Autoinhibition of Casein Kinase I epsilon  (CKIepsilon ) Is Relieved by Protein Phosphatases and Limited Proteolysis*

Aleksandra CegielskaDagger §, Kimberly Fish GietzenDagger §, Ann RiversDagger §, and David M. VirshupDagger par **

From the Dagger  Division of Molecular Biology and Genetics, Department of Oncological Sciences, Huntsman Cancer Institute,  Division of Hematology/Oncology, Department of Pediatrics, and the par  Program in Human Molecular Biology and Genetics, University of Utah, Salt Lake City, Utah 84112

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Casein kinase I epsilon  (CKIepsilon ) is a member of the CKI gene family, members of which are involved in the control of SV40 DNA replication, DNA repair, and cell metabolism. The mechanisms that regulate CKIepsilon activity and substrate specificity are not well understood. We report that CKIepsilon , which contains a highly phosphorylated 123-amino acid carboxyl-terminal extension not present in CKIalpha , is substantially less active than CKIalpha in phosphorylating a number of substrates including SV40 large T antigen and is unable to inhibit the initiation of SV40 DNA replication. Two mechanisms for the activation of CKIepsilon have been identified. First, limited tryptic digestion of CKIepsilon produces a protease-resistant amino-terminal 39-kDa core kinase with several-fold enhanced activity. Second, phosphatase treatment of CKIepsilon activates CKIepsilon 5-20-fold toward T antigen. Similar treatment of a truncated form of CKIepsilon produced only a 2-fold activation. Notably, this activation was transient; reautophosphorylation led to a rapid down-regulation of the kinase within 5 min. Phosphatase treatment also activated CKIepsilon toward the novel substrates Ikappa Balpha and Ets-1. These mechanisms may serve to regulate CKIepsilon and related forms of CKI in the cell, perhaps in response to DNA damage.

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The casein kinase I (CKI)1 gene family encompasses an increasing number of genes expressed in eukaryotes including yeast Caenorhabditis elegans and mammals. Two subgroups of the CKI family have been separated by functional analysis and complementation of mutations in yeast. One group encoding nuclear kinases appears in yeast to be involved in the response to DNA damage. Mutations in these genes, including HRR25 and YCK3 in Saccharomyces cerevisiae and hhp1 and hhp2 in Schizosaccharomyces pombe, lead to sensitivity to DNA damaging agents such as x-rays and methyl methanesulfonate (1-4). The mammalian genes encoding CKIdelta and CKIepsilon complement HRR25-deleted yeast, suggesting they too may be involved in DNA repair pathways (5). A second group in S. cerevisiae encodes prenylated membrane-bound isoforms involved in bud growth and includes YCK1 and YCK2 (6); deletions in these genes are complemented by the mammalian genes encoding CKIgamma (7).

The structure of the CKI family suggests several potential mechanisms for the regulation of activity. All family members contain a short amino-terminal domain of 9-76 amino acids, a highly conserved kinase domain of 284 amino acids, and a highly variable carboxyl-terminal domain that ranges from 24 to more than 200 amino acids in length. The carboxyl terminus of a CKI isoform may serve several functions, including regulation of substrate recognition, modulation of catalytic activity, and/or determination of kinase subcellular localization. Prenylation of the tail of the YCK1/YCK2 family has been shown to be of functional importance in yeast (3, 8, 9). Phosphorylation of CKI may also be an important regulatory mechanism. Most of the CKI proteins are phosphoproteins, and several of the yeast kinases can autophosphorylate on serine, threonine, and tyrosine residues (10). Studies using synthetic substrates and the artificial substrate casein have indicated that these phosphoryl residues may inhibit kinase activity (11, 12), although the location of these inhibitory groups remains unclear. Kuret and co-workers (11) found that an unphosphorylated truncation of Cki1 containing only the kinase domain was twice as active as the phosphorylated form, whereas Graves et al. (12) mapped a phosphorylation-dependent inhibitory domain in CKIdelta to a 26-amino acid domain in the carboxyl-terminal tail. Polyanions such as heparin can regulate kinase activity; activation by heparin appears dependent on the presence or absence of the carboxyl-terminal domain (12, 13). Additionally, membrane bound forms of the kinase may be regulated by phosphatidylinositol-4,5-bisphosphate (14).

One in vitro functional assay of CKI activity is its ability to phosphorylate simian virus 40 (SV40) large T antigen. We have previously shown that CKIalpha purified from HeLa cell extracts phosphorylates T antigen on physiologic sites and inhibits the initiation of viral DNA replication (15-17). As SV40 DNA replication is regulated in the cell cycle and by DNA damage (18-21), it was of interest to determine whether forms of CKI implicated in DNA repair pathways could also regulate in vitro DNA replication.

We have now overexpressed and purified active CKIepsilon . The data show that this form of CKI, although active on peptide substrates, is markedly hindered in its ability to phosphorylate and inhibit the origin unwinding function of T antigen. This decreased activity of CKIepsilon is apparently due to an inhibitory effect of the carboxyl-terminal domain not present in CKIalpha , since limited tryptic digestion of CKIepsilon released a catalytically active amino-terminal 39-kDa fragment able to both phosphorylate T antigen and inhibit its replication initiation function.

We also report that CKIepsilon is activated by dephosphorylation in a tail-dependent manner. Treatment of recombinant CKIepsilon with the catalytic subunits of PP1, PP2A, or PP2B (calcineurin) leads to as much as a 20-fold increase in activity toward T antigen, casein, and two novel substrates, the Ets-1 transcription factor and recombinant Ikappa Balpha . Activation of the kinase by phosphatases was transient and self-limited; reautophosphorylation of the kinase led to inactivation within 5 min. Activation was dependent on the presence of the carboxyl-terminal domain of the kinase; a truncation mutant of CKIepsilon was activated by phosphatase 4-fold less than was full-length kinase. These findings support the hypothesis that the carboxyl-terminal domain of CKIepsilon inhibits its activity on key protein substrates and suggests that in the cell, CKIepsilon may be regulated in a self-limited manner by phosphatases and in a more sustained manner by intracellular proteolysis.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Ni2+-nitrilotriacetate-agarose was obtained from Qiagen. Trypsin (T8642), calcineurin, and calmodulin were from Sigma. PP1c and inhibitor 2 were from New England Biolabs. Okadaic acid was from Life Technologies, Inc. Restriction enzymes were from Life Technologies, Inc. and New England Biolabs. Plasmids expressing Ikappa Balpha (22) were the generous gift of John Hiscott.

Cloning and Escherichia coli Expression of CKIepsilon -- The cDNA encoding wild type human CKIepsilon was isolated as a NcoI/SalI 1333-base pair fragment and ligated into NcoI/XhoI-digested pET16b (a T7-based expression vector from Novagen) as described previously (5). This construct (pKF115) removes the hexahistidine sequence present in the pET16b vector downstream from the NcoI site. The 1317-base pair NcoI/HindIII fragment from pKF115 was ligated into the same sites in pRSET-B (a T7-based expression vector from Invitrogen). This construct (pV71) encodes CKIepsilon with a 41-amino acid amino-terminal fusion that contains a hexahistidine tag and an enterokinase cleavage site. The d305 truncation mutant was created by the introduction of a stop codon after amino acid 305 in a derivative of pV71 (pKF162) by site-directed mutagenesis. All recombinant proteins were expressed in BL21(DE3) cells (23). Bacteria were grown in Luria broth containing 50 µg/ml carbenicillin at 37 °C to an A600 of 0.3 and induced overnight at 28 °C with 1 mM isopropyl-1-thio-beta -D-galactopyranoside.

Purification and Partial Proteolysis of Recombinant CKIepsilon -- Hexahistidine-tagged CKIepsilon was expressed at low levels in BL21(DE3) cells. Lysates in 20 mM Hepes, pH 7.5, 25 mM NaCl, 1 mM DTT, 1 mM EDTA, 0.02% Nonidet P-40, 10% sucrose (buffer B) with 0.1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, and 1 µg/ml pepstatin were applied to DEAE-cellulose and batch-eluted with the same buffer containing 200 mM NaCl. Kinase-containing fractions were dialyzed into buffer B with 10 mM NaCl and then applied to a S-Sepharose column equilibrated with the same buffer. Kinase activity was batch-eluted with buffer B with 250 mM NaCl (without EDTA or DTT) and loaded directly onto Ni2+-nitrilotriacetate-agarose from which it was eluted with buffer B (without EDTA or DTT) containing 80 mM imidazole. Untagged CKIepsilon was purified by a similar procedure except that the Ni2+-nitrilotriacetate-agarose step was omitted. The typical yield was 100 µg of CKIepsilon from a 2-liter culture. Hexahistidine-tagged CKIepsilon was used in all assays except where specifically indicated. Histidine-tagged and untagged kinase were found to behave identically in all assays tested.

Proteolysis of purified wild type and hexahistidine-tagged CKIepsilon was performed in the standard kinase reaction buffer (30 mM Hepes, pH 7.5, 7 mM MgCl2, 0.5 mM DTT for 30 °C for 15 min either without or with previous autophosphorylation of substrate. Trypsin was then inhibited by soybean trypsin inhibitor added to a final concentration of 10 µg/ml. Proteolysis with trypsin at 1 µg/ml gave almost quantitative scission of CKIepsilon into its major digestion products; this concentration of trypsin was routinely used for the production of the active tryptic fragment.

Immunoblot Analysis-- For immunoblot analysis, proteins and trypsin-generated peptides were separated by SDS-PAGE on a 17% gel and then transferred to nitrocellulose membrane in 12.5 mM Tris, 86 mM glycine, pH 8.3, 0.1% SDS, 20% methanol. After a 15-min fixation in 0.5% glutaraldehyde in phosphate-buffered saline, the membrane was blocked with 3% bovine serum albumin and then incubated with a 1:500 dilution of UT31 antiserum raised against the amino-terminal CKIepsilon peptide MELRVGNKYRLGC (5). Immunoreactive peptides were detected using an alkaline phosphatase-conjugated goat-anti-rabbit IgG (Bio-Rad) followed by incubation with bromo-chloro-indolylphosphate and nitroblue tetrazolium (24).

Kinase and Phosphatase Assays-- Kinase reactions were performed in buffer containing 30 mM Hepes, pH 7.5, 7 mM MgCl2, 0.5 mM DTT, 100 µg/ml bovine serum albumin, 150 or 250 µM ATP, and 1-5 µCi of [gamma -32P]ATP at 37 °C or as indicated. Reactions were stopped by the addition of SDS-PAGE sample buffer and analyzed by SDS-PAGE and autoradiography as described previously (15, 16).

Peptide phosphorylation reactions contained the synthetic 15-mer phosphopeptide substrate AHALS(P)VASLPGLKKK (termed 5P) that was synthesized with the serine in position 5 phosphorylated. This peptide contains a CKI consensus site and is phosphorylated by both CKIalpha and CKIepsilon with a Km of approximately 200 µM.2

Peptide kinase assays were terminated by the addition of 50 µl of 30% acetic acid/10 µl assay and quantitated by spotting the reaction mixture onto P81 phosphocellulose filters as described (25). All phosphatase reactions were performed in 30 mM Hepes, pH 7.5, 7 mM MgCl2, and 100 µg/ml bovine serum albumin unless otherwise noted. Protein concentration was determined by the method of Bradford using bovine serum albumin as a standard (26).

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Substrate Specificity-- In previous studies we purified CKIalpha based on its ability to phosphorylate SV40 large T antigen and thus inhibit the initiation of SV40 DNA replication (15, 16). In the process of characterizing human CKI genes, we cloned a novel member of the CKI family, CKIepsilon , that encodes a 48-kDa kinase (5). CKIepsilon was of interest because, unlike CKIalpha , it was able to functionally complement yeast deleted for the DNA repair-related kinase HRR25. In preliminary studies on recombinant CKIepsilon , we found virtually identical specific activity to CKIalpha when tested on peptide substrates (data not shown). However, although CKIalpha was found to readily phosphorylate T antigen, CKIepsilon was approximately 9-fold less active on this protein substrate (Fig. 1). Similar results were obtained when a partially purified non-tagged form of CKIepsilon was tested (data not shown), indicating the histidine tag has no significant effect on the ability of CKIepsilon to phosphorylate T antigen. Interestingly, CKIepsilon autophosphorylation was also consistently stimulated severalfold by the addition of T antigen (Fig. 1, lane 4 compared with lanes 5 and 6), suggesting a specific protein-protein interaction between the carboxyl-terminal domain and the substrate.


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Fig. 1.   CKIalpha and CKIepsilon vary markedly in their ability to phosphorylate SV40 large T antigen. 1 pmol each of CKIalpha purified from HeLa cells (lanes 1-3) and histidine-tagged recombinant CKIepsilon (lanes 4-6) were incubated with the indicated amount of immunoaffinity-purified T antigen (T ag) in the presence of 150 µM [gamma -32P]ATP for 30 min at 37 °C and then analyzed by SDS-PAGE and autoradiography. The arrow indicates the position of phosphorylated T antigen, the open circle indicates the position of autophosphorylated CKIepsilon , and the asterisk indicates the position of autophosphorylated CKIalpha . Sizes are indicated in kilodaltons.

Effect of Carboxyl-terminal Tail on CKIepsilon Activity-- CKIepsilon and CKIalpha are closely related in the kinase domain (86% similar and 74% identical) but differ in the length of the carboxyl-terminal tail. CKIalpha has only a 24-amino acid extension beyond the kinase domain, whereas CKIepsilon has a 123-amino acid tail. Recent studies of carboxyl-terminal domains in the related kinases CKIdelta and Cki1 have indicated that they perform a regulatory function (11, 27). The differing activities of the CKIalpha and CKIepsilon isoforms suggested that the carboxyl-terminal domain of CKIepsilon was responsible for the decreased activity of the kinase on T antigen and secondly, that this region might form a discrete structure separable from the kinase domain by limited proteolysis. To test this, intact autophosphorylated CKIepsilon was subjected to limited digestion with various concentrations (0-5 µg/ml) of trypsin. As demonstrated in Fig. 2, digestion of 32P-autophosphorylated CKIepsilon with increasing amounts of trypsin results in the production of a series of higher mobility species that are further digested to a relatively stable 39-kDa phosphoprotein that finally disappears during incubation at the highest trypsin concentration (5 µg/ml). This 39-kDa product appears to be activated CKIepsilon since (a) its appearance coincided with a 3-fold increase in kinase activity toward T antigen, (b) it reacts with polyclonal antibody UT31 that recognizes the amino terminus of CKIepsilon , and (c) the purified 39-kDa polypeptide retains kinase activity (Fig. 3 and data not shown). The 3-fold activation achieved by trypsin treatment may under-represent the actual activation since (a) there may have been partial loss of the kinase domain as well, and (b) proteolytic tail fragments may retain some inhibitory activity (data not shown). These results suggest that CKIepsilon contains a discrete inhibitory domain that blocks specific substrate phosphorylation.


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Fig. 2.   Activation of CKIepsilon toward T antigen by partial proteolysis. 32P-labeled CKIepsilon was incubated for 15 min at 30 °C with increasing concentrations (0, 0.01, 0.03, 0.05, 0.08, 0.10, 0.15, 0.20, 0.40, 1.00, and 5.00 µg/ml, lanes 1-11, respectively) of trypsin, followed by the addition of soybean trypsin inhibitor (100 µg/ml). In lane 12, soybean trypsin inhibitor was added before the addition of 5 µg/ml trypsin. At the end of proteolysis, aliquots of each digestion were used to phosphorylate 0.5 µg of T antigen in the presence of 150 µM [gamma -32P]ATP for 30 min at 37 °C and then analyzed by SDS-PAGE and autoradiography. The open circle indicates autophosphorylated full-length CKIepsilon , the filled circle indicates the stable 39-kDa tryptic fragment of CKIepsilon (CKIepsilon -39), and the arrow indicates T antigen. Sizes are indicated in kilodaltons.


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Fig. 3.   Tryptic removal of the carboxyl terminus partially restores the replication inhibitory activity of CKIepsilon . A, inhibition of unwinding by CKIalpha , CKIepsilon , and CKIepsilon -39. SV40 large T antigen was preincubated either alone or with the various forms of CKI and then assayed for its ability to unwind the SV40 origin of replication as described previously (16). The extent of unwinding of the origin fragment was quantitated with a PhosphorImager (Molecular Dynamics) and plotted as a function of the amount of added kinase. (black-triangle), CKIalpha ; (open circle ), CKIepsilon ; and (bullet ), CKIepsilon -39. B, bar graph of the reactions quantitated in panel A indicating the amount of each kinase needed to achieve 50% inhibition of origin unwinding. T ag, T antigen.

Functional Activity of the 39-kDa Amino-terminal Fragment of CKIepsilon Protein-- T antigen catalyzes the initial steps in SV40 DNA replication, the unwinding of the duplex origin of replication to single-stranded DNA. CKIalpha inhibits T antigen function by phosphorylating it on at least two inhibitory sites, serines 120 and 123 (15, 16). The finding that proteolytic cleavage of CKIepsilon stimulated its activity on T antigen led to the further functional characterization of the 39-kDa tryptic fragment. Specifically, we wished to determine whether its increased protein kinase activity translated into an increased ability to inhibit T antigen-catalyzed SV40 origin unwinding. CKIalpha , CKIepsilon , and the 39-kDa CKIepsilon fragment (CKIepsilon -39) were pre-incubated with T antigen and ATP, and then T antigen-dependent unwinding of the SV40 minimal origin of replication was assayed (Fig. 3). As previously demonstrated, phosphorylation of T antigen by CKIalpha inhibited its origin unwinding activity with 50% inhibition occurring at about 0.4 pmol of kinase/reaction (40 nM in the preincubation mixture). In contrast, CKIepsilon very inefficiently inhibited the origin unwinding activity of T antigen with 50% inhibition occurring at about 7 pmol of kinase/reaction (700 nM in preincubation mixture). The 39-kDa fragment of CKIepsilon was substantially more active than full-length CKIepsilon , producing 50% inhibition of T antigen origin unwinding activity at about 0.9 pmol of kinase/reaction (90 nM in preincubation mixture, Fig. 3B). These results indicate that the catalytic core of CKIepsilon is in fact similar in activity to CKIalpha and that proteolytic removal of the carboxyl terminus of CKIepsilon partially activates it to phosphorylate the same or similar inhibitory sites on T antigen.

Diverse Serine/Threonine Phosphatases Activate CKIepsilon toward T Antigen-- Recent studies on the related CKI isoform CKIdelta have demonstrated that the full-length kinase can be activated 2-5-fold toward casein or a synthetic peptide (D4) by treatment with the catalytic subunit of protein phosphatase 1 (PP1c) (27). We tested whether removal of phosphoryl groups from CKIepsilon stimulated its kinase activity toward SV40 large T antigen (Fig.4, A and B). Purified CKIepsilon was incubated with increasing amounts of the catalytic subunit of either PP1 or PP2A. At the end of the dephosphorylation reaction, okadaic acid was added to a final concentration of 250 nM, and the activity of the kinase on T antigen was assessed. As demonstrated in Fig. 4, A and B, pretreatment of CKIepsilon with either phosphatase stimulates its activity on T antigen up to 20-fold, with half-maximal activation occurring with less than 2 ng (~4 nM) of PP2Ac. The stimulation requires phosphatase catalytic activity, since inclusion of okadaic acid in the preincubation fully blocks the effect of PP2Ac (Fig. 4A, lane 7). The activation by 16 ng (~30 nM) of PP1c is only partially blocked by 250 nM okadaic acid, a not unexpected result since PP1c is 10-100-fold less sensitive than PP2Ac to inhibition by okadaic acid. Of note, alterations in the phosphorylation state of CKIepsilon had a significant effect on kinase mobility (Fig. 4A). This appears to be due to tail phosphorylation, as truncated CKIepsilon can autophosphorylate without a significant change in electrophoretic mobility (Fig. 7A and data not shown).


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Fig. 4.   Activation of CKIepsilon by protein serine/threonine phosphatases. A, CKIepsilon is activated toward T antigen by PP2Ac and PP1c. 250 ng of CKIepsilon was preincubated with between 0 and 8 ng of purified PP2Ac (lanes 1-7) or 0-16 ng PP1c (lanes 8-14) for 30 min at 37 °C. After the preincubation, phosphatase activity was inhibited by the addition of 250 nM okadaic acid, and kinase activity was assessed by the addition of 0.24 µg of T antigen and 100 µM [gamma -32P] ATP. The kinase reaction was allowed to proceed for 15 min at 37 °C and then analyzed by SDS-PAGE and PhosphorImager analysis. T antigen is indicated by an arrow, and CKIepsilon is indicated by an open circle. Note that phosphatase treatment produces a dramatic shift in the mobility of CKIepsilon . Various phosphorylation states of CKIepsilon are visible as a smear below the fully phosphorylated band. In the reactions shown in lanes 7 and 14, okadaic acid (O.A.) was added before the preincubation, demonstrating that activation of the kinase requires phosphatase activity. 250 nM okadaic acid completely inhibits PP2Ac but only partially inhibits 16 ng of PP1c. A, autoradiograph. B, quantitation of T antigen phosphorylation. T antigen (T Ag) phosphorylation by phosphatase-pretreated CKIepsilon was quantitated from the kinase reactions pictured in panel A. PP1c and PP2Ac pretreatment (indicated by open and filled circles, respectively) gave similar results. C, CKIepsilon autophosphorylates to approximately 12 mol/mol. CKIepsilon autophosphorylation after phosphatase pretreatment was quantitated from the kinase reactions pictured in panel A. PP1c and PP2Ac pretreatment (indicated by open and filled circles, respectively) gave similar results.

Autophosphorylation Rapidly Inactivates CKIepsilon -- CKIepsilon is capable of autophosphorylation of up to approximately 12 mol/mol (Fig. 4C and see below). Since phosphatase treatment of the bacterially expressed kinase leads to marked activation toward protein substrates, we asked how rapidly reautophosphorylation of the kinase occurs and whether this reautophosphorylation correlates with a decrease in protein kinase activity. CKIepsilon was dephosphorylated with PP2Ac and then allowed to reautophosphorylate in the presence of okadaic acid. T antigen was either added at the same time as [gamma -32P]ATP (time 0) or at the indicated times after ATP addition. As Fig. 5 demonstrates, CKIepsilon reautophosphorylation was complete within 5 min, coincident with the re-suppression of kinase activity. Thus, CKIepsilon appears to re-autophosphorylate and autoinhibit itself rapidly in the absence of phosphatase activity. Dilution studies and experiments with CKIepsilon kinase dead mutants indicate that CKIepsilon autophosphorylation is entirely intramolecular (data not shown). Of note, neither activation nor autoinhibition of kinase activity toward a peptide substrate was seen in similar time course reactions (data not shown), suggesting autoinhibition is effective toward protein but not peptide substrates.


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Fig. 5.   Rapid reautophosphorylation of CKIepsilon leads to inhibition of kinase activity within 5 min. A and B, CKIepsilon was preincubated alone (lane 1) or activated by preincubation with 4 ng of PP2Ac (lanes 2-6). At the end of the preincubation, okadaic acid (OA) and [gamma -32P]ATP were added to all reactions to final concentrations of 250 nM and 100 µM, respectively. T antigen (T ag) was added either at the end of the preincubation (time 0, lanes 1 and 2) or at the indicated times after ATP and okadaic acid (OA) addition (lanes 3-6). The T antigen kinase reaction proceeded for 15 min. T antigen phosphorylation was quantitated by SDS-PAGE and PhosphorImager analysis. A, autoradiograph of the kinase reaction. T antigen is indicated by an arrow, CKIepsilon is indicated by an open circle, and duration of the autophosphorylation reaction (min) is denoted at the top of each lane. B, quantitation of T antigen phosphorylation. CKIepsilon activity toward T antigen was activated by PP2Ac but was markedly diminished by a 5-min autophosphorylation reaction with ATP and okadaic acid.

Bacterially-produced CKIepsilon Is Heavily Autophosphorylated-- Dephosphorylation of bacterially expressed CKIepsilon had a significant activating effect on the kinase. To determine the number of phosphates on CKIepsilon that contribute to kinase inhibition, CKIepsilon autophosphorylation was quantitated after pretreatment with increasing amounts of PP2Ac or PP1c (Fig. 4C). Untreated kinase was able to add about 2.5 mol of phosphate/mol of kinase, whereas PP2Ac-treated kinase added about 12 mol/mol. These data suggest that the kinase was close to maximally autophosphorylated at approximately 10 mol/mol when it was purified from E. coli. Half-maximal activation of the kinase toward T antigen was seen when the kinase added approximately 8 mol/mol, suggesting that 4 mol/mol of phosphate had not been removed by the pretreatment. Similar results were seen with PP1c.

Phosphoamino acid analysis performed on CKIepsilon pretreated with PP2Ac and autophosphorylated in the presence of [gamma -32P]ATP indicates that autophosphorylation occurs on both serine and threonine residues (data not shown). Although CKI family members have been shown to be able to autophosphorylate on tyrosine as well as on serine and threonine (10), the autophosphorylation experiments in this study address only serine and threonine phosphorylation as the phosphatases used are serine-/threonine-specific. The data indicate that removal of phosphoryl groups from serine and threonine but not tyrosine residues is responsible for the activation of the kinase.

PP2A Activates CKIepsilon Toward an Array of Protein Substrates-- To determine whether dephosphorylation of CKIepsilon activated it toward multiple protein substrates, the kinase was tested using casein, replication protein A (the gift of Marc Wold, U. Iowa), and recombinant Ets-1 (the gift of Barbara Graves, U. Utah) as substrates. As demonstrated in Fig. 6A, CKIepsilon is activated toward T antigen, casein, and Ets-1 but not replication protein A (RP-A) by PP2Ac treatment. Similarly, treatment of CKIepsilon with PP1, PP2A, or PP2B catalytic subunit activated the kinase toward GST-Ikappa Balpha (Fig. 6B). The activation of CKIepsilon by phosphatases is therefore a general phenomenon. Whereas CKI substrate specificity on peptides can be determined by phosphoryl groups, we note that the increase in activity occurs toward unphosphorylated (bacterially produced Ets-1 and GST-Ikappa Balpha ) as well as phosphorylated (T antigen) proteins.


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Fig. 6.   Dephosphorylation activates CKIepsilon toward diverse substrates. A, CKIepsilon is activated toward T antigen, casein, and Ets-1. CKIepsilon (125 ng) was preincubated without (lanes 1, 3, 5, and 7) or with (lanes 2, 4, 6, and 8) 4 ng of PP2Ac and then incubated with okadaic acid, 100 µM [gamma -32P] ATP, and either 0.24 µg of T antigen (T, 90 kDa, lanes 1 and 2), 1 µg of casein (C, 27 kDa, lanes 3 and 4), 1 µg of replication protein A (RP-A) (R1, 70 kDa, and R2, 32 kDa, lanes 5 and 6) or 1 µg of recombinant Ets-1 (E, 50 kDa, lanes 7 and 8). An open circle denotes autophosphorylated CKIepsilon . B, CKIepsilon is activated toward Ikappa Balpha by PP2A, PP1, and PP2B. CKIepsilon (20 ng) was preincubated alone (lanes 1, 3, and 5) or with PP2Ac (30 ng, lane 2), PP1c (0.5 units, lane 4), or calcineurin (10 units, lane 6) for 15 min at 37 °C in buffer containing 7 mM MgCl2, 30 mM Hepes, pH 7.5, 100 µg/ml bovine serum albumin, 100 µM CaCl2, and 2 units of calmodulin. After the preincubation, kinase activity was assayed for 5 min at 37 °C in the presence of 3 µg of GST-Ikappa Balpha and 100 µM [gamma -32P]ATP with 100 ng of inhibitor 2, 250 nM okadaic acid, and 1 mM EGTA. Reactants were separated by 10% SDS-PAGE and analyzed by PhosphorImager. The expected migration of GST-Ikappa Balpha is indicated by an arrow and that of CKIepsilon is indicated by an open circle. C, carboxyl-terminal truncation mutants of Ikappa Balpha are not substrates for CKIepsilon . PP2Ac-treated CKIepsilon was incubated with 400 ng of wildtype or carboxyl-terminal truncation forms of GST-Ikappa Balpha for 5 min at 37 °C. Reactants were separated by 8% SDS-PAGE, silver-stained, and analyzed by PhosphorImager. WT, full-length GST-Ikappa Balpha ; Delta 2, deletion of amino acids 269-317; Delta 3, deletion of amino acids 288-317; Delta 4, deletion of amino acids 296-317 (nomenclature of Ref. 22). Removal of the terminal 22 amino acids (mutant Delta 4) was sufficient to abrogate CKIepsilon phosphorylation of Ikappa Balpha , suggesting carboxyl-terminal structure is essential for kinase recognition of the substrate. autorad, autoradiography.

CKIepsilon Phosphorylates the Carboxyl Terminus of Ikappa Balpha -- Although genetic studies have suggested a role for CKIepsilon -related proteins in DNA damage response pathways, few physiologic substrates of CKIepsilon have been identified. As Ikappa Balpha is phosphorylated and degraded in response to many signals including DNA damage and it contains sequences in the amino and carboxyl termini similar to CKI phosphorylation consensus sites found in peptide substrates, we tested whether CKIepsilon could be activated to phosphorylate Ikappa Balpha . As shown in Fig. 6B, CKIepsilon phosphorylates GST-Ikappa Balpha in a phosphatase-activable manner. The serine/threonine phosphatases PP2Ac, PP1c, and PP2B/calcineurin were all able to activate CKIepsilon to phosphorylate recombinant Ikappa Balpha .

Two regions of phosphorylation of Ikappa Balpha have been identified; serines 32 and 36 in the amino terminus are essential for the ubiquitin-mediated degradation of Ikappa Balpha bound to NF-kappa B, whereas phosphorylation of extreme carboxyl-terminal residues in a PEST region regulates proteolysis of free Ikappa Balpha (22, 28, 29). To determine whether CKIepsilon phosphorylated the carboxyl terminus of Ikappa Balpha , the ability of activated kinase to phosphorylate full-length and carboxyl-terminal truncation mutants (the generous gift of John Hiscott) was assayed. As shown in Fig. 6C, CKIepsilon phosphorylated full-length GST-Ikappa Balpha (lanes 1 and 5) but had no activity on mutants lacking the carboxyl-terminal 22 residues or greater (lanes 2-4, 6-8) (22). Note that although CKIepsilon phosphorylates Ikappa Balpha on both serine and threonine (data not shown), the nonphosphorylated Delta 4 truncation removes two threonine but no serine residues. Studies with point mutants suggest serines 288 and 293 are phosphorylated by CKIepsilon (data not shown). The Delta 4 mutant (truncated at amino acid 296) has been shown to be a substrate for the unrelated kinase, casein kinase II, indicating that the truncation does not lead to denatured protein (22). The data therefore suggest that removal of the carboxyl-terminal 22 amino acids disrupts the local structure enough to interfere with CKIepsilon but not CKII activity on Ikappa Balpha . These results are consistent with our previous data (15) indicating that CKI activity on protein (but not peptide) substrates is highly dependent on the intact tertiary structure of the substrate.

Full Activation by Phosphatases Requires the Carboxyl Terminus of CKIepsilon -- One model to explain both the inhibitory effect of the carboxyl-terminal tail and the stimulatory effect of dephosphorylation is that phosphate groups on the carboxyl terminus of CKIepsilon interact with the kinase domain, leading to inhibition of protein substrate binding. This model suggests that a tail-less CKIepsilon should not be activated by phosphatases. To test this, a stop codon was introduced after amino acid 305 in CKIepsilon , and the truncated histidine-tagged protein (denoted CKIepsilon -d305) was expressed in E. coli and purified on Ni2+-nitrilotriacetate-agarose. Roughly equal amounts of full-length and truncated CKIepsilon were used to phosphorylate T antigen, either without or with prior phosphatase treatment. As shown in Fig. 7, CKIepsilon was activated 9-fold by PP2Ac, whereas CKIepsilon -d305 was activated only 2-fold. Similarly, CKIalpha (76% identical to CKIepsilon over the kinase domain and lacking a carboxyl-terminal tail) was not activated by PP2Ac (data not shown). The data suggest that much of the observed autoinhibition of CKIepsilon requires the carboxyl-terminal tail, but that an inhibitory phosphoryl group on the CKIepsilon kinase domain also contributes to autoinhibition. These results are consistent with those seen by Kuret and co-workers (11) on the S. pombe homolog Cki1-d298 but differ slightly from those of Graves and Roach (27) who found that truncated CKIdelta was not activated by phosphatase treatment.


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Fig. 7.   The carboxyl terminus of CKIepsilon is the major determinant of PP2Ac-mediated activation. A and B, PP2Ac activated CKIepsilon 9.1-fold and CKIepsilon -d305 2.0-fold toward T antigen. 85 ng each of CKIepsilon (lanes 1 and 2) or CKIepsilon -d305 (lanes 3 and 4) lacking the carboxyl-terminal 111 amino acids of CKIepsilon were incubated without (lanes 1 and 3) or with (lanes 2 and 4) 8 ng of PP2Ac for 15 min at 37 °C before the addition of okadaic acid, T antigen, and [gamma -32P]ATP for a 3-min kinase reaction. Reaction products were separated by SDS-PAGE and quantitated by PhosphorImager analysis. T antigen is indicated by an arrow, and CKIepsilon and CKIepsilon -d305 are indicated by open circles. A, autoradiograph; B, PhosphorImager quantitation. C, comparison of activation of CKIepsilon by PP2Ac toward T antigen (closed circles, solid line) and the 5P peptide (open circles, dashed line). The kinase reactions were identical to those above but lasted only 2 min.

Previous work demonstrated that the activity of CKIepsilon toward peptide substrates was not significantly influenced by the presence of the carboxyl-terminal domain. We next tested whether phosphatase activation of the kinase had an effect on activity toward peptide, as opposed to protein, substrates. As shown in Fig. 7C, PP2Ac selectively activated CKIepsilon toward T antigen but not the 5P peptide substrate.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The casein kinase I family is characterized by a conserved core kinase domain and a series of variable carboxyl-terminal extensions. This study indicates that one major function of the carboxyl terminus is to regulate the activity of CKI on protein substrates. The ability of the carboxyl terminus to inhibit protein but not peptide phosphorylation suggests that this tail region interacts with the substrate binding face of the kinase, as illustrated in Fig. 8, rather than directly in the catalytic cleft. The data suggest CKI autoinhibition may be relieved by at least three mechanisms; (a) proteolytic cleavage of the tail (this study), (b) dephosphorylation of the kinase (this study and Refs. 11 and 27), and (c) binding of heparin (and presumably other polyanions) to the tail (27). Which, if any, of these mechanisms function in vivo is the subject of ongoing investigation. One reason for the diversity in CKI carboxyl-terminal domains may be to allow distinct activation mechanisms for the different family members. Alternatively, the function of the tail may be to constitutively restrict access to the catalytic cleft to all but a limited number of substrates, and no further regulation of the kinase may occur in vivo. Differentiation between these mechanisms will require the demonstration and characterization of CKI activation in vivo. Given the functional similarity between CKIepsilon and HRR25, one possibility is that agents that trigger the DNA repair response will lead to CKIepsilon activation.


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Fig. 8.   Model. Regulation of CKIepsilon activity by autophosphorylation and an inhibitory tail. The tail of autophosphorylated CKIepsilon interacts with the kinase domain to block access to the active site of protein but not peptide substrates. Full autoinhibition requires the presence of both the tail and inhibitory phosphoryl groups that are accessible to multiple phosphatases. The data suggest that at least one inhibitory phosphoryl group is on the body of the kinase.

Activation of forms of CKI lacking a carboxyl-terminal extension by dephosphorylation suggests that at least one inhibitory phosphoryl group is present on the core kinase domain and may interact directly with the kinase tail. Additionally, the finding that multiple phosphatases can activate CKIepsilon raises the possibility that the kinase may be activated by diverse signal transduction pathways. Since the inhibitory phosphoryl groups are placed in an intramolecular autophosphorylation reaction, kinase activation by dephosphorylation allows a very tight temporal control over the duration of kinase activity. As we have demonstrated, once the activating phosphatase is removed, autophosphorylation leads to rapid kinase inactivation. Such self-limited bursts of kinase activity allow rapid down-regulation of kinase activity, a mechanism that may be required to turn off a pathway once its inciting agent is removed. Alternatively, if CKIepsilon itself activates a phosphatase, for example by CKI phosphorylation of the protein phosphatase 1 inhibitor 2 (data not shown and Ref. 30), then a positive feedback loop may be established that can further increase the activity of CKIepsilon .

The observation that trypsin can activate CKIepsilon supports the model (shown in Fig. 8) that the inhibitory tail is much more accessible to proteolysis than is the kinase domain itself. Cleavage of CKIepsilon by regulated intracellular proteolysis would lead to a long lived activation of CKIepsilon in response to the appropriate stimulus. Proteolytic activation has been demonstrated for an increasing number of intracellular proteins, including protein kinase C, mitogen-activated protein kinase kinase kinase 1 (MEKK1), and NF-kappa B (31-33). DNA damage resulting in protease activation might lead to CKIepsilon cleavage. Notably, caspases with DEVD sequence specificity (34) may be activated during apoptosis and cleave in the acidic region of CKIepsilon at the beginning of the carboxyl terminus. Such cleavage might either activate CKIepsilon or release it from its normal anchoring site.

Previous studies have suggested that CKI recognizes peptide and protein substrates differently. CKI phosphorylates acidic peptides, with the best peptide substrates containing either phosphorylated or acidic residues amino-terminal to the target site (35, 36). In this case, peptide recognition requires interaction of the phosphorylated region with the catalytic fold (37, 38). However, this localized charge interaction does not appear operative in the case of T antigen phosphorylation. We have previously shown that (a) CKIalpha can phosphorylate bacterially expressed (and hence unphosphorylated) T antigen and (b) CKIalpha does not recognize its target site in the amino terminus of T antigen unless the carboxyl-terminal residues of T antigen are present (15). Thus, CKIalpha could not phosphorylate a T antigen-derived peptide, an amino-terminal tryptic fragment of T antigen, nor T259, an active recombinant amino-terminal fragment of T antigen that contains the target sites and the DNA binding domain. Similarly, in the current study we found that removal of the carboxyl terminus of Ikappa Balpha blocks phosphorylation of upstream residues. These results are consistent with a more complex picture of kinase-substrate interactions, where the tertiary structure of the substrate interacts with multiple surface features of the kinase. Therefore, additional structural elements of the kinase may interfere with docking of substrate proteins at sites distant from the target residues. One function of the CKIepsilon tail may be to restrict the access of protein substrates to the active site until the kinase is activated by transient dephosphorylation or limited proteolysis. However, these results do not exclude the possibility that in vivo, the inhibitory effect of the tail observed in vitro could also be overcome by constitutive active dephosphorylation or additional mechanisms such as noncovalent binding of regulatory molecules.

Identification of potential cellular substrates of CKI has been problematic. CKI isoforms can phosphorylate SV40 large T antigen (15, 16), p53 (39), inhibitor-2 (30), and glycogen synthase, among others. The present study extends the list by identifying the carboxyl terminus of Ikappa Balpha as an in vitro substrate. Whether CKI is as important as CKII in the phosphorylation of Ikappa Balpha in vivo is unclear. In very few cases has a clear functional role for the mammalian CKI family been defined. One approach to this question has been to overexpress kinases in cells or organisms and examine them for changes in phosphorylation patterns. An implication of this current study is that overexpression of CKIepsilon and related CKI family members may produce inactive, autoinhibited kinase with no phenotype or effect on in vivo substrates unless point mutations, truncations, or activating conditions are first introduced. One possible route to the identification of additional in vivo-specific substrates of CKIepsilon is therefore the development of autophosphorylation site point mutants that retain an intact regulatory domain but are constitutively active.

    ACKNOWLEDGEMENTS

We thank John Hiscott for Ikappa Balpha constructs and Erica Vielhaber, Paola Dal Santo, Mylynda Schlessinger, and Brent McCright for help along the way.

    FOOTNOTES

* Oligonucleotide synthesis was supported by NCI, National Institutes of Health Grant 3P30 CA42014, and the research was supported by National Institutes of Health Grant CA71074.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Contributed equally to this work.

** To whom correspondence should be addressed: University of Utah, 15 North 2030 East, Room 2100, Salt Lake City, UT 84112-5330. E-mail: David.Virshup{at}genetics.utah.edu.

1 The abbreviations used are: CKI, casein kinase I; SV40, simian virus 40; PP2A, PP1, and PP2B, protein phosphatase 2A, 1, and 2B, respectively; DTT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase.

2 A. Cegielska, E. Vielhaber and D. M. Virshup, unpublished results.

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Abstract
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Materials & Methods
Results
Discussion
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