shemerly, D. Hess, and Y. Nagamine
Serine/Threonine Phosphorylation of ShcA. REGULATION OF PROTEIN-TYROSINE PHOSPHATASE-PEST BINDING AND INVOLVEMENT IN INSULIN SIGNALING
J. Biol. Chem., August 9, 2002; 277(33): 30144 - 30152.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
S. Le, T. J. Connors, and A. C. Maroney
c-Jun N-terminal Kinase Specifically Phosphorylates p66ShcA at Serine 36 in Response to Ultraviolet Irradiation
J. Biol. Chem., December 14, 2001; 276(51): 48332 - 48336.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Pharmacol.Home page
H. M. McDaid and S. B. Horwitz
Selective Potentiation of Paclitaxel (Taxol)-Induced Cell Death by Mitogen-Activated Protein Kinase Kinase Inhibition in Human Cancer Cell Lines
Mol. Pharmacol., August 1, 2001; 60(2): 290 - 301.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
J.-i. Okano and A. K. Rustgi
Paclitaxel Induces Prolonged Activation of the Ras/MEK/ERK Pathway Independently of Activating the Programmed Cell Death Machinery
J. Biol. Chem., May 25, 2001; 276(22): 19555 - 19564.
[Abstract] [Full Text] [PDF]

<
This Article
Right arrow Abstract Freely available