COT Kinase Proto-oncogene Expression in T Cells

COT/Tpl-2 proto-oncogene encodes a serine/threonine kinase implicated in cellular activation. In this study we have identified the human COT gene promoter region and three different human COT transcripts. These transcripts, with the same initiation site, display heterogeneity in their 5′ untranslated regions and in their subcellular localization. Activation of Jurkat T cells with either calcium ionophore A23187 or αCD3 and a phorbol ester increases the levels of the different COT transcripts. Analysis of the 5′ flanking region of the human COT gene reveals a unique transcription initiation site and a TATA element 20 nucleotides upstream. Transient expression of COT promoter constructs containing a reporter gene indicates that the transcriptional activity of the 5′ flanking region of the COT gene is regulated by T cell-activating signals. Cotransfection of a dominant negative version of SEK-2 abolishes the inducible transcriptional activity ofCOT promoter, indicating that the inducible expression of the COT gene by T cell activating signals is mediated by the JNK/SAPK signal transduction pathway. All these data indicate stringent regulation of COT kinase proto-oncogene expression.

Several COT/Tpl-2 cDNAs comprising the complete coding sequence and 3Ј UTR have been reported: two different human cDNAs (GenBank TM accession numbers Z14138 and D14497) (14,15), two rat cDNAs (GenBank TM accession numbers M94454 and L15358) (16), and one murine COT cDNA (Gen-Bank TM accession number D13759) (17). Identities between human, rat, and murine COT cDNAs in their coding sequences and 3Ј UTRs are 85 and 75%, respectively. The 5Ј UTR of the human, rat, and murine COT cDNAs did not reveal any homology, with the exception of the 23 nt upstream from the first COT/Tpl-2 ATG codon.
The human COT gene is a single copy locus (14,18) localized on the short arm of chromosome 10 at band p11.2 (14). Ohara et al. (17) proposed that the human COT gene contains nine exons, of which the last seven are coding exons. COT kinase was first identified in a truncated form in transformed foci induced in SHOK cells by transfection of the genomic DNA of a human thyroid carcinoma cell line (18,19). This rearrangement occurs in the penultimate coding exon and provides transformation capacity (15). The rat homologue of the human COT gene (Tpl-2) was identified as an oncogene associated with the progression of Moloney murine leukemia virus-induced T cell lymphomas in rats (4,16). As with the human and murine homologues, the disruption of the last coding exon of Tpl-2 by insertion of the Moloney murine leukemia virus enhances mRNA levels and appears to unmask the oncogenic potential of the protein (4,15,20,21). It has been suggested recently that an amplification of the genomic locus of the COT gene plays a role in human breast cancer (22).
In this paper we have studied the expression of the human COT gene in T cells. We have identified three different human COT mRNAs and the 5Ј region flanking the transcription initiation site of the COT gene. We also provide evidence that T cell activation up-regulates the levels of the three COT kinase mRNAs and increases the transcriptional activity of the human COT gene promoter through the JNK/SAPK signal transduction pathway. * This work was supported by Plan Nacional, Comunidad de Madrid, and Europharma. 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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM /EBI Data Bank with accession number(s) AF133211.
DNA Isolation and COT Promoter-Luciferase Reporter Vector Constructs-Genomic DNA was obtained as described previously (23). A 6.1-kb DNA fragment containing the 5Ј region flanking the translation initiation site of COT kinase was obtained with the human genomic DNA PromoterFinder TM DNA walking kit (CLONTECH), using two specific reverse primers deduced from the sequence of the first COT coding exon. Different DNA fragments of the 5Ј flanking region of the COT gene were generated by PCR with different direct primers (Ϫ8D, Ϫ7D, Ϫ6D, Ϫ5D, Ϫ4D, Ϫ3D, Ϫ2D, and Ϫ1D) and PR as reverse primer. The Ϫ778⌬ PCR product was performed with Ϫ7D and upTATA primers. Purified PCR products were cloned in pMOS Blue T-vector (Amersham Pharmacia Biotech). Single clones were selected, and their sequences were compared with the original template. From these constructs, the KpnI/HindIII fragments were cloned in the pGL3-Basic Luc-reporter vector (Promega). Sequencing, using specific oligonucleotides and Sequenase (U. S. Biochemical Corp.), was performed by the Sanger method (24). Gene Jockey II, DNAstrider 1.1, MacPattern Folder, and Blast programs were used to analyze the DNA sequences.
To perform the polysome fractionation, 8 ϫ 10 7 cells were lysed as described previously (25) and centrifuged at 10,000 ϫ g for 10 min at 4°C. The supernatant was spun in a 10 -50% linear sucrose gradient buffered with 20 mM HEPES (pH 7.3), 250 mM KCl, 20 mM MgCl 2 , 2 mM dithiothreitol, and 500 g/ml heparin at 36,000 rpm for 2 h at 4°C in a SW 41 rotor (Beckman). Fractions of 1 ml were collected, and ethanol was precipitated. RNA was extracted from the pellets by using the SV total RNA isolation system kit (Promega).
Northern Blot and Dot Blot Analysis-By using Ultraspec (Biotecx Laboratories), 20 g of total RNA was extracted from Jurkat cells stimulated with 50 ng/ml PDBu, 0.25 M calcium ionophore, and 100 nM okadaic acid for different times. RNA was electrophoresed and blotted onto a nylon membrane (Nytran, NY 13N, Renner GmbH). Filters were hybridized with a random primer-labeled cDNA (Ͼ10 9 cpm/g) comprising the entire human COT open reading frame (14). Membranes were exposed to x-ray film for 10 days at Ϫ70°C.
Multiple tissue human poly(A) ϩ RNA Northern blot (CLONTECH) was hybridized with different probes as described above. Stripping was performed by boiling the membrane for 30 min in 0.1 ϫ SSC and 0.5% SDS. Control autoradiographs were performed after each stripping.
Dot blots performed with total RNA and RNA isolated from different subcellular fractions (25) were hybridized with the COT coding probe, with probe C (3837-4646 nt), and with the COT promoter probe (Ϫ778 to Ϫ30 nt). The autoradiograph was exposed for 7 days at Ϫ70°C.
RT-PCR and Primer Extension Assays-Two g of total RNA treated with 2.5 units of DNase I (Life Technologies, Inc.) as in Ref. 9 were subjected to the RT reaction (9). One l of this reaction was used for PCR analysis. Control samples treated with DNase I and not exposed to RT were also subjected to PCR. To perform the RT-PCRs from leukocyte mRNA, 1 l of human leukocyte Marathon-ready cDNA library (CLON-TECH) was used. Controls without a template, with genomic DNA as a template, and with one single primer were performed.
Seven pmol of the 5Ј-end-labeled oligonucleotide PE, complementary to nt 123-140 of the three COT transcripts, was annealed to 7 g of poly(A) ϩ RNA from PDBu-and calcium ionophore-stimulated Jurkat cells, and a RT reaction was carried out. After synthesis, nucleic acids were precipitated, and reaction products were analyzed on 6% polyacrylamide gels containing 8 M urea.
RNase Protection and S1 Nuclease Protection Assays-A 434-nt labeled RNA probe (50 -80 ϫ 10 4 cpm/fmol), of which 409 nt were complementary to nt 5820 -6229 of COT-1 that corresponds to a region of the coding sequence, was obtained using the Maxiscript kit (Ambion). RNase A protection assay was performed with the radioimmune precipitation buffer II kit (Ambion). Dried gels were exposed to x-ray films at Ϫ70°C for 2 days. For the S1 nuclease analysis a radiolabeled DNA probe (probe D (589 -2784 nt), 10 -30 ϫ 10 5 cpm/fmol) was obtained using the Prime-A-Probe kit (Ambion) according to the manufacturer's instructions, except that 0.9% alkaline agarose gels were used to purify the labeled probe. Nuclease S1 protection assay was performed with the Multi-NPA kit (Ambion). Agarose gels (0.9%) were exposed to x-ray films at Ϫ70°C for 4 days.

Identification of Three Human COT Transcripts-Hybridiza-
tion of a leukocyte poly(A) ϩ RNA Northern blot with a coding COT probe yielded two hybridization signals at 3.0 and 7.3 kb (Fig. 1A). To investigate whether the occurrence of these transcripts was due to the different length of the 5Ј UTR, we cloned and sequenced a 6.1-kb DNA genomic fragment containing the 5Ј flanking region of the COT translation initiation site. Several probes from this region were generated by PCR (Fig. 1A). Analysis of the poly(A) ϩ RNA Northern blot with these probes revealed a hybridization signal only at 7.3 kb (Fig. 1A), indicating that this COT mRNA species has a large 5Ј UTR.
To determine the sequence of the 5Ј UTR of the different COT mRNAs, RT-PCR analysis was performed. The direct and reverse primers used were deduced from the sequence of the 6.1-kb genomic fragment containing the 5Ј flanking region of the COT translation initiation site. PCR of overlapping fragments was performed using as a template reverse transcribed mRNA of Jurkat cells. Different PCRs were performed with the combinations of each direct primer and all the different 3Јlocated reverse primers. Control samples treated with DNase I and not exposed to RT were also subjected to PCR. Controls without a template, with genomic DNA as a template, and with one single primer were also performed. The different PCR products obtained (Fig. 1B) were analyzed by restriction mapping and by sequencing (data not shown). The same overlapping PCR products were obtained when human leukocyte cDNA was used as a template (data not shown). This analysis revealed that the COT gene is transcribed with three different 5Ј UTRs. The transcription start site of these three 5Ј UTRs was delimited to the same 30-nt region by PCR analysis, using as a template cDNA from Jurkat cells as well as human leukocyte cDNA (data not shown). (See "Determination of the Transcription Start Site of the Human COT Gene" for the location of the exact start transcription site.) To establish the 5Ј UTR of COT-1 by a method other than RT-PCR, S1 nuclease analysis was performed. A DNA probe (probe D) complementary to the 589 -2784-nt sequence of the 5Ј UTR of COT-1 was hybridized with RNA obtained from Jurkat cells and incubated with nuclease S1 (Fig. 1C). This probe contains the entire sequence of probe B and is extended to the DNA sequence of probe A. The intron/exon boundaries of the three different 5Ј UTRs of COT transcripts are shown in Table I.
We also investigated the possibility of alternative splicing in the coding sequence and 3Ј UTR of COT transcripts. RT-PCR analysis revealed no alternative splicing in these regions. The coding sequence and 3Ј UTR region of COT transcripts have a size of 2.5 kb (data not shown). Considering the length of the 5Ј UTR of the three COT transcripts, the 7.3-kb COT mRNA species detected in the leukocyte poly(A) ϩ RNA Northern blot (Fig. 1A) should correspond to COT-1, and the 3.0-kb signal should correspond to COT-2 and COT-3. The three COT transcripts were detected by RT-PCR in all human tissues tested (Fig. 1D), indicating that none of the different COT transcripts is tissue-specific, although the relative amounts seem to vary between the different tissues. When the PCRs were performed as described in the legend to Fig. 1D, the ratio of COT-2 to COT-1 oscillated between 0.5 for liver or pancreas and 1.6 for muscle. The ratio of COT-3 to COT-2 varied from 9.8 for lung to 1.2 for pancreas.
Determination of the Transcription Start Site of the Human COT Gene-The transcription start site of the COT gene was delimited by RT-PCR analysis to a 30-nt region (data not shown). The exact transcription start site of the COT gene was determined by primer extension on poly(A) ϩ RNA from stimulated Jurkat cells with a primer complementary to primer 1D (PE primer). This sequence is complementary to the three COT transcripts. As shown in Fig. 2, a single product corresponding to a 140-base extended fragment was detected. The first transcribed base has been designated ϩ1, to facilitate numbering of the different COT transcripts. Sequence analysis revealed a putative TATA box located at position Ϫ20 nt (Figs. 2 and 3A), which is in agreement with the preferred position occupied by this element in a typical eukaryotic promoter (26).
To confirm that the DNA region 5Ј flanking the defined transcription start site of the human COT gene has promoter activity, transient expression experiments with the pGL3-Luc basic vector linked to different fragments of this DNA region were carried out. Jurkat cells were transfected with pGL3, pGL3-778 (5Ј-3Ј), and pGL3-778⌬ as well as with the pGL3-778 (3Ј-5Ј) construct, and luciferase activity was measured (Fig.  3B). The pGL3-778 (5Ј-3Ј) construct, containing nt Ϫ778 to ϩ115 of the COT gene, exhibited a transcriptional activity 20-fold higher than that of the empty pGL3 vector. The Ϫ30 to ϩ115-nt region is essential in maintaining this increase, because deletion of the Ϫ30 to ϩ115-nt fragment, in the pGL3-778⌬ construct, resulted in transcriptional activity similar to pGL3. No promoter activity over background levels was detected with the pGL3-778 (3Ј-5Ј) construct, indicating that this region contains the transcription start site of the COT gene and not only cis-response elements.   (Fig. 4A).

Regulation of Human COT Promoter Activity by T Cell-Reg
Comparison of the relative promoter activities of the different constructs indicated that the progressive removal of 5Ј sequences up to Ϫ650 did not significantly affect the COT promoter activity in unstimulated cells (Fig. 4B). Deletion of the Ϫ466 to Ϫ650 DNA fragment significantly decreased the transcriptional activity. Further deletion of nt Ϫ340 to Ϫ229 further decreased the activity of the COT promoter (Fig. 4B). However, the pGL3-229 and pGL3-102 constructs still exhibited a transcriptional activity 7-fold higher than vector pGL3 (data not shown).
To determine whether COT promoter activity is regulated by T cell-activating signals, Jurkat cells were transfected with the different COT promoter-derived constructs and stimulated with PDBu (50 ng/ml) and calcium ionophore (0.25 M) or with ␣CD3 (10 g/ml) and PDBu (50 ng/ml). Comparison of the luciferase activities produced by each different plasmid in unstimulated and stimulated cells showed that deletion of the sequences located between positions Ϫ1082 to Ϫ340 did not significantly affect the 3-fold induction relative to the unstimulated activity of each construct. Further removal of the sequences from position Ϫ340 to Ϫ229 abolished the 3-fold induction by these signals (Fig. 4B).
One AP-1 binding site (27,28) is found at position Ϫ327 nt of the 5Ј flanking region of the COT gene (Fig. 3A). The AP-1 transcription factor is up-regulated in T lymphocytes activated with PDBu and calcium ionophore or with ␣CD3 and PDBu, and the JNK/SAPK signal transduction pathway mediates its activation. To determine whether this signal transduction pathway regulates, at least in part, activation of the COT promoter, Jurkat cells were cotransfected with pGL3-340 or pGL3-788 together with the dominant negative form of JNK kinase, DN-SEK-2 (MKK7-KL), that inhibits the activation of c-Jun. Cells were stimulated or not with PDBu (50 ng/ml) and calcium ionophore (0.25 M) or with ␣CD3 (10 g/ml) and PDBu (50 ng/ml). As shown in Fig. 4C, the expression of the DN-SEK-2 abolished the increase of the promoter-driven transcription of the pGL3-340 and pGL3-778 constructs by T-cell activating signals.
To further analyze the signal transduction mechanism by which the COT promoter is activated, transient transfection experiments with the pGL3-340 construct were performed. Addition of PDBu or calcium ionophore by itself did not increase the luciferase activity, indicating that an integration of both signals has to occur to activate COT promoter-driven transcription (Fig. 4D). Transfected cells were also incubated with different inhibitors or activators of protein kinases or protein phosphatases. The transfected cells were incubated with PDBu (50 ng/ml) and calcium ionophore (0.25 M) in the presence or absence of cyclosporin A (100 ng/ml), MEK inhibitor (20 M), HOG inhibitor (20 M), okadaic acid (100 ng/ml), or 8-Br-cAMP (0.5 mM) at doses that have already been reported to regulate the activation of Jurkat T cells (9). Because addition of PDBu and calcium ionophore to Jurkat T cells increases the phosphorylation state of many proteins involved in the signal transduction mechanism, the addition of okadaic acid, an inhibitor of protein phosphatases 1 and 2A, to these activated Jurkat cells could induce a further increase in the phosphorylation state of the proteins. According to the results obtained in Fig. 4D, the addition of okadaic acid did not increase the luciferase activity. Addition of cyclosporin A prior to activation of the cells reduced the luciferase activity, indicating that calcineurin (protein phosphatase 2B) is at least partially involved in the activation of the COT promoter. MEK inhibitor, which blocks the ERK signal pathway, and HOG/p38 mitogen-activated protein kinase inhibitor hardly diminished the stimulatory signal of PDBu and calcium ionophore. Activation of cAMPdependent protein kinase by the addition of 8-Br-cAMP increased the luciferase activity by about 1.7-fold (Fig. 4D).
Up-regulation of COT mRNA Levels-An RNase protection assay was performed to determine whether the increase in the transcriptional activation of the 5Ј flanking region of the human COT gene by T cell activating signals correlates with an increase in COT mRNA levels after T lymphocyte stimulation. A riboprobe from the COT coding sequence was hybridized with RNA isolated from Jurkat cells stimulated with soluble ␣CD3 (10 g/ml) and PDBu (50 ng/ml) for different times. As shown in Fig. 5A, ␣CD3 and PDBu stimulation transiently increased COT mRNA levels (ϳ4-fold). A similar increase in the level of COT transcripts was detected by RT-PCR analysis of mRNA of Jurkat cells stimulated with PDBu and calcium ionophore for different times, using primers that amplified a COT coding sequence fragment. As a control, a 187-bp fragment of ␤-actin was also amplified in each reaction (Fig. 5B).
To distinguish COT-1 from COT-2 and COT-3, a Northern blot with total RNA from Jurkat cells stimulated for different times with PDBu (50 ng/ml), calcium ionophore (0.25 M), and okadaic acid (100 nM) was hybridized with the coding COT probe (Fig. 6A). Whereas a hybridization signal of 3.0 kb, corresponding to COT-2 and COT-3, was detected 3 h after stimulation, the COT-1 message was first detected 6 h after stimulation. In agreement with the Northern blot analysis, COT-1 transcript levels determined by RT-PCR were not increased at 4 h after stimulation of Jurkat cells with the stimuli described above (Fig. 6B). At this time of stimulation, COT-2 and COT-3 levels were increased to an equal extent (Fig. 6B).
Subcellular Distribution of COT Transcripts-Next, we decided to investigate the subcellular distribution of COT-1, COT-2, and COT-3. RNA was isolated from the cytoplasmic fraction and the nuclear fraction of intact Jurkat cells. A dot blot performed with these RNAs was hybridized with a probe specific for the 5Ј UTR of COT-1 (probe C), the COT coding probe, and a probe containing the Ϫ778 to Ϫ30 nt sequence of the COT promoter (Fig. 7A). Comparison of the hybridization signals obtained with the different probes and RNA fractions indicated that the COT-1 transcript is mainly located in the nuclear fraction.
We next decided to study the distribution of the different COT messengers to polysomes. The postmitochondrial supernatant of Jurkat cells was subjected to sucrose gradient fractionation, and RNA was isolated from the different fractions.
The levels of the different COT transcripts were measured by RT-PCR analysis. As shown in Fig. 7B, the fraction of COT-1 located in the cytoplasmic fraction is not associated with polysomes. In addition, only a small fraction of COT-2 was not loaded with ribosomes. The fact that both COT-2 and COT-3 were detected in fractions corresponding to small polysomes indicates a low translation efficiency of these transcripts. As a control of polyribosome-associated mRNA, we assayed the same fractions for ␤-actin messenger (Fig. 7B). Stimulation of Jurkat cells with PDBu and calcium ionophore did not change the distribution of these transcripts in the different fractions (data not shown).

DISCUSSION
In this paper we have identified the promoter region of the human COT gene and demonstrated that its activity is inducible by T cell-activating signals. We have also identified three different human COT mRNAs, COT-1, COT-2, and COT-3, with different lengths in the 5ЈUTR but a common transcription initiation site. This site is located 4748 nt upstream of the translation initiation site of COT kinase. The first exon of COT-1 (denominated exon 1) comprises these 4748 nt and the first 336 nt of the coding sequence of COT kinase (see Table I and Fig. 1). The lack of splicing in the 5ЈUTR of COT-1 mRNA species results in a predominantly nuclear distribution. The physiological significance of this finding remains to be established. Nevertheless, the possibility that COT-1 mRNA is stored in the cell nucleus and a later processing of its 5Ј UTR triggers the transport of the generated transcript to the cytoplasm should not be excluded. A similar situation has been described for other mRNAs (29). The 5Ј UTR of the COT-2 transcript is generated when the 5Ј region flanking the translation initiation site of human COT kinase undergoes splicing, and this UTR comprised of exons 1a, 1b, and 1c. This 5Ј UTR contains 444 nt upstream of the coding sequence of COT kinase and exhibits a putative open reading frame located in exon 1b. COT-3 mRNA, with a 215-nt 5Ј UTR, is comprised of exons 1a and 1c and does not have any open reading frame upstream of the translation initiation site of COT kinase. The occurrence of upstream open reading frames has only been detected in about 10% of vertebrate mRNAs, and their physiological role is still unclear. Interestingly, the majority of these mRNA species code for proteins involved in signal transduction (30, 31 and references therein).
Toyoshimo and co-workers (15,17) reported a genomic structure of the human COT gene with 9 exons, from which the 2 upstream exons are noncoding, and defined a 5Ј UTR of the COT transcript (GenBank TM accession number D14497) with 366 nt; this sequence corresponds to part of the 5Ј UTR of the COT-2 transcript defined here. Chan et al. (14) reported a COT transcript (GenBank TM accession number Z14138) (denominated est in their study) with a 159-nt long 5Ј UTR; this sequence corresponds to part of the 5Ј UTR of COT-1 defined here.
It has recently been reported that an increase in COT mRNA levels, determined by RT-PCR analysis, of a 136-nt fragment of the COT coding sequence plays a role in human breast cancer (22). Based on the data obtained here, demonstration of this role in tumorigenesis would require analysis of the expression of the different COT transcripts in tumoral versus normal tissues, because the study of the subcellular and polysome distribution of the different COT transcripts indicates that COT-1 is not attached to polysomes but is mainly located in the nucleus and is, therefore, probably not translated.
We did not find any evidence of additional splicing variants in the protein coding exons of the described human COT gene (data not shown). This is probably because COT kinase does not have any of the known signaling modules, and consequently changes in the coding sequence (with the exception of the last exon) would result in the modification of at least one of the XI regions necessary for a functional protein kinase (32). Deletion of the last exon provides transformation capacity to COT kinase (4,15,20,21).
The COT gene is induced during T cell activation. Both combinations of stimuli (PDBu and calcium ionophore or PDBu and ␣CD3) induce an increase in COT mRNA levels as well as activation of the COT promoter-driven transcription. A number of consensus sequences reported to bind specific trans-acting factors regulated by activating T signals are present in the 5Ј flanking region of the COT gene (Fig. 3). Thus, three AP-1 binding sites (27,28) were found at positions Ϫ79, Ϫ327, and Ϫ637 nt. One PEA-3 motif (33) was located at Ϫ267 nt. The analysis also revealed one consensus recognition motif for ets (34) at position Ϫ808 nt and four consensus binding sequences for OCT-1 (35) at positions Ϫ293, Ϫ501, Ϫ708, and Ϫ1037 nt. The ets and OCT-1 response elements have been reported to be regulated by phorbol esters (36). Two cAMP-response element (CRE)-like sequences (36) are found at positions Ϫ41 and Ϫ164 nt, which could account for the increased COT promoter activity observed upon 8-Br-cAMP treatment. This finding also suggests that different signal transduction pathways can mediate COT regulation. The relative activities of the different COT promoter constructs in the presence and absence of T cell activating signals indicated that a PDBu and calcium ionophore response element is located in the Ϫ340 to Ϫ229-nt region of the COT promoter. Activation of the AP-1 response elements in T cells requires an integration of both PDBu and calcium ionophore signals, is independent of ERK pathway activation, is sensitive to cyclosporin A, and is up-regulated by the JNK/SAPK signal transduction pathway (9, 37). The same FIG. 7. Distribution of COT transcripts. A, a dot blot performed with total RNA, RNA isolated from the cytoplasmic fraction, and RNA isolated from the nuclear fraction was hybridized with probe C (nt 3837-4646), the COT coding probe, and a probe generated from the COT promoter region (nt Ϫ778 to Ϫ30). B, distribution of COT transcripts in polysomes. The figure shows the relative absorbance corresponding to the different sucrose fractions. The 28 S and 18 S RNAs from 6 l of each fraction were separated on a 2.2 M formaldehydeagarose denaturing gel and photographed after ethidium bromide staining. RT-PCR analysis of the different fractions was performed. COT-1 levels were determined with primers 8D (1 M) and 8R (1 M), COT-2 levels were determined with primers 6D (1 M) and 7R (1 M), and COT-3 levels were determined with primers 1D (1 M) and 7R (1 M). Primers for ␤-actin detection were used at a concentration of 0.1 M. requirements are needed for activation of the Ϫ340 COT promoter-driven transcription, indicating that at least the AP-1 binding site present at Ϫ327 nt could play a role in the PDBu and calcium ionophore-triggered COT promoter activation.
COT kinase activity is crucial for the transduction mechanism of activating signals in T cells during G 0 /G 1 transition (1, 8 -12). The data presented here indicate that the expression of the COT gene is regulated by these same signals.