©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Requirements for Calcium and Calmodulin in the Calmodulin Kinase Activation Cascade (*)

(Received for publication, November 20, 1995)

Hiroshi Tokumitsu Thomas R. Soderling (§)

From the Vollum Institute, Oregon Health Sciences University, Portland, Oregon 97201

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We have previously purified and cloned rat brain Ca/calmodulin-dependent protein kinase kinase (CaM-KK), and the 68-kDa recombinant CaM-KK activates in vitro both CaM-kinase IV (CaM-K IV) and CaM-K I (Tokumitsu, H., Enslen, H., and Soderling, T. R.(1995) J. Biol. Chem. 270, 19320-19324). In the present study we have determined that activation of CaM-K IV through phosphorylation of Thr by CaM-KK is triggered by elevated intracellular Ca in intact cells and requires binding of Ca/CaM to both enzymes. An expressed fragment of CaM-K IV (CaM-K IV), which contains the activating phosphorylation site (Thr) but not the autoinhibitory domain or the CaM-binding domain, still required Ca/CaM for phosphorylation by wild-type CaM-KK. A truncated mutant of CaM-KK (CaM-KK) phosphorylated CaM-K IV in a Ca/CaM-independent manner, but this constitutively active CaM-KK required Ca/CaM for phosphorylation and activation of wild-type CaM-K IV. These results demonstrate that binding of Ca/CaM to both CaM-K IV and CaM-KK is required for the CaM-kinase cascade. Both CaM-KK and CaM-K IV appear to have similar Ca/CaM requirements with EC values of approximately 100 nM. Studies using co-expression of CaM-K IV with CaM-KK in COS-7 cells demonstrated that CaM-KK rapidly activated both total and Ca/CaM-independent activities of wild-type CaM-K IV, but not the Thr Ala mutant, upon ionomycin stimulation.


INTRODUCTION

Ca/calmodulin-dependent protein kinase IV (CaM-K IV)(^1)(1, 2, 3) , a member of the CaM-kinase family, mediates Ca-dependent transcriptional activation through phosphorylation of transcription factors such as cAMP response element binding protein (4, 5, 6, 7) and serum response factor(8, 9) . Originally, it was reported that purified CaM-K IV from rat brain undergoes regulatory autophosphorylation which enhances Ca/CaM-dependent (total) and -independent activities of the enzyme(10, 11) . However, since recombinant CaM-K IV exhibits little autophosphorylation and activation (4, 8) , it was likely that the purified brain CaM-K IV contained a contaminating activator. Recently several groups have identified and purified brain CaM-kinase kinases (CaM-KK) that phosphorylate and activate not only CaM-K IV (12, 13) but also CaM-K I(14) . These results suggested the existence of a CaM-kinase cascade analogous to other kinase cascades such as cAMP-dependent protein kinase/phosphorylase kinase(15) , MAP-kinase(16) , and AMP-kinase (17) .

The cDNA for a 68-kDa rat brain CaM-KK has been cloned, and the expressed recombinant CaM-KK in vitro activates CaM-K I and CaM-K IV but not CaM-K II(18) . CaM-KK is abundant in brain and detectable in thymus and spleen, similar to the tissue distribution of CaM-K IV (19) but more restricted than the distribution of CaM-K I (20) . CaM-KK binds Ca/CaM by the gel overlay technique, and its activation of CaM-K IV and CaM-K I requires Ca/CaM(18) . However, it is not clear whether the required Ca/CaM is binding to CaM-KK, CaM-K IV or both. Since there are no known substrates of CaM-KK which do not also bind Ca/CaM, we decided to examine the phosphorylation of a fragment of CaM-K IV that contains its activating phosphorylation site. Both CaM-K I and IV, but not CaM-K II, contain Thr residues in putative ``activation loops'' similar to the MAP-kinases and cdc kinases which are also activated by kinase cascades(21, 22) . Thus, we suspected that Thr in this activation loop of CaM-K IV may be the phosphorylation site for CaM-KK. Recently it has been shown that purified 52-kDa porcine brain CaM-K I kinase increases CaM-K IV total activity through phosphorylation of Thr(23) , but effects of the Thr Ala mutation on the generation of Ca-independent activity of CaM-K IV were not reported. Since the 52-kDa CaM-K I kinase has not been cloned, it is difficult to compare it with our 68-kDa CaM-KK. The 52-kDa CaM-KK may represent the porcine brain isoform of rat brain 68-kDa CaM-KK.

The above studies suggest the existence of a unique protein kinase cascade regulating CaM-K I and IV, but this system needs further confirmation in intact cells. Although we showed that co-transfection of CaM-KK with CaM-K IV gave a 14-fold enhancement of transcriptional activation of a reporter gene(18) , the direct activation of CaM-K IV by transfected CaM-KK has not been demonstrated in intact cells. In this report, we demonstrate in COS-7 cells the activation of CaM-K IV by CaM-KK and its requirement for elevation of intracellular Ca. Furthermore, we establish in vitro that binding of Ca/CaM to both CaM-K IV and CaM-KK is required for the CaM-K activation cascade.


EXPERIMENTAL PROCEDURES

Materials

CaM-KK cDNA (18) (accession no. L42810) was cloned from a rat brain cDNA library as described previously. Recombinant CaM-K IV was expressed in Sf9 cells and purified as described previously(4, 13) . CaM was purified from bovine brain(24) . All other chemicals were from standard commercial sources.

Construction of Plasmids

His-tagged wild-type CaM-K IV construct (pME-His-CaM-K IV) was made as follows. The PCR fragment of the 5`-end of CaM-K IV, amplified using 5`-GCG GCT AGC ATG CTC AAA GTC ACG-3` and 5`-CCT CCT CTT GCA-3` as primers, was digested with NheI and BstBI and subcloned into pRSETB vector (Invitrogen). After digesting this plasmid with NdeI and filling in, a 0.32-kilobase pair His-tagged (Met-Arg-Gly-Ser-His(6)-Gly-Ala-Ser) fragment was obtained by digestion with BstBI and subcloning into pME18s plasmid (DNAX Research Institute) with the 3`-portion of CaM-K IV (BstBI-XbaI fragment)(13) . Thr Ala mutants were made by using a site-specific plasmid DNA mutagenesis kit (5 Prime 3 Prime, Inc.) and pME-His-CaM-K IV as a target plasmid with a mutagenic oligonucleotide (5`-AAG TGC TCA TGA AGG CAG TGT GTG GAA CCC CGG G-3`). Mutant plasmids were selected by nucleotide sequencing. For expression of CaM-KK in E. coli we made a HIS-tagged CaM-KK construct (pRSET-CaM-KK). The 5` portion of CaM-KK was made by PCR to create a unique NheI site using 5`-ACT GCT AGC ATG GAG CGC AGT CCA-3` and 5`-TTC AGG ATC AGG TCT TT-3`. The NheI-XbaI fragment was subcloned into NheI/NcoI sites of pRSETB vector with the 3` portion of CaM-KK (XbaI-NcoI fragment). Truncated His-tagged CaM-KK construct (pRSET-CaM-KK) was made by replacement of BstXI-EcoRI fragment of pRSET-CaM-KK and BstXI-NotI fragment of pME-CaM-KK construct which was made by PCR using 5`-TTG GCG CCG CTC ACA CCT CCT CCT CAG TCA CCT CT-3` and 5`- AGG AAA GAC CAG CGG AAA-3` and subcloning of the PCR fragment (XbaI/NotI digest) into XbaI/NotI digested pME-CaM-KK (wild type)(18) . For expression of CaM-K IV and CaM-K IV, these fragments were amplified by PCR using combination of 5`-AAG GAT CCG AAA ATT GCT GAT TTT GGA CTT3` and 5`-GTA AAG CTT TCA TCC TAC AGA CCA CAT GTC-3`, and 5`-AAG GAT CCG ATG CTC AAA GTC ACG GTG CCC-3` and 5`-GGT AAG CTT TCA TTT GCA TCT GTA CAC AAT-3`, respectively, as PCR primers followed by digestion with BamHI/HindIII and subcloning into BamHI/HindIII sites of pRSETB vector. Mutant fragments were made by site-directed mutagenesis as described above and selected by sequencing.

Transient Expression and Stimulation of COS-7 Cells

COS-7 cells were maintained in Dulbecco's modified Eagle medium containing 10% fetal calf serum. Cells were subcultured in 6-cm dishes for 12 h before transfection. The cells were then transferred to serum-free medium and treated with a mixture of either pME18s plasmid DNA (4 µg, mock) or plasmid containing His-tagged CaM-K IV cDNA (4 µg, pME-His-CaM-K IV), either with or without 0.8 µg of pME-CaM-KK and 30 µg of LipofectAMINE reagent (Life Technologies, Inc.) in 2.4 ml of serum-free medium. After 5 h of incubation, 2.4 ml of medium containing 20% fetal calf serum were added to the cells, and the incubation was continued for another 20 h. After changing to fresh medium, cells were cultured an additional 20 h. Prior to stimulation by ionomycin, the cells were cultured for 2 h in serum-free medium (Dulbecco's modified Eagle's medium, Life Technologies, Inc.). At the indicated times (1-10 min), stimulation was terminated by aspirating the medium, and the cells were immediately frozen with liquid N(2). The COS cells were lysed for 30 min at 4 °C with 1 ml of lysis buffer A (150 mM NaCl, 20 mM Tris-HCl, pH 7.5, 10% glycerol, 1% Nonidet P-40, 10 mM pyrophosphate, 50 mM NaF, 1 mM sodium vanadate, 1 µM microcystin-LR, 0.5 mM PMSF, 10 mg/liter trypsin inhibitor, 10 mg/liter leupeptin, 10 mg/liter antipain, 10 mg/liter aprotinin and 1 mM benzamidine). After centrifugation at 10,000 times g for 20 min, 20 µl of Ni resin (50% slurry, ProBond resin (Invitrogen)) were added to the supernatant, and the mixture was incubated for 1 h at 4 °C. After precipitation of the resin by centrifugation and removal of the supernatant, the resin was washed three times with 1 ml of lysis buffer and once with buffer A containing 0.3 M NaCl. The resin was transferred into 0.45-µm filter unit (ULTRAFREE-MC, Millipore Corp.), and CaM-K IV was eluted with 50 µl of 20 mM Tris-HCl (pH 7.5), 0.15 M NaCl, 250 mM imidazole, 1 mM EDTA, and 1 mM EGTA. Five µl and 15 µl of eluted CaM-K IV were used for the protein kinase assay as described below and for the immunoblotting, respectively.

Expression and Purification of Recombinant CaM-KKs and CaM-K IV Fragments

Escherichia coli (JM109) transformed with pRSET-CaM-KK (wild type), -CaM-KK, -CaM-K IV, or -CaM-K IV were grown in ampicillin-containing SOB medium (200 ml) to an OD = 0.3. After adding 1 mM isopropyl-1-thio-beta-Dgalactopyranoside, the cells were grown for an additional 1 h, infected with M13/T7 phage at an multiplicity of infection of 5 plaque-forming units/cell, and cultured for 5 h. Cells were collected by centrifugation at 10,000 times g for 10 min and frozen in liquid N(2). The cell pellet was resuspended in 15 ml of lysis buffer A as described above, sonicated, and centrifuged at 10,000 times g for 15 min. Two ml of Ni resin were added to the supernatant, and the mixture was incubated for 1 h on ice and then applied to a Poly-Prep chromatography column (Bio-Rad). After washing the column extensively with lysis buffer A and buffer with 0.3 M NaCl, elution was carried out with 50 mM Tris-HCl (pH 7.5), 250 mM imidazole, 1 mM EGTA, 1 mM EDTA, 0.1 mM PMSF, and 1 mM benzamidine. After dialysis against 50 mM Tris-HCl (pH 7.5), 0.2 mM EGTA, 0.2 mM EDTA, 0.2 mM PMSF, eluates were applied to Q-Sepharose (0.5 ml) and eluted with 0.1 M NaCl containing buffer. Recombinant CaM-K IV and CaM-K IV was stored frozen at -20 °C. Recombinant wild-type CaM-KK was further purified by CaM-Sepharose column chromatography as previous described(13) . The truncation mutant CaM-KK was further purified by Q-Sepharose chromatography as follows. The fractions eluted from the Ni column were dialyzed against buffer B (50 mM Tris-HCl (pH 7.5), 1 mM EGTA, 1 mM EDTA, 0.1 mM PMSF, 1 mM benzamidine) and applied to Q-Sepharose (1-ml column) which was equilibrated with buffer B. After washing the column with buffer B, elution was carried out by a linear NaCl gradient (0-0.6 M NaCl). Truncated CaM-KK was eluted at 0.2 M NaCl. Recombinant CaM-KKs were dialyzed against 40% glycerol, 10% ethylene glycol, 0.5 mM EDTA, 0.5 mM EGTA, 1 mM benzamidine, and 2 mM DTT and stored at -20 °C.

In Vitro CaM-K IV Activation and Protein Kinase Assay

Recombinant CaM-K IV (5.4 µM) was incubated with the recombinant CaM-KKs (36 nM) at 30 °C in 50 mM HEPES (pH 7.5), 10 mM magnesium acetate, 1 mM DTT, 400 µM ATP, and either 1 mM CaCl(2), 10 µM CaM or 1 mM EGTA. The reaction was terminated at 5 min by a 25-fold dilution at 5 °C with 50 mM HEPES (pH 7.5), 2 mg/ml bovine serum albumin, 10% ethylene glycol, and 1 mM EDTA. CaM-K IV activity was measured at 30 °C in a 25-µl assay containing 50 mM HEPES (pH 7.5), 10 mM magnesium acetate, 1 mM DTT, 1 µM microcystin-LR, 400 µM [-P]ATP (1000-2000 cpm/pmol), 40 µM syntide-2, 43 nM recombinant CaM kinase IV (or 5 µl of Ni resin purified CaM-K IV), and either 1 mM CaCl(2), 1 µM CaM (total activity) or 1 mM EGTA (Ca/CaM-independent activity). The reaction was initiated by the addition of CaM-K IV and terminated at 5 min by spotting aliquots (15 µl) onto phosphocellulose paper (Whatman P-81) followed by washing in 75 mM phosphoric acid(25) .

In Vitro Phosphorylation of CaM-K IV Fragments

Either recombinant CaM-K IV-wild type, CaM-K IV, or CaM-K IV was incubated at 30 °C for 15 min with either 36 nM of recombinant CaM-KK or 22 nM of recombinant CaM-K IV in a solution containing 50 mM HEPES (pH 7.5), 10 mM magnesium acetate, 1 mM DTT, 100 µM [-P]ATP (400 µM for phosphorylation of recombinant CaM-K IV-wild type) and either 1 mM CaCl(2), 1 µM CaM (10 µM CaM for phosphorylation of recombinant wild-type CaM-K IV) or 1 mM EGTA. Reactions were terminated by adding 2.5 µl of SDS-PAGE sample buffer, and the samples were immediately subjected to SDS-PAGE. After staining and destaining, the gels were exposed to autoradiography. The bands corresponding to the fragment were excised and P incorporation was measured.

Other Methods

Anti-human CaM-K IV rabbit IgG (26) was raised against the COOH-terminal sequence (GLAEEKLTVEEA) of human CaM-K IV(27) . SDS-PAGE was carried out with either Tris-glycine (28) or the Tricine system(29) . Protein concentrations were measured by the Bradford (30) method using bovine serum albumin as a standard. The protein concentration of partially purified truncated CaM-KK was adjusted by immunoreactivity against anti-CaM-K II antibody (18) using purified recombinant CaM-KK as a standard.


RESULTS

CaM-KK Is a Ca^2/CaM-dependent Enzyme That Phosphorylates Thr in CaM-K IV

We previously demonstrated the activation of CaM-K IV and CaM-K I by an extract of COS-7 cells expressing CaM-KK(18) . To further characterize CaM-KK, we have expressed it in E. coli and purified it on CaM-Sepharose. The recombinant CaM-KK, like the brain CaM-KK (13) , bound to CaM-Sepharose in the presence of Ca and was eluted in the presence of EGTA (not shown) to yield a highly purified 68-kDa protein (Fig. 1A). Incubation of the purified CaM-KK with recombinant CaM-K IV in the presence of Mg/ATP enhanced by 10-fold or more both total and Ca-independent activities of CaM-K IV (Fig. 1B). This activation of CaM-K IV by CaM-KK was totally dependent on the presence of Ca/CaM.


Figure 1: Activation of CaM-K IV by purified recombinant CaM-KK in vitro. A, Purified E. coli-expressed CaM-KK (2 µg, right lane), Sf9-expressed CaM-K IV (1.5 µg, center lane), and M(r) standard proteins (left lane) were separated on SDS-10% PAGE and stained with Coomassie Brilliant Blue R250. B, CaM-K IV (5.4 µM) or kinase buffer was incubated at 30 °C for 5 min with either purified, recombinant CaM-KK (36 nM) or buffer in a kinase cascade activation reaction (see ``Experimental Procedures'') with either 1 mM EGTA or 1 mM CaCl(2), 10 µM CaM as indicated. After terminating the reaction, CaM-K IV activity was measured using 40 µM syntide-2 in the presence of 1 mM EGTA (open bars) or 1 mM CaCl(2), 1 µM CaM (closed bars) under standard assay conditions. CaM-KK activity toward syntide-2 was also measured in the absence of exogenous CaM-K IV under the same conditions, but it was negligible (right four bars). The mean ± S.E. of three independent experiments is shown.



In order to determine whether the required Ca/CaM was binding to CaM-KK, CaM-K IV, or both, we needed a substrate of CaM-KK which itself does not bind Ca/CaM. We constructed a poly(His)(6)-tagged fragment of CaM-K IV (residues 178-246) which contains the activating phosphorylation site (i.e. Thr) (23) but not the AID or CaM-binding domain (residues 304-328). The His/CaM-K IV was expressed in E. coli and purified for in vitro phosphorylation by CaM-KK. Using this fragment of CaM-K IV eliminates any ambiguity due to autophosphorylation by activated CaM-K IV. As shown in Fig. 2A (lower panel), His/CaM-K IV was phosphorylated by recombinant CaM-KK in a completely Ca/CaM-dependent manner. Phosphoamino acid analysis (not shown) showed that this phosphorylation was exclusively on Thr, and mutation of Thr to Ala abolished approximately 90% of P incorporation into His/CaM-K IV by CaM-KK (Fig. 2A). These results demonstrate that recombinant CaM-KK is Ca/CaM-dependent and directly phosphorylates Thr of CaM-K IV. The His/CaM-K IV was not phosphorylated by CaM-K IV itself (data not shown).


Figure 2: Ca/CaM-dependent phosphorylation of His/CaM-K IV by CaM-KK and of His/CaM-K IV by CaM-K IV. A, wild-type or Thr Ala mutant of His/CaM-K IV was expressed, purified, and incubated (40 µg/ml) at 30 °C for 15 min with 36 nM of recombinant CaM-KK (or buffer) and 100 µM [-P]ATP in the presence of either 1 mM CaCl(2)/1 µM CaM or 1 mM EGTA as indicated. Reactions were analyzed by SDS-18% PAGE (Tricine system) and either Coomassie Blue stain (top panel) or autoradiography (bottom panel). B, His/CaM-K IV was expressed, purified, and incubated with either CaM-KK (36 nM, lanes 1 and 2), CaM-K IV (22 nM, lanes 3 and 4) activated by CaM-KK as in Fig. 1B, or nonactivated CaM-K IV (22 nM, lanes 5 and 6) for 15 min at 30 °C in the presence of either 1 mM CaCl(2), 1 µM CaM or 1 mM EGTA as indicated. Samples were analyzed by SDS-PAGE and autoradiography as in A.



It has also been reported that phosphorylation of Ser residues in the NH(2) terminus of CaM-K IV by purified rat brain CaM-KK is responsible for activation of CaM-K IV(31) . We tested the ability of CaM-KK to phosphorylate this domain by expressing a His/CaM-K IV construct. However, His/CaM-K IV was not significantly phosphorylated by CaM-KK, but it was phosphorylated by CaM-K IV, especially after its activation by CaM-KK (Fig. 2B). These results suggest that the observed phosphorylation of the NH(2)-terminal Ser residues in CaM-K IV may have been due to autophosphorylation subsequent to activation of CaM-K IV by CaM-KK.

Characterization of a Constitutively Active CaM-KK

To further examine the Ca/CaM dependence of CaM-KK, we attempted to construct a constitutively active species of CaM-KK by truncating the putative AID and CaM-binding domain. Members of the CaM-kinase family usually contain adjacent or overlapping AID and CaM-binding domain COOH-terminal of the catalytic domain(32) . Truncation of the COOH-terminal 71 amino acid residues (CaM-KK) and expression in E. coli generated a recombinant 60-kDa protein on SDS-PAGE (not shown). This CaM-KK mutant phosphorylated Thr in His/CaM-K IV in a Ca/CaM-independent manner (Fig. 3A, lower panel). However, when wild-type CaM-K IV was the substrate, its phosphorylation (Fig. 3B, insert) and activation (Fig. 3B) by CaM-KK required Ca/CaM. The above results establish that the CaM-kinase cascade requires binding of Ca/CaM to both CaM-KK and CaM-K IV, and they also show that CaM-KK has an AID and CaM-binding domain COOH-terminal of residue 434.


Figure 3: Phosphorylation and activation of CaM-K IV by truncated CaM-KK. A, CaM-KK (36 nM) or buffer were assayed (30 °C, 15 min) for their abilities to phosphorylate wild-type His/CaM-K IV (lanes 1-4), the Thr196Ala mutant of His/CaM-K IV (lanes 5 and 6) or buffer (lanes 7 and 8) in the presence of either 1 mM EGTA or 1 mM CaCl(2), 1 µM CaM as indicated. Reactions were analyzed by SDS-PAGE and either stained with Coomassie Blue (top panel) or subjected to autoradiography (bottom panel). B, CaM-K IV (5.4 µM) was incubated (30 °C, 5 min) with either 36 nM of partially purified recombinant CaM-KK or buffer in a kinase activation reaction (see ``Experimental Procedures'') with either 1 mM EGTA or 1 mM CaCl(2), 10 µM CaM as indicated and either 400 µM ATP or 100 µM [-P]ATP (inset). After terminating the activation, the reaction mixture was analyzed by SDS-PAGE autoradiography (inset) or CaM-K IV activity was measured using 40 µM syntide-2 in the presence of 1 mM EGTA (open bars) or 1 mM CaCl(2), 1 µM CaM (closed bars) under standard assay conditions. Kinase activity toward syntide-2 of the recombinant CaM-KK was also measured in the absence of exogenous CaM-K IV under the same conditions, but it was negligible (right four bars). The mean ± S.E. of three independent experiments is shown. For the insert: a and b = CaM-K IV - CaM-KK -/+ Ca/CaM, respectively; c and d = CaM-K IV + wild-type CaM-KK -/+ Ca/CaM, respectively; e and f = CaM-K IV + CaM-KK -/+ Ca/CaM, respectively. The arrow denotes the position of CaM-K IV.



Since binding of Ca/CaM to both CaM-KK and CaM-K IV was required for the CaM-kinase cascade to operate, we tested whether CaM-KK and CaM-K IV had different requirements for Ca/CaM. If CaM-KK needed much higher concentrations of Ca/CaM than CaM-K IV, it was possible that small elevations of intracellular Ca might selectively activate CaM-K IV, and higher elevations of Ca would be required to trigger the CaM-kinase cascade. However, it appears that the Ca/CaM requirement (EC) of both CaM-KK and CaM-K IV is approximately 100 nM (Fig. 4).


Figure 4: Ca/CaM activation of CaM-KK and CaM-K IV in vitro. Nonactivated CaM-K IV (22 nM, circle), CaM-K IV (22 nM, bullet) activated by CaM-KK, or CaM-KK itself (36 nM, up triangle) were assayed for their activities with 1 mM CaCl(2) and the indicated concentrations of CaM (other conditions as given under ``Experimental Procedures''). The requirement for CaM of activated CaM-K IV was determined by subtraction of its Ca-independent activity from total activity. For nonactivated and activated CaM-K IV, 40 µM syntide-2 was used as substrate, whereas His/CaM-K IV (40 µg/ml) was the substrate for CaM-KK.



Ca^2-dependent Activation of CaM-K IV by CaM-KK in COS-7 Cells

It is well established that CaM-K IV can be activated by CaM-KK in vitro(12, 13, 23) . To directly demonstrate that CaM-KK can activate CaM-K IV in intact cells, we transfected His-tagged CaM-K IV with or without co-transfected CaM-KK into COS-7 cells. At various times after ionomycin stimulation to allow Ca influx, cells were frozen in liquid N(2), lysed, and His-tagged CaM-K IV was purified from the cell lysate by a Ni resin. As shown in Fig. 5A, both total and Ca-independent activities of CaM-K IV were elevated upon ionomycin stimulation when CaM-KK was co-transfected (circles). In the absence of CaM-KK co-transfection, ionomycin treatment did not alter CaM-K IV activities. The increases in total (3-fold) and Ca-independent (6-fold) activities by CaM-KK and ionomycin stimulation occurred within 4 min but returned to near basal values within 10 min. This biphasic activation phenomena was not due to changes of CaM-K IV expression levels (Fig. 5B).


Figure 5: Ca-dependent activation of CaM-K IV by CaM-KK in COS-7 cells. A, CaM-K IV cDNA which was fused with HIS-tag (4 µg of pME-His-CaM-K IV) was co-transfected into COS-7 cells with either wild-type CaM-KK (0.8 µg of pME-CaM-KK-wild, circles) or plasmid alone (0.8 µg of pME18s, triangles). Cells were stimulated with 1 µM ionomycin and at the indicated times frozen with liquid N(2). Cells were lysed, expressed His/CaM-K IV was partially purified by Ni resin, and CaM-K IV activity was measured either in the presence of 1 mM CaCl(2), 1 µM CaM (closed symbols) or 1 mM EGTA (open symbols) using 40 µM syntide-2 as a substrate. B, expressed His/CaM-K IV (15 µl of Ni column eluate) in the experiment of Panel A was quantitated at the indicated time points after ionomycin stimulation by Western blot analysis (chemiluminescent detection) using anti-CaM-K IV antibody.



It was recently reported that mutation of Thr to Ala in CaM-K IV blocks the increase in its total activity normally generated in vitro by purified procine brain CaM-K I kinase(23) . The data of Fig. 2confirm that Thr in HIS/CaM-K IV was phosphorylated by CaM-KK. To test whether Thr is also the phosphorylation/activation site in intact cells, we introduced His-tagged CaM-K IV mutant into COS-7 cells with CaM-KK. Fig. 6shows that the Thr Ala mutant had basal Ca/CaM-dependent activity, but it was not activated by CaM-KK in response to ionomycin treatment.


Figure 6: Mutation of Thr Ala abolished CaM-K IV activation by CaM-KK in COS-7 cells. Either wild-type or Thr Ala mutant of CaM-K IV cDNA (4 µg), which are fused with HIS-tag, were transfected into COS-7 cells with wild-type CaM-KK (0.8 µg of pME-CaM-KK). After stimulation with or without 1 µM ionomycin for 4 min, cells were frozen with liquid N(2) and lysed. Expressed His/CaM-K IV (wild-type or mutant) was partially purified by Ni resin, and CaM-K IV activity was measured in the presence of either 1 mM CaCl(2)/1 µM CaM (solid bars) or 1 mM EGTA (open bars) using 40 µM syntide. The mean ± S.E. of three experiments using three independent transfections is shown.



Mutation of Thr to Ala not only prevents the increase in total activity of CaM-K IV generated by CaM-KK(23) , but it also blocked the increase in Ca-independent activity (Fig. 6). It is possible that phosphorylation of Thr is directly responsible for increasing both total and Ca-independent activities. Alternatively, phosphorylation of Thr may be a prerequisite for phosphorylation of some other site, either by CaM-KK or through autophosphorylation by activated CaM-K IV, which generates Ca-independent activity. Since Thr of CaM-K IV is analogous to the phosphorylation site (Thr) in CaM-K II which generates Ca-independent activity, Thr was considered a likely candidate. Furthermore, mutation of HMDT to DEDD converts CaM-K IV into a Ca-independent species(13) . We therefore made CaM-K IV mutants Thr Ala and Ser Ala and examined their in vitro activation by CaM-KK. Both mutant CaM-K IV species showed normal increases in both total and Ca-independent activities upon phosphorylation by CaM-KK (data not shown).


DISCUSSION

Extensive in vitro studies have documented the phosphorylation and activation of CaM-K IV by CaM-KK(12, 13, 23) . This report extends these studies by 1) establishing a requirement for binding of Ca/CaM to both the CaM-KK and the substrate CaM-K IV and 2) demonstrating in COS-7 cells with transfected CaM-KK the Ca-dependent activation of co-transfected CaM-K IV through phosphorylation of Thr.

The requirement for binding of Ca/CaM to CaM-KK is consistent with the previous observation that CaM-KK can be purified on CaM-Sepharose (13) and that the expressed CaM-KK binds Ca/CaM using the gel overlay technique(18) . However, since both CaM-KK and CaM-K IV are apparently CaM-dependent, it was not clear whether the requirement for Ca/CaM in the CaM-kinase cascade was due to binding of Ca/CaM to either CaM-KK, CaM-K IV, or both. By constructing a His-tagged construct containing the phosphorylation site in CaM-K IV (Thr) but lacking the AID and CaM-binding domain (residues 304-328), we demonstrated that phosphorylation of Thr in this CaM-independent substrate by wild-type CaM-KK still required Ca/CaM (Fig. 2). This indicates a requirement for Ca/CaM in this reaction for the activation of CaM-KK. This conclusion was substantiated by using a truncated form of CaM-KK, CaM-KK, lacking the putative AID and CaM-binding domain. This CaM-KK phosphorylated the His/CaM-K IV in a Ca/CaM-independent manner (Fig. 3), confirming the requirement for Ca/CaM of wild-type CaM-KK. Furthermore, the requirement for Ca/CaM in the phosphorylation of wild-type CaM-K IV by CaM-KK confirmed the requirement for Ca/CaM-binding to the substrate CaM-K IV. Thus, binding of Ca/CaM to both CaM-KK and CaM-K IV is required for this kinase cascade. The binding of Ca/CaM to CaM-KK is presumably required for neutralization of an AID COOH-terminal of residue 434. Studies are currently in progress to further define the AID and CaM-binding domain in CaM-KK. Extensive studies have established the existence of adjacent and sometimes overlapping AIDs and CaM-binding domains in numerous other kinases activated by Ca/CaM(32) . Presumably Ca/CaM must bind to CaM-K IV and remove its AID to expose the activation loop Thr which is probably within the catalytic cleft.

Our studies confirm with recombinant CaM-KK the observation made with purified porcine brain CaM-K I kinase that mutation of Thr to Ala blocks the increase in total activity of CaM-K IV(23) . We also observed that the increase in Ca-independent activity was also absent in this mutant (Fig. 6). It is interesting that this single site mutation blocks the increases in both total and Ca-independent activities of CaM-K IV, whereas phosphorylation of the equivalent site (Thr) in CaM-K I produces only an increase in total activity(34) . Both of these phosphorylation sites are within the ``activation loops'' that require phosphorylation for activation of numerous protein kinases. One possibility is that the AID in CaM-kinase IV lies within the catalytic cleft and makes an inhibitory interaction with the activation loop. This could explain why removal of the AID through binding of Ca/CaM is required to expose Thr for phosphorylation by CaM-KK. Phosphorylation of Thr could prevent the inhibitory interaction of the activation loop with the AID in the absence of Ca/CaM, thereby generating Ca-independent activity. Another possibility is that phosphorylation of Thr in CaM-K IV generates elevated total kinase activity which then allows autophosphorylation on another site to account for the elevated Ca-independent activity. If such were the case, a phosphorylation site in the AID would seem likely since introduction of negative charge in the AID generates Ca-independent activity of CaM-K IV(13) . To test this possibility we mutated Thr, which is equivalent to the autophosphorylation site in CaM-K II (Thr) that generates Ca-independent activity. However, CaM-KK increased both total and Ca-independent activities of the Thr Ala mutant. Similar results were obtained with the Ser Ala mutant. Thus, it is still not clear whether phosphorylation of Thr alone is sufficient for increasing Ca-independent activity of CaM-K IV.

A major purpose of this study was to test whether this CaM-kinase cascade, which has been demonstrated in vitro, could also be observed in intact cells. When COS-7 cells were transfected with His-tagged CaM-K IV alone, there was no effect of Ca-mobilization through ionomycin treatment on the His/CaM-K IV activity subsequently assayed in vitro. However, when CaM-KK was co-transfected with His/CaM-K IV, then ionomycin-treatment resulted in a 3-6-fold increases in total and Ca-independent His/CaM-kinase IV activities (Fig. 5A). These changes in CaM-K IV activities were not due to changes in amounts of expressed CaM-K IV (Fig. 5B). Interestingly, the activation of CaM-K IV by ionomycin was transient, peaking at 4 min and returning to near basal values at 10 min. This biphasic nature is consistent with phosphorylation of CaM-K IV followed by dephosphorylation. That phosphorylation was required for the activation was demonstrated by the fact that the Thr Ala mutant of CaM-K IV was not activated by CaM-KK upon ionomycin treatment (Fig. 6). The fact that only a 3-4-fold activation of CaM-K IV was observed in the intact cells compared to the 10-fold or greater activation in vitro is probably due to the endogenous protein phosphatases in the COS-7 cells that limit both the extent and duration of activation. These results are similar to the activation of CaM-K IV in Jurkat cells upon stimulation of the CD3 receptor(33) . The CD3-mediated activation of CaM-K IV is due to its phosphorylation since this activation can be reversed in vitro by treatment with protein phosphatase 2A but not protein phosphatase 1(26) , the same specificity as for reversal of in vitro activation of CaM-K IV by CaM-KK. Furthermore, CD3-mediated activation of CaM-K IV is transient, presumably due to endogenous protein phosphatase 2C which can inactivated CaM-K IV(33) , and the in situ activation can be further augmented 2-3-fold by subsequent in vitro treatment with purified CaM-KK(26) . From these studies we concluded that the CD3-dependent activation of CaM-K IV in Jurkat cells is probably mediated by CaM-KK.

It is puzzling why adjacent steps in a kinase cascade should both require the same activator, i.e. Ca/CaM. Our initial thought was that perhaps the CaM-KK would have a much lower affinity for activation by Ca/CaM. Thus, low levels of elevated intracellular Ca might selectively activate CaM-K IV and CaM-K I, whereas much higher level of Ca would be required for activation of CaM-KK to initiate the cascade. However, the data of Fig. 4show that both kinases have very similar requirements for Ca/CaM. Of course, the effect of activation of different substrates of CaM-KK may confer differences in their Ca dependencies. CaM-K IV, which has been activated by CaM-KK, can maintain sustained activity in the absence of continued Ca because of its considerable Ca-independent activity. This is not true for CaM-K I which does not generate Ca-independent activity upon activation by CaM-KK. While this manuscript was under review, a paper appeared which shows a requirement for binding of Ca/CaM to both CaM-KI and CaM-KI kinase and for binding of AMP to both AMP-kinase and AMP-kinase kinase in those cascades(35) . Lastly, it is possible there may be unidentified CaM-KK substrates which themselves are not regulated by Ca/CaM. We are currently searching for additional physiological pathways which may be regulated by CaM-KK.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grant GM 41292. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Vollum Institute L474, Oregon Health Sciences University, 3181 S.W. Sam Jackson Park Rd., Portland, OR 97201. Tel.: 503-494-6931; Fax: 503-494-6934; soderlit{at}OHSU.edu.

(^1)
The abbreviations use are: CaM, calmodulin; CaM-K, Ca/CaM-dependent protein kinase; CaM-KK, Ca/CaM-dependent protein kinase kindase; AID, autoinhibitory domain; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; DTT, dithiothreitol; PMSF, phenylmethylsulfonyl fluoride; MAP, mitogen-activated protein; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.


ACKNOWLEDGEMENTS

We thank Dr. Hervé Enslen for helpful discussions and performing phosphoamino acid analysis and Dr. Debra Brickey for critical reading of the manuscript.


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