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The Journal of Immunology, 2001, 166: 6084-6090.
Copyright © 2001 by The American Association of Immunologists

p27kif Protein Levels and E2F Activity Are Targets of Cot Kinase During G1 Phase Progression in T Cells1

Ana Velasco-Sampayo and Susana Alemany2

Instituto de Investigaciones Biomédicas, Consejo Superior de Investigaciones Cientificas, Facultad. Medicina Universidad Autónoma de Madrid, Madrid, Spain


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Cot/Tpl-2 kinase, homologous to members of mitogen-activated protein kinase kinase kinase, was initially discovered by its capacity to promote cell transformation. Cot/Tpl-2 mRNA levels are increased during G0 to G1 phase progression in T lymphocytes, suggesting a role for this kinase later on in the cell cycle. The IL-2-dependent CTLL-2 cells were used to investigate the role of Cot kinase in G1 to S phase transition. Transient expression of Cot kinase in CTLL-2 cells increases DNA synthesis triggered by IL-2 and the transient expression of a dominant negative form of Cot kinase in CTLL-2 markedly reduces the DNA synthesis triggered by this cytokine. Cell cycle analysis of synchronized CTLL-2 stabling overexpressing Cot kinase indicates that this kinase contributes to the passage to S and G2-M phases of the cell cycle. Cot kinase reduces the levels of the cyclin kinase inhibitor p27kip, whereas bcl-xL expression is unaffected. Cot kinase also increases E2F transcriptional activity in a phosphatidylinositol 3 kinase-independent way and acts in synergy with this kinase. These data give evidence, for the first time, of the regulation of different G1 progression events by Cot kinase.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Interleukin 2 receptors activation leads to the up-regulation of antiapoptotic as well as proliferative pathways (1, 2, 3) and culminates in the assembly of the G1 to S phase cell cycle transition machinery. Activity of the cyclin-dependent kinases is up-regulated through a number of mechanisms including posttransductional modifications and the dissociation of complexes with the cyclin-dependent kinase inhibitors (4, 5). These inhibitors act stoichiometrically, and oscillations in their levels can have a profound effect on cell proliferation (6, 7). The p27kip is present at high levels in quiescent cells and down-regulated by mitogenic stimulation (7, 8). One of the essential events in IL-2-induced G1 progression is the up-regulation of the E2F transcription factor activity by phosphatidylinositol 3-kinase (PI3-kinase)3 (9). The increment in the E2F activity in the mid- to late-G1 phase allows the expression of E2F target genes and entry into the S phase (for a review, see Ref. 10). IL-2 deprivation or an inaccurate regulation of the different events implicated in the G1 to S phase transition promotes apoptosis (1, 11, 12).

The Cot kinase gene was first cloned in a truncated form in transformed foci induced in the embryonic cells of a Syrian hamster Osaka Kanazawa (SHOK) cells (13). The first 397 aa of the normal human cellular homologue are identical with the truncated form, and the remaining 69 aa from the carboxyl terminus are replaced by 18 aa in the truncated form (13, 14). This rearrangement gives the kinase its transformation capacity (13, 14, 15), although higher expression of the normal gene also confers transformed phenotype in fibroblasts (15). It has been proposed that an amplification of the genomic locus of Cot gene plays a role in human breast cancer (16). The same gene was identified as an oncogene associated with the progression of Moloney leukemia virus-induced T cell lymphomas in rats (Tpl-2) (17). Transgenic mice expressing the truncated oncogenic protein in thymocytes develop T cell lymphomas (18).

The Cot/Tpl-2 protein is homologous to members of the mitogen-activated protein (MAP) kinase kinase kinase (MAP3K) and regulates the activity of several transduction pathways that converge into the activation of several MAP kinases: extracellular signal-regulated kinase (ERK) 1, c-Jun N-terminal kinase, ERK6 (p38{gamma}), and ERK5 (19, 20, 21, 22). Cot kinase up-regulates the activity of the AP-1 and NF-{kappa}B transcription factors (16, 21, 23, 24). Cot kinase also participates in the IL-2 and TNF-{alpha} secretion in T lymphocytes (23, 25, 26). All of these data support a role for Cot kinase in the G0 to G1 phase transition of T lymphocytes.

Cot mRNA levels are up-regulated by signals that induce G0 to G1 phase transition in T lymphocytes (27). This suggests that Cot kinase also could be involved in the G1 to S phase transition of the cell cycle in T lymphocytes.

In this paper, we have evaluated the role of Cot kinase in G1 phase progression, with a T cell line (CTLL-2) as a cell model, which is dependent on IL-2 to perform the G1 to S phase transition and in which the absence of this cytokine promotes apoptosis (28). DNA synthesis induced by IL-2 in T lymphocytes is partially inhibited by blocking endogenous Cot kinase activity and ectopic expression of Cot or truncated Cot kinase contributes to cell cycle progression. We also show that Cot kinase reduces the expression of the cyclin kinase inhibitor p27kip and induces E2F activity in a PI3 kinase-independent way. These data give evidence of different targets of Cot kinase in the contribution to the G1/S transition in T lymphocytes.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Constructs

The pEF-BOS Cot, pEF-BOS trunc-Cot, and pEF-BOS inac-Cot constructs have been described previously (23). Hemagglutinin (HA)-Cot and HA-trunc-Cot were obtained by PCR:HA tagging was introduced into Cot kinase (15), trunc-Cot kinase (13), or inac-Cot (23) constructs after the second translation initiation site. An EcoRI site 5' from the first initiation site and a SalI site 3' from the stop codon were generated in the PCR. The different HA constructs were cloned in the EcoRI Sal1 sites of the pCDNA3.1 vector (Invitrogen, San Diego, CA). DNA sequencing was performed to verify the constructs. Dr. J. Downward (Imperial Cancer Research, London, U.K.) generously provided the pSG5 p110 CAAX. The E2A Luciferase (Luc) construct (pGL2-3xwt) and mutated version (mt) E2A Luc construct (pGL2-3xmt) were generously provided by Dr. Lam (Ludwig Institute for Cancer Research, London, U.K.). The pEGFP-N1 construct was obtained from Clontech Laboratories (Palo Alto, CA).

Cell sorting of transiently transfected cells and thymidine incorporation

CTLL-2 cells (20 x 106) were electroporated with pEGFP-N1 (10 µg) and pEF-BOS (30 µg), pEF-BOS Cot (30 µg), or pEF-BOS inac-Cot (30 µg), and resuspended in basal medium with 50 U/ml rIL-2. After 4 h of incubation, viable cells were purified by a Ficoll-Hystopaque 1077 gradient, as indicated in Ref. 29 . Cells were further incubated for 16 h in basal medium in the absence of IL-2. After this time, the green fluorescent protein (GFP)-positive cells were selected by cell sorting in a FACStars plus (Becton Dickinson, Mountain View, CA). The same gate was used to isolated the GFP-positive transfected cells. Between 90 and 95% of the selected cells were positive for GFP. A thymidine incorporation assay with the selected cells was performed. Cells (10,000 cells/well) were incubated with different concentrations of IL-2 for 24 h. Four hours before harvesting, 1 µCi/well of [3H]thymidine was added. Measurement of radioactivity incorporated into DNA was performed as indicated in Ref. 25 .

Isolation of CTLL-2 cells constitutively expressing HA, HA-Cot, or HA-trunc-Cot

CTLL-2 were maintained in basal medium (RPMI 1640 (Life Technologies, Rockville, MD) medium supplemented with 10% (v/v) FBS, 50 µg/ml gentamicin, and 50 µM 2-ME) supplemented with 50 U/ml rIL-2 (generously provided by Hoffman-La Roche, Nutley, NJ) at a concentration of 2–4 x 105 cells/ml. To transfect CTLL-2 cells, 20 x 106 cells were resuspended in 0.5 ml of RPMI and electroporated with 20 µg of pCDNA3.1 HA-Cot, pCDNA3.1 HA-trunc-Cot, or pCDNA3.1 HA with a "Gene Pulser (Hoeffer, Barcelona, Spain), as described in Ref. 23 . Then, cells were resuspended in basal medium containing 50 U/ml rIL-2. After 48 h, the intact CTLL-2 transfected with HA-Cot, HA-trunc-Cot, or HA CTLL-2 were separated by Ficoll-Hystopaque 1077 (Sigma, St. Louis, MO) gradient centrifugation as described for peripheral blood mononuclear cell isolation (29). Selection of the cells expressing the different transfected plasmids (HA-Cot CTLL-2, HA-trunc-Cot CTLL-2, and HA CTLL-2) was done in basal medium with 50 U/ml rIL-2 and 1 mg/ml G418 (Calbiochem, La Jolla, CA), and maintained at 1–4 x 105 cells/ml. To avoid the obtention of single clones that could not have a representative behavior, selection of the G418-resistant cells was conducted in pools of 100 ml. The HA-Cot CTLL-2, HA-trunc-Cot CTLL-2, and HA CTLL-2 cells were maintained in basal medium supplemented with 50 U/ml rIL-2. Cells were periodically incubated in the presence of 1 mg/ml G418 to elude the expansion of cells sensitive to this antibiotic.

RT-PCR assay

Four weeks after transfection, HA-Cot CTLL-2, HA-trunc-Cot CTLL-2, and HA CTLL-2 cells (5 x 106) were pelleted and total RNA was isolated. A RT-PCR was performed as described previously (23). Primers 5'-TATGATGTTCCTGATTATGCT-3' (62–82 nt, corresponding to the HA sequence) and 5'-GAGAACATCGGAATCTATTT-3' (355–373 nt) were used to examine the levels of HA-Cot or HA-trunc-Cot. {beta}-Actin levels were determined with primers 5'-AGCACAATGAAGATCAAGAT-3' (sense) and 5'-ACATTGCGTTGATTCAGTAT-3' (antisense). PCR was amplified for 30–35 denaturation cycles at 94°C for 1 min, annealing at 56°C for 1 min, and extension at 72°C for 1 min. Amplified fragments were separated by 1% agarose gel electrophoresis and bands were visualized by ethidium bromide staining.

Cell cycle analysis

Exponentially growing HA-Cot CTLL-2, HA-trunc-Cot CTLL-2, and HA CTLL-2 cells were washed twice with PBS and resuspended at a concentration of 5 x 105 cells/ml in basal medium in the absence of IL-2 for 24 h. After this incubation period (0 h), the majority of the cells were found in G1 phase. Synchronized cells then were incubated in the absence or in the presence of different concentrations of IL-2. After the incubation time, cells (5 x 105) were fixed and permeabilized with 70% ethanol at 4°C for 5 min and then with 0.2 M NaH2PO4, 4 mM citric acid, pH 7.8, for 10 min at 37°C. Cells then were resuspended in PBS with 100 µg/ml RNase A (Boehringer Mannheim, Indianapolis, IN) and 40 µg/ml propidium iodide (Sigma) and incubated for 30 min at 37°C. Fixed and stained cells were analyzed by flow cytometry for light scattering properties and DNA content with a FACScan flow cytometer (Becton Dickinson). The percentages of cells in different stages of the cell cycle were calculated with the help of the computer program MOD FIT LT and CellQuest (Becton Dickinson).

Preparation of cell lysates and immunoblot analysis

G1 phase-synchronized HA-Cot CTLL-2, HA-trunc-COT CTLL-2, and HA CTLL-2 cells were incubated or not for 10 h with IL-2 (1.5 or 20 U/ml). Cells then were collected by centrifugation, washed twice with ice-cold PBS, and frozen in dry ice. Pellets were resuspended in lysis buffer containing 20 mM Tris-HCl, 10 mM EDTA, 100 mM NaCl, 1% Triton X-100, 1 mM NaF, 1 mM {beta}-glycerophosphate, 1 mM EGTA, 5 mM NaH2PO4, pH 7.4, and 1 tablet/10 ml of the cocktail inhibitors of Boehringer Mannheim. Protein concentration was determined in the supernatant by using the Dc protein assay (Bio-Rad, Richmond, CA) and 30 µg of protein were resolved by using 12% SDS-PAGE, before transfer to membranes. Incubation of the membranes with the p27kip and bcl-xL Abs has been previously described in Ref. 30 . Isabel Mérida (30) generously provided Abs.

Transfections and Luc assay

CTLL-2 cells (20 x 106) were transfected as described above with, unless otherwise indicated, 30 µg of pEF-BOS Cot, pEF-BOS trunc-Cot, pEF-BOS, and/or pSG5 p110 CAAX constructs together with 5 µg/ml of pGL2-3xwt E2A Luc or pGL2-3xmt E2A Luc. Transfected cells were resuspended for 36 h in basal medium in the presence or absence of IL-2, or wortmannin (1 µM; Biomol, Plymouth Meeting, PA), or MAP/ERK (MEK) inhibitor (10 µM; Calbiochem). After this incubation time, cells were collected by centrifugation, and Luc activity was determined by the luciferase assay kit (Promega, Madison, WI), according to the manufacturer’s instructions. Cell extracts were normalized by protein measurements with the Dc protein assay (Bio-Rad).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Regulation of IL-2-induced DNA synthesis by transient expression of Cot kinase or Cot kinase-deficient mutant in CTLL-2 cells

To investigate the role of COT kinase in the G1 to S phase of T lymphocytes we used the CTLL-2 cell line as a cell system, which is dependent on IL-2 to progress to the S phase of the cell cycle. CTLL-2 cells were transiently transfected with pEGFP-N1 (10 µg) and pEF-BOS (30 µg) or pEF-BOS inac-Cot (30 µg). After 20 h of transfection, GFP-positive cells were selected to perform a thymidine incorporation assay. The positive GFP cells (90–95%) expressing Cot kinase, inactive Cot kinase, or just cotransfected with pEF-BOS were subjected to stimulation with different concentrations IL-2 for 24 h, and thymidine incorporation was measured. Cells expressing Cot kinase incorporated 10-fold more thymidine than control cells (Fig. 1Go). Transient transfection of Cot kinase also regulated IL-2-induced thymidine incorporation in BAF-BO3 {alpha}{beta} cells (data not shown), a murine pro-B cell line that constitutively express the three subunits of the IL-2 receptor (30). In agreement with these data, in the selection of single CTLL-2 clones constitutively expressing HA, HA-Cot, or HA-trunc-Cot, the number of positive single clones after 10 days of transfection was significantly higher in pCDNA3.1 HA-Cot or pCDNA3.1 HA-trunc-Cot transfected cells than in cells transfected with pCDNA3.1 HA: HA-Cot CTLL-2, 12 clones; HA-trunc-Cot CTLL-2, 19 clones; and HA CTLL-2, 2 clones (data not shown).



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  		FIGURE 1. Thymidine incorporation in Cot kinase and Cot kinase-deficient mutant CTLL-2 cells. Cells (10 x 103) transiently expressing Cot kinase, inactivated Cot kinase, or not were incubated with different doses of IL-2 for 24 h, and 1 µCi/well of [3H]thymidine was added 4 h before harvesting. The graph shows the mean induction of two different experiments performed in triplicate.

 
Cells transiently expressing Cot kinase-deficient mutant (inac-Cot) incorporated less thymidine than control cells, indicating that the blockage of the endogenous Cot activity decreased IL-2-triggered DNA synthesis (Fig. 1Go). The degree of inhibition in the thymidine incorporation in Cot kinase-deficient mutant transfected vs control cells increased systemically at high doses of IL-2.

Cell cycle analysis of CTLL-2 expressing Cot and trunc-Cot kinase

To perform cell cycle analysis of CTLL-2 expressing Cot kinase, we obtained three different CTLL-2 cell lines that constitutively express protooncogenic HA-Cot kinase (HA-Cot), oncogenic HA-Cot kinase (HA-trunc-Cot), and HA (HA-Cot CTLL-2, HA-trunc-Cot CTLL-2, and HA CTLL-2 cells, respectively). After G418 cell selection, RT-PCR analysis was performed to confirm the expression of transfected Cot kinase. The 62–373 nt fragments of HA-Cot or HA-trunc-Cot were detected in HA-Cot CTLL-2, and in HA-trunc-Cot CTLL-2 cells, respectively (Fig. 2GoA).



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  		FIGURE 2. Cell cycle analysis of exponentially growing HA CTLL-2, HA-Cot CTLL-2, and HA-trunc-Cot CTLL-2 cells. A, Expression of HA-Cot and HA-trunc-Cot in HA-Cot CTLL-2 and HA-trunc-Cot CTLL-2 cells. Total RNA from the different stable CTLL-2 cell lines were used for RT-PCR analysis. Data shown corresponds to 30 cycles, and the amount of amplified products (62–373 nt of HA-Cot or 62–373 nt of HA-trunc-Cot, or 1292–1479 of {beta}-actin) are proportional to the abundance of the starting material. B, Cell cycle analysis of 5–10 x 103 exponentially growing HA CTLL-2, HA-Cot CTLL-2, and HA-trunc-Cot CTLL-2 cells was performed as indicated in Material and Methods. The figure shows one representative experiment, and the table indicates the means of five different experiments performed with two different sets of stable HA CTLL-2, HA-Cot CTLL-2, and HA-trunc-Cot CTLL-2 cells. Results are expressed as arithmetic mean ± SD of the mean. Statistical analysis was performed by Student’s t test for HA-Cot CTLL-2 or HA-trunc-Cot CTLL-2 vs HA CTLL-2 samples (*, p < 0.05; **, p < 0.02).

 
To determine the implication of Cot kinase in the cell cycle progression of T lymphocytes, cell cycle analysis of the different CTLL-2 lines was performed. Exponentially growing HA-Cot CTLL-2, HA-trunc-Cot CTLL-2 displayed a slight increase in the percentage of cells in S phase of the cell cycle. This increase is statistically significant in HA-trunc-Cot CTLL-2 cells vs HA CTLL-2 (p < 0.05). The percentage of cells in sub-G1 phase also was significantly diminished in HA-Cot CTLL-2 (p < 0.02), HA-trunc-Cot CTLL-2 (p < 0.02) when compared with HA CTLL-2 cells (Fig. 2GoB).

The withdrawal of IL-2 for 24 h in the HA-Cot CTLL-2, HA-trunc-Cot CTLL-2, or HA CTLL-2 cells resulted in an accumulation of the cells in the G1 phase of the cell cycle (Fig. 3Go, 0 h). The withdrawal of IL-2 for 24 h more to the different G1 phase-synchronized CTLL-2 cells promoted apoptosis independently of whether they express or do not express HA-Cot or HA-trunc-Cot (Fig. 3Go, 24 h).



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  		FIGURE 3. Cell cycle analysis of synchronized HA CTLL-2, HA-Cot CTLL-2, and HA-trunc-Cot CTLL-2 cells. The different CTLL-2 cell lines were synchronized in the G1 phase of the cell cycle (0 h) and stimulated with rIL-2 (0, 1.5, or 20 U/ml) for 24 h. Analysis of the cell cycle was performed with 5–10 x 103 cells. The figures show one representative experiment, and the tables indicate the means of at least four different experiments performed with two different sets of stable HA CTLL-2, HA-Cot CTLL-2, and HA-trunc-Cot CTLL-2 cells. Results are expressed as arithmetic mean ± SD of the mean. Statistical analysis was performed by Student’s t test for HA-Cot CTLL-2 or HA-trunc-Cot CTLL-2 vs HA CTLL-2 samples (*, p < 0.05; **, p < 0.02).

 
Addition of 20 U/ml rIL-2 to G1 phase-synchronized HA CTLL-2, HA-Cot CTLL-2, and HA-trunc-Cot CTLL-2 cells commit them to proceed through the cell cycle, but a different distribution pattern between the different cell lines was observed after 24 h of stimulation. HA-Cot CTLL-2 and HA-trunc-Cot CTLL-2 cells exhibited 2.7 times more cells in S phase than HA CTLL-2 cells (p < 0.02; Fig. 3Go, 24 h, 20 U/ml IL-2) CTLL-2 cells exhibited a similar cell cycle distribution than HA CTLL-2 (data not shown).

Stimulation of G1 phase-synchronized HA CTLL-2 cells with 1.5 U/ml of IL-2 for 24 h exhibits a distribution of 40.5% of the cells in sub-G1 phase, 5.6% in S phase, and 1.3% in G2-M phase. In the same stimulation conditions, the expression of HA-Cot or HA-trunc-Cot in CTLL-2 cells decreased the number of cells in subG1, with the consequent increase of cells in G1, S, and G2-M phases of the cell cycle. The increase of HA-trunc-Cot CTLL-2, and HA-Cot CTLL-2 cells vs HA-CTLL-2 cells in G1 and S phases of the cell cycle is statistically significant with p < 0.02 for HA-trunc-Cot cells and with p < 0.05 for HA-Cot cells. The decrease of HA-trunc-Cot CTLL-2 and HA-Cot CTLL-2 vs HA-CTLL-2 cells in sub-G1 phase of the cell cycle also is statistically significant (p < 0.02; Fig. 3Go, 24 h, 1.5 IL-2 U/ml). CTLL-2 cells exhibited a similar cell cycle distribution than HA CTLL-2 (data not shown).

Regulation of p27kip levels by Cot kinase

IL-2 regulates the expression of proteins involved in antiapoptotic signaling, like bcl-xL (1, 3). An IL-2 dose dependent up-regulation of bcl-xL was observed in G1 phase-synchronized HA CTLL-2 cells after 10 h of stimulation (Fig. 4GoA). The expression of HA-Cot or HA-trunc-Cot in CTLL-2 did not modify its expression levels (Fig. 4GoA). IL-2 also down-regulates the expression of p27kip cyclin-dependent kinase inhibitor that arrest cell cycle progression (8). HA-Cot CTLL-2 and HA-trunc-Cot CTLL-2 cells expressed lower levels of p27kip than HA CTLL-2 cells (Fig. 4GoB). This decrease in the expression of p27kip was already observed in G1 phase-synchronized HA-Cot CTLL-2 and HA-trunc-Cot CTLL-2 cells. Stimulation of the CTLL-2 cell lines with 1.5 U/ml of IL-2 further decreased its expression. When HA-Cot CTLL-2 or HA-trunc-Cot CTLL-2 cells were stimulated with 20 U/ml of IL-2, p27kip could not be detected (Fig. 4GoB).



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  		FIGURE 4. Expression of bcl-xL and p27kip in HA CTLL-2, HA-Cot CTLL-2, and HA-trunc-Cot CTLL-2 cells. The different G1 phase-synchronized CTLL-2 cell lines (0 h) were stimulated with rIL-2 (1.5 or 20 U/ml) for 10 h, and the expression of bcl-xL (A) and p27kip (B) were analyzed by Western blot analysis with specific Abs. Similar results were observed with two different sets of stable HA CTLL-2, HA-Cot CTLL-2 and HA-trunc-Cot CTLL-2 cells.

 
Cot kinase induces E2F transcriptional activity

E2F promotes the expression of genes involved in DNA synthesis, and its activity is crucial for the G1 to S phase transition (9, 10). Therefore, we next decided to analyze whether Cot kinase enhanced E2F activity. The E2A Luc construct or the mutated version of E2A Luc construct ((mt) E2A Luc) were transiently cotransfected together with pEF-BOS, pEF-BOS trunc-Cot, or pEF-BOS Cot, and Luc activity was measured. Cot or trunc-Cot transiently transfected cells exhibited a threefold higher Luc activity than the CTLL-2 cells expressing only the E2A Luc construct (Fig. 5Go). Cot kinase was not able to induce Luc activity when cotransfected with the mutated form of E2A Luc (Fig. 5Go). Cot kinase also induced E2F activity in COS fibroblasts cells (data not shown), indicating that the E2F up-regulation by Cot kinase is not specific for T cells. Incubation of the Cot or trunc-Cot transiently transfected CTLL-2 cells with 20 U/ml IL-2 also increased the E2F activity (Fig. 5Go). PI3-kinase, implicated in cell survival (for a review see Ref. 31), regulates the E2F activity in T lymphocytes as well as in other cell systems (9, 32). In agreement with these data, expression of the constitutively activated PI3-kinase p110 CAAX in CTLL-2 also increased E2F activity (Fig. 6GoA). PI3-kinase activated E2F transcriptional activity to a similar extent as COT kinase, but cotransfection of both kinases together with the E2A Luc construct increased Luc activity by ~60- to 70-fold (Fig. 6GoA). This synergism in E2F activation could indicate that both kinases regulate E2F through different signal transduction pathways. To test this hypothesis, E2F activation by Cot kinase or by p110 CAAX was measured in the presence of wortmannin or MEK inhibitor. Cot kinase activation of E2F activity was independent of the addition of 1 µM of wortmannin to the incubation medium. The same dose of wortmannin inhibited the PI3-kinase-induced E2A transcriptional activation by 64% (Fig. 6GoB). MEK inhibitor, added at a concentration of 10 µM, was able to reduce E2A Luc activity by 70% when cotransfected with Cot kinase and did not reduce E2F activity when stimulated with p110 CAAX (Fig. 6GoB). Coexpression of a Cot kinase-deficient mutant together with E2A Luc diminished by 55–60% the E2F activity in the presence of IL-2 and did not change E2F activation when cotransfected with p100 CAAX (Fig. 6GoC).



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  		FIGURE 5. E2F activation by Cot and trunc-Cot in CTLL-2 cells. CTLL-2 cells were cotransfected with E2A Luc construct (5 µg/ml) or (mt) E2A Luc construct (5 µg/ml) together with 30 µg/ml of pEF-BOS Cot, pEF-BOS trunc-Cot, or pEF-BOS. After stimulation or not with 20 U/ml IL-2 for 36 h, Luc activity was measured. The graph shows the mean ± SD of three experiments performed in triplicate.

 


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  		FIGURE 6. Cot kinase synergizes with PI3 kinase in the E2F activation. A, CTLL-2 were cotransfected with E2A Luc construct (5 µg/ml) together with pEF-BOS COT (30 µg/ml), or pSG5 p110 CAAX (30 µg/ml), or pEF-BOS (30 µg/ml), or together with pEF-BOS Cot (15 µg/ml) and pSG5 p110 CAAX (15 µg/ml), and Luc activity was measured after 36 h of incubation. The graph shows the fold induction of three different experiments performed in duplicate ± SD, giving a value of 1 to cells electroporated with E2A Luc construct and pEF-BOS. B, The E2A Luc construct was cotransfected with pSG5 p110 CAAX or with pEF-BOS Cot in CTLL-2 for 36 h, and Luc activity was measured. Twenty hours after transfections, the incubation medium was replaced to avoid the decrease in the concentrations of wortmannin (1 µM) and for MEK inhibitor (10 µM) due to break down. Cot kinase or p110 CAAX-transfected cells without addition of inhibitors were given a Luc activity of 100%. The graph shows the mean ± SD of three different experiments performed in duplicate. C, CTLL-2 cells were cotransfected with E2A Luc construct (5 µg/ml) together with pSG5 p110 CAAX (15 µg/ml), or pEF-BOS (15 µg/ml) and pEF-BOS inac-Cot (15 µg/ml) or pEF-BOS (15 µg/ml). Cells not transfected with pSG5 p110 CAAX were incubated with 20 U/ml of IL-2. Luc activity was measured after 36 h of incubation. The value of 100% of Luc activity are given to the cells transfected without pEF-BOS inac-Cot. The graph shows the mean ± SD of three different experiments performed in duplicate.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The increase of COT/Tpl-2 kinase mRNA levels during G0 to G1 phase transition in T lymphocytes suggested an implication of Cot/Tpl-2 kinase later on in the cell cycle (17, 27). The biological system used to analyze an implication of Cot kinase in G1 phase progression was CTLL-2 cells, which need IL-2 to progress in the cell cycle. In the absence of this cytokine, these cells remain in the G1 phase, and if the IL-2 default persists, CTLL-2 cells undergo apoptosis. Here, we demonstrate that overexpression of Cot kinase contributes to G1 phase progression of T lymphocytes. The notion that Cot kinase plays a physiological role in G1 to S phase transition is supported by the finding that a kinase-deficient mutant of Cot kinase partially blocks IL-2-induced DNA synthesis. The finding that Cot kinase-deficient mutant more efficiently inhibits thymidine incorporation at high doses of IL-2 could indicate that the implication of the different intracellular signal pathways involved in cell cycle progression of T lymphocytes varies with IL-2 concentration.

We have reported previously that Cot kinase, in conjunction with signals that increase cytoplasmatic calcium concentration, controls IL-2 secretion in Jurkat T cells (25), indicating that Cot kinase contributes to the G1 to S phase transition of T lymphocytes by regulating the secretion of this cytokine.

The possibility that regulation of both p27kip levels and E2F activity by Cot kinase is IL-2-secretion-mediated should be excluded, because HA-COT-CTLL-2 and HA-trunc-Cot-CTLL-2 do not produce IL-2 (data not shown). In fact, the withdrawal of exogenous IL-2 promotes apoptosis in these cell lines. In contrast, reduction of DNA synthesis by the blockage of endogenous Cot activity is not abolished by the incubation of these cells with high concentrations of IL-2. Furthermore, IL-2, but not Cot kinase, regulates bcl-xL levels.

Cot kinase has been classified as an MAP3K that activates the ERK1, c-Jun N-terminal kinase, p38{gamma} (ERK6), and ERK5 signal transduction pathways (19, 20, 21, 22) and up-regulates the activity of NF-{kappa}B and AP-1 transcription factors (21, 24, 33, 34). The contribution of the different signal pathways regulated by Cot kinase in promoting E2F activation has not been elucidated. However, the fact that Cot kinase-induced E2F activation is partially inhibited by the MEK inhibitor indicates that activation of MEK/ERK-1 signal transduction pathway by Cot kinase is implicated. In this regard, RAF kinase, which activates the MEK/ERK1 signal pathway, also synergizes with the PI3 kinase pathway in E2F activation in NIH 3T3 cells (35); both signal pathways also synergize in NIH 3T3 cell transformation (32).

Truncation of the last coding exon of the human, rat, and mouse COT/Tpl-2 gene provides transformation (13, 14, 17, 33, 36), and higher expression of the normal gene also is capable of conferring the transformed phenotype (15, 30). Recent data gave evidence for increased Cot mRNA levels in 40% of human breast cancer, and a role of Cot kinase in the development of this tumor has been proposed (16). Regulation of the p27kip protein levels could be one of the mechanisms by which the Cot gene is implicated in tumorigenesis. In fact, the protein level of p27kip is an excellent prognostic indicator of survival of human cancer patients with tumors in breast, colon, ovary ,and lung (37).

The increase of Cot/Tpl-2 mRNAs levels kinase during G0 to G1 phase transition in T lymphocytes suggested an implication of Cot/Tpl-2 kinase later on in the cell cycle (17, 27). The data reported here give evidence for Cot kinase contribution to the G1 phase progression through the cell cycle.


   Acknowledgments
 
We thank Abelardo López Rivas and Alberto Alvarez for their help with the cell cycle analysis; Isabel Mérida, Victor Calvo, and Abelardo López Rivas for critical reading of the manuscript, and Joaquin Pérez for technical assistance. We also thank Dr. J. Downward for the pSG5 p110 CAAX construct and Dr. Lam for the pGL2-3xwt Luc and pGL2-3xmt Luc constructs.


   Footnotes
 
1 This work was supported by Plan Nacional SAF99-0067, Comunidad de Madrid and Europharma. A.V. is the recipient of a fellowship from the Comunidad de Madrid. Back

2 Address correspondence and reprint requests to Dr. Susana Alemany, Instituto de Investigaciones Biomédicas, Consejo Superior de Investigaciones Cientificas, Arturo Duperier 4, 28029 Madrid, Spain. Back

3 Abbreviations used in this paper: PI3-kinase, phosphatidylinositol 3-kinase; MAP, mitogen-activated protein; MAP3K, MAP kinase kinase kinase; ERK, extracellular signal-regulated kinase; MEK, MAP-ERK kinase; GFP, green fluorescent protein; Luc, luciferase; HA, hemagglutinin. Back

Received for publication September 25, 2000. Accepted for publication March 12, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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