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. 2003 Oct;23(20):7377-90.
doi: 10.1128/MCB.23.20.7377-7390.2003.

The COOH-terminal domain of wild-type Cot regulates its stability and kinase specific activity

Maria Luisa Gándara  1 , Pilar López, Raquel Hernando, José G Castaño, Susana Alemany
Affiliations

The COOH-terminal domain of wild-type Cot regulates its stability and kinase specific activity

Maria Luisa Gándara et al. Mol Cell Biol. 2003 Oct.

Abstract

Cot, initially identified as an oncogene in a truncated form, is a mitogen-activated protein kinase kinase kinase implicated in cellular activation and proliferation. Here, we show that this truncation of Cot results in a 10-fold increase in its overall kinase activity through two different mechanisms. Truncated Cot protein exhibits a lower turnover rate (half-life, 95 min) than wild-type Cot (half-life, 35 min). The degradation of wild-type and truncated Cot can be specifically inhibited by proteasome inhibitors in situ. The 20S proteasome also degrades wild-type Cot more efficiently than the truncated protein. Furthermore, the amino acid 435 to 457 region within the wild-type Cot COOH-terminal domain confers instability when transferred to the yellow fluorescent protein and targets this fusion protein to degradation via the proteasome. On the other hand, the kinase specific activity of wild-type Cot is 3.8-fold lower than that of truncated Cot, and it appears that the last 43 amino acids of the wild-type Cot COOH-terminal domain are those responsible for this inhibition of kinase activity. In conclusion, these data demonstrate that the oncogenic activity of truncated Cot is the result of its prolonged half-life and its higher kinase specific activity with respect to wild-type Cot.

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Figures

FIG. 1.
FIG. 1.
Expression of wt Cot and trunc-Cot in HEK293 transfected cells. (A and B) Cells were transfected with pcINEO HA-wt Cot (10 μg), pcINEO HA-trunc-Cot (10 μg), or pcINEO HA (10 μg), and 24 h after transfection wt Cot and trunc-Cot protein and mRNA levels were analyzed. wt Cot and trunc-Cot protein levels were determined by Western blot analysis using the anti-Cot monoclonal antibody (Calbiochem) (A). The same PVDF membrane was probed with an anti-PDI antibody as a control of protein loading. The data shown correspond to 30 PCR cycles, and the amount of product amplified (62 to 373 nucleotides [nt] of HA-wt-Cot, 62 to 373 nt of HA-trunc-Cot, and 1,292 to 1,479 nt of β-actin) was proportional to the abundance of the starting material (B). (C) Quantification of the expression of wt Cot and trunc-Cot in total extracts at different times after transfection (12, 24, 36, and 44 h), as well as in soluble and insoluble 1% NP-40 fractions of HEK293 cells transfected with pcINEO HA-wt Cot (10 μg) or pcINEO HA-trunc-Cot (10 μg). wt Cot and trunc-Cot were detected by Western blot analysis with anti-Cot antibodies either generated in the laboratory (6) or commercially available (Calbiochem). As a control of protein loading, membranes were also probed with an anti-PDI antibody. The pixel intensities of the different wt Cot and trunc-Cot bands were quantified with GelDoc 200 (Bio-Rad) and corrected to the pixel intensity of PDI. The graphs shows the mean ± standard deviation of the intensities of the different wt Cot and trunc Cot bands from three different experiments, assuming a value of 1 for the intensity obtained for wt Cot 24 h after transfection. ♦, wt Cot; ▪, trunc-Cot.
FIG. 2.
FIG. 2.
Degradation of wt Cot and trunc-Cot in HEK293 transfected cells. Immunoprecipitation of 35S-labeled wt Cot and trunc-Cot from HEK293 cells transfected with 10 μg of pcINEO HA-wt Cot or 10 μg of pcINEO HA-trunc-Cot after a 20-min pulse-label and a chase for the times indicated (20, 40, 60, 120, and 240 min). The proteins immunoprecipitated were resolved by SDS-PAGE and visualized by autoradiography. The radioactivity incorporated was quantified using an Instant Imager. The figure shows one experiment, and the graph shows the mean ± standard deviation of the radioactivity incorporated in four experiments. The radioactivity incorporated into wt Cot or trunc-Cot after the 20-min pulse was considered 100%. The degradation of both proteins correlated with one-phase exponential equations fit to each data set, with R2 = 0.975 for wt Cot and R2 = 0.9736 for trunc-Cot. The graph also shows a representation of the decay as a log plot. ▴, wt Cot; ▪, trunc-Cot.
FIG. 3.
FIG. 3.
Levels of wt Cot and trunc-Cot expression after treatment of HEK293 transfected cells with different protease inhibitors. (A) Total extracts of HEK293 cells 14.5 h after transfection with 10 μg of pcINEO HA-wt Cot or 10 μg of pcINEO HA-trunc-Cot were further incubated for 7.5 h in the presence of different protease inhibitors (10 μM Z-AAF-CMK, a tripeptidyl peptidase inhibitor; 20 μM LLMet, a calpain II inhibitor; 20 μM MG132, a proteasome inhibitor; 20 μM phosporamidon, a metalloprotease inhibitor; 1 mM prefabloc AEBSF, a serine protease inhibitor; or 20 μM E64, a cysteine protease inhibitor) as indicated in Materials and Methods. Total extracts were analyzed by Western blotting using anti-Cot (Calbiochem) and anti-PDI antibodies. The figure shows representative results of one of the three experiments performed. (B) Cells transfected as indicated for panel A and incubated in the presence or absence of 10 μM lactacystin were lysed in lysis buffer, and both the 1% NP-40-soluble and -insoluble fractions were analyzed by Western blotting with an anti-Cot antibody (Calbiochem). (C) Total extracts of cells 14.5 h after transfection with 5 μg of pcDNA3.1 HA-wt Cot or 5 μg of pcDNA3.1 HA-trunc-Cot together with 5 μg of pCMV-His-ubiquitin, which were further incubated for 7.5 h in the presence or absence of 10 μM lactacystin, were analyzed by Western blotting with anti-Cot antibody (Calbiochem) or with antiubiquitin antibody. Both wt Cot and trunc-Cot were immunoprecipitated from the same transfected cells and analyzed by Western blotting with antiubiquitin antibody and anti-Cot antibody. The figure shows representative results of one of the three experiments performed. (D) Accumulation of newly synthesized wt Cot and trunc-Cot after lactacystin (10 μM) incubation. 35S-labeled wt Cot and trunc-Cot were immunoprecipitated from HEK293 cells transfected with 10 μg of pcINEO HA-wt Cot or 10 μg of pcINEO HA-trunc-Cot after a 20-min pulse and 3-h chase (wt Cot) or 4-h chase (trunc-Cot) in the presence or absence of 10 μM lactacystin. The proteins were resolved by SDS-PAGE and visualized by autoradiography. The figure shows representative results of one of the three experiments performed.
FIG. 4.
FIG. 4.
Degradation of wt Cot and trunc-Cot by the 20S proteasome. Transcribed and translated wt Cot and trunc-Cot were incubated for the indicated times with 20S proteasome in the presence or absence of 10 μM lactacystin. The proteins were then resolved by SDS-PAGE and visualized by autoradiography, and the incorporated radioactivity was quantified by using an Instant Imager. The figure shows one representative experiment, and the graph shows the mean ± standard deviation of two experiments performed in duplicate.
FIG. 5.
FIG. 5.
The C terminal of wt Cot triggers its degradation by the proteasome. (A) HEK293 cells 14.5 h after transfection with 10 μg of pEYFP-Cot390-467 or 10 μg of pEYFP or cotransfected with 5 μg of pEYFP-Cot390-467 together with 5 μg of pEYFP were further incubated with 100 μg of cycloheximide/ml for different times (0, 30, 60, 120, 240, and 480 min). The total extracts were Western blotted and probed with the anti-EYFP antibody. The figure shows one of two experiments performed in duplicate. (B) Total extracts of cells 14.5 h after transfection with 5 μg of pEYFP or 5 μg of pEYFP-Cot390-467 together with 5 μg of pCMV-His-ubiquitin and further incubated for 7.5 h in the presence or absence of 10 μM lactacystin. The extracts were Western blotted and probed with anti-EYFP. Similar results were obtained in two different experiments performed in duplicate. (C) Recombinant GST-Cot390-467 was subjected to in vitro degradation by the 20S proteasome for 20 and 40 min. GST-Cot390-467 was also incubated with 20S proteasome in the presence of 20 μM MG132 or 10 μM lactacystin. The different samples were resolved by SDS-PAGE and Western blotted to be probed with an anti-C-terminal Cot antibody. Similar results were obtained in three experiments performed in duplicate. Recombinant GST was subjected to in vitro degradation for 20, 40, 60, and 80 min by the 20S proteasome. The different samples were resolved by SDS-PAGE followed by Western blotting, which was probed with anti-GST antibody. Similar results were obtained in two experiments performed in duplicate.
FIG. 6.
FIG. 6.
The amino acid 435 to 457 region within the C terminal of wt Cot confers susceptibility to proteasome degradation and is independent of PKB activity. (A) HEK293 cells were cotransfected with 5 μg of EYFP-Cot390-467 together with 15 μg of HA-PKB-DD or empty vector, and 14.5 h after transfection the cells were further incubated with 100 μg of cycloheximide/ml in the presence or absence of 10 μM lactacystin as described in Materials and Methods. The figure is representative of the three experiments performed. (B) Two hours after the transfection of HEK293 cells with 10 μg of EYFP-Cot390-467, the cells were incubated with 50 μM LY 294002 and the medium was then supplemented with 100 μg of cycloheximide/ml and 50 μM LY 294002, with and without 10 μM lactacystin, for a further 8 h. The figure shows representative results of the three experiments performed. (C) HEK293 cells were transfected with 10 μg of pEYFP-Cot390-467, pEYFP-Cot390-457, pEYFP-Cot390-446, pEYFP-Cot390-435, or pEYFP-Cot390-424, and after 14.5 h cells were treated as described in the legend for Fig. 5B. Total extracts were Western blotted and probed with anti-EYFP and anti-PDI antibodies; the figure shows representative results of one of the three experiments. (D) HEK293 cells transfected for 14.5 h with pEYFP-Cot414-457, pEYFP-Cot426-457, pEYFP-Cot435-457, and pEYFP-Cot445-450 were treated as explained above for panel C. The figure shows one of the two experiments performed in duplicate.
FIG. 7.
FIG. 7.
trunc-Cot exhibits a higher transformation capacity and kinase activity than wt Cot. (A) Western blots of P-Erk in total extracts of HEK293 cells 20 h after transfecting with pcINEO HA-wt Cot (10 μg), pcINEO HA-trunc-Cot (10 μg), or pcINEO HA (10 μg). As a control of protein loaded, total Erk-2 levels were tested. The expression of wt Cot and trunc-Cot was also determined with the anti-Cot antibody (Calbiochem). Similar results were obtained in three different experiments. (B) Different amounts of pEF-BOS wt Cot, pEF-BOS trunc-Cot, or pEF-BOS (0.01, 0.03, 0.1, or 0.3 μg) together with 0.15 μg of −73 pcol-Luc were cotransfected in HEK293 cells, and Luc activity was measured 20 h after transfection. The graph shows the means ± standard deviations of three different experiments performed in duplicate. (C) NIH 3T3 cells were transfected with 10 μg of pcINEO HA-wt Cot, pcINEO HA-trunc-Cot, or pcINEO HA. Cells were cultured for 2 weeks with 10% filtered calf serum, fixed, and then stained as described in Materials and Methods. The figure shows a representative experiment of the three performed. (D) Cot activity per milligram of protein extract and Cot specific activity of wt Cot and trunc-Cot immunoprecipitated from HEK293 cells transfected as described for panel A. Cot activity indicates the milliunits of Cot kinase activity corresponding to 1 mg of transfected extract, with 1 U being 1 nmol of phosphate incorporated into myelin basic protein in 1 min. The specific activity indicates the milliunits of Cot kinase activity divided by the pixel intensity obtained from the scanned Western blots of the immunoprecipitated wt Cot or trunc-Cot, with a value of 1 being attributed to the specific activity of wt Cot divided by the pixel intensity of immunoprecipitated wt Cot. The graphs show the means ± standard deviations of three different experiments performed in triplicate.
FIG. 8.
FIG. 8.
Identification of the region within the C-terminal domain of wt Cot that inhibits Cot specific activity. (A) HEK293 cells were cotransfected with 0.15 μg of pcINEO HA-wt Cot(467), pcINEO HA-Cot457, pcINEO HA-Cot446, pcINEO HA-Cot435, pcINEO HA-Cot424, or pcINEO HA-trunc-Cot together with 0.15 μg of −73 pcol-Luc, and Luc activity was measured 20 h after transfection. The graph shows the means ± standard deviations of two different experiments performed in duplicate. (B) HEK293 cells were transfected with 10 μg of pcINEO HA-wt Cot(467), pcINEO HA-Cot457, pcINEO HA-Cot446, pcINEO HA-Cot435, or pcINEO HA-Cot424. Western blotting of the total extracts of HEK293 cells 20 h after transfection with the different pcINEO Cot constructs was performed as described in the legend for Fig. 1A. The figure shows one representative result of the three experiments performed. As a control of protein loaded, total PDI levels were also assessed. Cot activity and Cot specific activity of the different Cot constructs [wt Cot(467), Cot 457, Cot446, Cot435, and Cot424] were determined as described in the legend for Fig. 7D. The graphs also show the means of three experiments ± standard deviations.
FIG. 9.
FIG. 9.
Representation of the different regions within C-terminal wt Cot. The analysis of the putative PEST sequences was performed with the pestfind program located at http://www.at.embnet.org/embnet/tools/bio/PESTfind/.
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References

    1. Alessi, D. R., P. Cohen, A. Ashworth, S. Cowley, S. J. Leever, and C. J. Marshall. 1995. Assay and expression of mitogen-activated protein kinase, Map kinase kinase, and RAF. Methods Enzymol. 255:279-290. - PubMed
    1. Aoki, M., T. Akiyama, J. Miyoshi, and K. Toyoshima. 1991. Identification and characterization of protein products of the cot oncogene with serine kinase activity. Oncogene 6:1515-1519. - PubMed
    1. Arribas, J., P. Arizti, and J. G. Castano. 1994. Antibodies against the C2 COOH-terminal region discriminate the active and latent forms of the multicatalytic proteinase complex. J. Biol. Chem. 269:12858-12864. - PubMed
    1. Arribas, J., and J. G. Castano. 1993. A comparative study of the chymotrypsin-like activity of the rat liver multicatalytic proteinase and the ClpP from Escherichia coli. J. Biol. Chem. 268:21165-21171. - PubMed
    1. Arribas, J., and J. G. Castano. 1990. Kinetic studies of the differential effect of detergents on the peptidase activities of the multicatalytic proteinase from rat liver. J. Biol. Chem. 265:13969-13973. - PubMed

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