©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Characterization of a Ca/Calmodulin-dependent Protein Kinase Cascade
MOLECULAR CLONING AND EXPRESSION OF CALCIUM/CALMODULIN-DEPENDENT PROTEIN KINASE KINASE (*)

(Received for publication, May 26, 1995)

Hiroshi Tokumitsu Hervé Enslen Thomas R. Soderling (§)

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

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Recent studies have demonstrated that Ca/calmodulin-dependent protein kinase IV (CaM-kinase IV) can mediate Ca-dependent regulation of gene expression through the phosphorylation of transcriptional activating proteins. We have previously identified and purified a 68-kDa rat brain CaM-kinase kinase that phosphorylates and increases total and Ca-independent activities of CaM-kinase IV (Tokumitsu, H., Brickey, D. A., Gold, J., Hidaka, H., Sikela, J., and Soderling, T. R.(1994) J. Biol. Chem. 269, 28640-28647). Using a partial amino acid sequence of the purified brain kinase, a CaM-kinase kinase cDNA was cloned from a rat brain cDNA library. Northern blot analysis showed that CaM-kinase kinase mRNA (3.4 kilobases) was expressed in rat brain, thymus, and spleen. Sequence analyses revealed that the cDNA encoded a 505-amino acid protein, which contained consensus protein kinase motifs and was 30-40% homologous with members of the CaM-kinase family. Expression of the cDNA in COS-7 cells yielded an apparent 68-kDa CaM-binding protein, which catalyzed in vitro activation in the presence of Mg/ATP and Ca/CaM of CaM-kinases I and IV but not of CaM-kinase II. Co-expression of CaM-kinase kinase with CaM-kinase IV gave a 14-fold enhancement of cAMP-response element-binding protein-dependent gene expression compared with CaM-kinase IV alone. These results are consistent with the hypothesis that CaM-kinases I and IV are regulated through a unique signal transduction cascade involving CaM-kinase kinase.


INTRODUCTION

The Ca/calmodulin-dependent protein kinase family (CaM-kinases I-V) (^1)is involved in many cellular responses that are triggered by elevated intracellular Ca concentration(1) . CaM-kinase IV was first identified as a multifunctional Ser/Thr protein kinase that has two monomeric isoforms; alpha (63 kDa) and beta (67 kDa)(2) . Although several in vitro substrates for CaM-kinase IV have been reported(3, 4) , the physiological function(s) of CaM-kinase IV remains to be established. Three groups have reported that CaM-kinase IV is involved in Ca-dependent transcriptional activation through the phosphorylation of cAMP-response element-binding protein (CREB) (5, 6, 7) and of the serum response factor(4, 32) , and this would be consistent with its nuclear localization(8) . However, the specific activity of recombinant CaM-kinase IV for phosphorylation of CREB or other substrates is about 20-fold lower than that of other protein kinases(5) . This observation prompted studies of mechanisms, in addition to Ca/CaM, for activating CaM-kinase IV.

The original studies on purified brain CaM-kinase IV indicated that its autophosphorylation results in 20-fold increases in Ca-independent and total kinase activities(9) . However, since recombinant CaM-kinase IV exhibits very slow autophosphorylation, which at best increases total activity only 2-fold(4, 10) , this suggested that the purified brain CaM-kinase IV may contain a contaminating activator protein. Indeed, it has been reported that rat brain extract contains a protein, which increases the total activity of bacterially expressed CaM-kinase IV(11) . More recently, we (10) and another group (12) purified a 66-68-kDa protein from rat brain, which in the presence of Mg/ATP and Ca/CaM gives time-dependent increases in total and Ca/CaM-independent activities of recombinant CaM-kinase IV. This activation of CaM-kinase IV correlates with P incorporation and could be reversed by subsequent treatment with protein phosphatase 2A(10) , so the activator protein is referred to as CaM-kinase kinase. CaM-kinase IV, which has been activated by CaM-kinase kinase, exhibits kinetics for the in vitro phosphorylation of Ser in CREB that are comparable with CREB phosphorylation by cAMP-kinase(13) . These results suggest that CaM-kinase IV may be involved in transcriptional regulation through phosphorylation of CREB and perhaps other transcriptional activators as part of a unique kinase cascade pathway involving CaM-kinase kinase. Interestingly, a 52-kDa activating factor, which is probably a protein kinase, for CaM-kinase I has been highly purified from rat brain(14) . We therefore set out to clone and characterize the 68-kDa CaM-kinase kinase and to test its specificity for activating CaM-kinases.


EXPERIMENTAL PROCEDURES

Materials

CaM-kinase IV kinase was purified from rat brain as described previously(10) . Recombinant wild type and double mutant (F316D,N317D) of CaM-kinase IVs and alpha CaM-kinase II were expressed in Sf9 cells and purified as described previously(10, 15) . Recombinant CaM-kinase I, which was expressed in Sf9 cells and purified, was kindly provided by Drs. P. Sun and R. A. Maurer (Oregon Health Sciences University). Rat brain gt10 cDNA libraries were provided by Dr. A. Yamakawa (Dana Farber Cancer Institute). Calmodulin was purified from bovine brain.

Protein Sequencing of CaM-kinase Kinase

Purified rat brain CaM-kinase kinase (10) was separated by 7.5% SDS-PAGE and electrotransferred onto PVDF membrane. After staining with Amido Black, the protein band (100 pmol) was excised and digested with trypsin (2 µg). Digested peptides were separated by C18 reverse phase chromatography (Applied Biosystems 130A high pressure liquid chromatography) and subjected to automatic gas-phase protein sequencing (Applied Biosystems 470A equipped with 120A PTH analyzer).

Cloning of CaM-kinase Kinase cDNA

Two degenerate primers corresponding to tryptic peptide 1 (IADFGVSNQFEGNDAQLSST) of rat brain CaM-kinase kinase were synthesized: sense primers, 5`AT(TCA)GC(TCAG)GA(TC)TT(TC)GG3` based on IADFG, and antisense primers, 5`A(AG)(TC)TG(TCAG)GC(AG)TC(AG)TT3` based on NDAQL. Forty cycles of PCR amplification were performed using rat brain random-primed, first-stranded cDNA as a template. The 50-bp PCR product was cloned into pBluescript, confirmed by sequencing, and used as the authentic probe. About 4.8 10^5 plaques from a rat brain oligo(dT)-primed cDNA library in gt10 were screened with the 50-bp probe labeled with [alpha-P]dCTP. The 5` end EcoRI-StuI 0.3-kbp fragment of clone 7AL was used as a hybridization probe for screening a rat brain random-primed gt10 cDNA library (4.7 10^5 plaques) to obtain clone R5.

Amplification of CaM-kinase Kinase cDNA 5` End

Rat brain mRNA (0.94 µg) was reverse transcribed with 10 pmol of a gene-specific primer (5`AGCATCATTCCCCTCAAACTGGTT3`) using the Invitrogen Fast Track cDNA kit. After removing excess primer by Centricon 100, the tailing reaction was carried out with 15 units of terminal deoxytransferase (Life Technologies, Inc.) and dCTP (0.2 mM). The PCR reaction was performed using poly(dC)-tailed first stranded DNA, a nested primer 1 (5`TGCTGACACCAAAGTCGGCGATCTT3`, 0.5 µM), and an anchor primer (5`ATCGAATTCCGGGIIGGGIIGGGIIG3`, 0.5 µM) under the following conditions: 1st cycle, 94 °C for 4 min, 42 °C for 2 min, 72 °C for 3 min; 2nd to 36th cycles, 94 °C for 1 min, 52 °C for 1 min, 72 °C for 2 min. After digestion with XhoI and EcoRI, the PCR fragment was subcloned into pBluescript. Another set of PCR reactions was performed using a nested primer 2 (5`GCGAGGAAAGCCATACT3`, 0.5 µM) and an anchor primer under conditions as described above. The PCR product was digested with XbaI and EcoRI and then subcloned into pBluescript. We sequenced both strands of cDNAs using a Sequenase version 2 kit (U. S. Biochemical Corp.). Sequence data are being submitted to GenBank.

Northern Blot Analysis

Total cellular RNA from rat tissues was isolated by the guanidine thiocyanate method, and rat brain poly(A) RNA was selected by an oligo(dT)-cellulose column. The total RNAs (30 µg) were electrophoresed on a formaldehyde-containing 1% agarose gel and then blotted onto Hybond-N (Amersham Corp.). The blot was hybridized in rapid hybridization buffer (Amersham Corp.) with a [alpha-P]dCTP random primed XbaI-XhoI fragment (540 bp) from P32 clone as a probe and washed in 0.2 SSC, 0.1% SDS at 65 °C. The blotted membrane was dried and autoradiographed.

Transfection of COS Cells

COS-7 cells were maintained in Dulbecco's modified Eagle's medium containing 10% fetal calf serum. Cells were subcultured in 10-cm dishes 12 h before transfection. The cells were then transferred to serum-free medium and treated with a mixture of either 20 µg of pME18s plasmid DNA (DNAX Research Institute) (mock transfection) or CaM-kinase kinase full-length cDNA containing plasmid DNA (20 µg, pMECaMKK) and 80 µg of LipofectAMINE Reagent (Life Technologies, Inc.) in 10 ml of medium. After a 20-h incubation, the cells were collected and homogenized with 1 ml of lysis buffer (50 mM NaCl, 50 mM Tris-HCl, pH 7.5, 1 mM EGTA, 1 mM EDTA, 0.1 mM PMSF, 1 mM benzamidine, 5 mg/liter leupeptin, and 5 mg/liter pepstatin A) using a Potter-Elvehjem homogenizer at 4 °C.

CaM-kinase Activation

The kinase activation reaction contained 5.7 µM CaM-kinase IV (wild-type or double mutant(10) ) or CaM-kinase IV buffer (control blank) and 50 mM HEPES (pH 7.5), 10 mM magnesium acetate, 1 mM dithiothreitol, 0.4 mM ATP, and 1 µM microcystin-LR plus EGTA, Ca, and/or CaM as indicated in Fig. 3legend. CaM-kinases I or II were used at 2 µM. The activation reactions were terminated by dilution in buffer containing EDTA, and kinase activity (46 nM of CaM-kinase IV or 15 nM of either CaM-kinases I or II) was determined at 30 °C for 1-5 min using standard assay conditions as described previously (10) with 40 µM syntide-2 as substrate and either 1 mM EGTA (Ca-independent activity) or 1 mM CaCl(2) plus 1 µM CaM (total activity).


Figure 3: Expression of CaM-kinase kinase and its activation of CaM-kinase IV. A, COS-7 cells were transfected (see ``Experimental Procedures'') with plasmid encoding CaM-kinase kinase (CaMKK) or plasmid alone (Mock), the cells were lysed, and lysate (6 µg) was separated by 10% SDS-PAGE and electrotransferred onto PVDF membrane. The membrane was subjected to Western blotting using CaM-kinase II peptide 132-146 antiserum (1/500 dilution, leftpanel) or to a biotinylated CaM (0.5 µg/ml) overlay (rightpanel). Molecular mass markers are shown to the left. B, Sf9 cell-expressed and purified wild-type or double mutant (F316D,N317D (10) ) CaM-kinase IV or kinase buffer (K-buffer) was incubated at 30 °C for 5 min with either the lysate buffer (Buffer) or lysate from COS-7 cells (180 ng) transfected with plasmid (Mock) or plasmid-expressing CaM-kinase kinase (CaMKK) in a kinase activation reaction (see ``Experimental Procedures'') with either 1 mM EGTA, 1 mM CaCl(2), or 10 µM CaM, as indicated. After terminating the activation, CaM-kinase IV activity was measured using 40 µM syntide-2 in the presence of either 1 mM EGTA (Ca-independent activity, openbar) or 1 mM CaCl(2) plus 1 µM CaM (total activity, closedbar) under standard assay conditions. Kinase activity toward syntide-2 of the lysate from COS-7 cells transfected with CaM-kinase kinase was also measured in the absence of exogenous CaM-kinase IV under the same conditions, but it was negligible (rightsixlanes). The mean ± S.E. of three experiments using three independent transfections is shown.



Transcriptional Activation

COS-7 cells (8 10^5 cells/10-cm dishes) were transfected with 2 µg of GAL4 or a GAL4-CREB fusion protein (16) and 5 µg of a reporter plasmid (5) and a combination of plasmids (pME18 s) encoding CaM-kinase IV (1 µg) and/or CaM-kinase kinase (5 µg). After incubation for 36 h, the cells were treated with 5 µM ionomycin and 10 mM CaCl(2) for 2 h. The cells were collected and lysed, and luciferase activity was measured by the luciferase assay kit (Promega). Luciferase activity for the mock transfected cells has been subtracted as a blank, and transfection with GAL4 without the CREB fusion protein gave insignificant luciferase activities.

Others

Western blotting was performed using anti-CaM-kinase II peptide(132-146) antiserum (1/500 dilution), and immunoreactive protein was visualized by chemiluminescence (DuPont NEN). Biotinylated CaM overlay was carried out as described previously(10) .


RESULTS AND DISCUSSION

Isolation and Analysis of cDNA Clones Coding for CaM-kinase Kinase

In order to clone CaM-kinase kinase cDNA, we purified the 68-kDa rat brain enzyme and obtained an amino acid sequence from two of its tryptic peptides. The amino acid sequence of tryptic peptide 1 (IADFGVSNQFEGNDAQLSST) confirmed that this protein contained a motif (DFG) that is conserved among the protein kinase family. The PCR-amplified DNA fragment (50 bp) coding for peptide 1 was prepared using degenerate oligonucleotide primers (see ``Experimental Procedures''), and this 50-bp fragment was used as a probe to screen a rat brain oligo(dT)-primed cDNA library, resulting in isolation of a 2.7-kbp clone 7AL (Fig. 1A). The 7AL clone contained sequences for peptide 1, other highly conserved protein kinase motifs, a TGA stop codon, and a 3` noncoding sequence including a poly(A) tail but no sequence for peptide 2. Screening of a rat brain random primed gt10 cDNA library yielded a 2.9-kbp clone (R5), which contained the sequence for peptide 2 (Fig. 1B) but no consensus ATP-binding motif (GXGXX(G/S)). To complete the 5` sequence we used 5`-RACE PCR to obtain three overlapping clones (P32, P122, and P22). Clones P32 and P122 contained the ATP-binding motif (GKGAYG), and clone P22 had an initiation ATG that was consistent with a Kozak consensus sequence(17) . These overlapping clones gave an open reading frame of 1515 nucleotides coding for a protein of 505 amino acids with a calculated molecular mass of 56 kDa (Fig. 1B).


Figure 1: Cloning and tissue mRNA analyses of rat brain CaM-kinase kinase. A, clone 7AL was isolated from oligo(dT)-primed rat brain library using a PCR product encoding tryptic peptide 1 of purified rat brain CaM-kinase kinase, and clone R5 was obtained from a random-primed rat brain cDNA library in gt10 using the 5` end EcoRI-StuI fragment of clone 7AL as the probe. Clones P32, P122, and P22 were obtained as 5`-RACE PCR products (see ``Experimental Procedures'' for cloning details). B, the nucleotide and deduced amino acid sequences are shown for CaM-kinase kinase with the two tryptic peptides (peptide 1 and peptide 2) obtained from purified rat brain CaM-kinase kinase underlined; the ATP-binding site is indicated by a dashedunderline. Only part of the 3` noncoding sequence is shown. C, total RNA (30 µg) from rat tissues was hybridized with a XbaI-XhoI fragment (540 bp) from clone P32. The estimated size of the major band (arrow) is about 3400 nucleotides.



Northern Analysis

Northern analyses of rat tissues were performed using a 0.54-kbp XbaI-XhoI fragment of clone P32. This probe hybridized to a 3.4-kb RNA, which was very abundant in forebrain, weaker in cerebellum, and detectable in thymus and spleen. This tissue distribution is similar to that of CaM-kinase IV except that CaM-kinase IV is more abundant in cerebellum than forebrain(18) .

Homology to Other CaM-kinases

Homology analyses of the deduced amino acid sequence of cloned CaM-kinase kinase cDNA revealed it was a unique protein that had considerable sequence identity (30-40%) between residues 121 and 408 with the catalytic domains of Ser/Thr protein kinases, especially with the CaM-kinase family (Fig. 2). Interestingly, CaM-kinase kinase had an insert of 22 residues, which is rich in Pro, Arg, and Gly, between the ATP-binding motif and the DGF kinase motif. An insert in this location is present in a few other protein kinases such as STE11(19) , a yeast homologue of Raf. Like CaM-kinase IV, the COOH terminus of CaM-kinase kinase was quite acidic with residues 374-505 being 24% Asp/Glu and only 13% Arg/Lys. Other than the conserved protein kinase region, no other conserved motifs were detected in CaM-kinase kinase.


Figure 2: Comparison of amino acid sequences of catalytic domains of CaM-kinase kinase with other protein kinases. Residues 121-408 of CaM-kinase kinase (CaMKK) are aligned with the catalytic domains of rat CaM-kinase II alpha subunit (CaMKII(28) ), rat CaM-kinase IV (CaMKIV (29)), rat CaM-kinase I (CaMKI(30) ), and rat cAMP-dependent protein kinase (cAPK(31) ). Identical residues are shown in boldface. The ATP-binding site and catalytic motifs are boxed. The peptide sequence in CaM-kinase II against which an antibody was generated is doubleunderlined.



Characterization of Expressed CaM-kinase Kinase

When the cDNA for CaM-kinase kinase was expressed in COS cells, a new 68-kDa protein was detected by Western blotting (Fig. 3A) using an antibody against residues 132-146 of CaM-kinase II (underlined in Fig. 2). This sequence in CaM-kinase II is highly conserved in Ser/Thr protein kinases including CaM-kinase kinase (Fig. 2). We next tested whether expressed CaM-kinase kinase bound Ca/CaM since the purified rat brain CaM-kinase kinase was purified by EGTA elution from CaM-Sepharose, and activation of CaM-kinase IV by the brain CaM-kinase kinase required Ca/CaM(10) . Gel overlay of the expressed CaM-kinase kinase with biotinylated CaM revealed strong binding of CaM to the 68-kDa protein. Although no obvious CaM-binding domain was detected by the homology search, it is possible that residues 444-457, which contain hydrophobic and basic residues, may constitute a basic amphipatic alpha-helix characteristic of CaM-binding domains.

Activation of CaM-kinase IV

The most important test of the cloned putative CaM-kinase kinase was whether the expressed protein activated CaM-kinase IV. In Fig. 3B the COS cell extract was incubated with Mg/ATP for 5 min in the presence of purified recombinant CaM-kinase IV and either EGTA, Ca, or Ca/CaM. The reaction was terminated by dilution and then assayed to determine the activity of CaM-kinase IV in the presence of EGTA (Ca-independent activity, openbars) or Ca/CaM (total activity, solidbars). In the absence of CaM-kinase kinase (i.e. buffer or mock-transfected COS cells) the total CaM-kinase IV activity was about 2.5 pmol/min, and it increased to about 4 pmol/min upon autophosphorylation in the presence of Ca/CaM. This small 1.5-2-fold activation upon autophosphorylation is consistent with previous reports for Sf9 cell-expressed CaM-kinase IV(4, 10) . However, when CaM-kinase kinase was expressed in the COS cells there was a 6-fold increase in total CaM-kinase IV activity and a 100-fold increase in its Ca-independent activity (Fig. 3B). Similar activation was observed with either wild-type CaM-kinase IV or a double mutant in which the overlapping CaM-binding and autoinhibitory domains were disrupted by mutation of Phe and Asn to Asp. This mutant no longer required Ca/CaM for activity, and it bound Ca/CaM very poorly(10) . The results of Fig. 3B, which were essentially identical to those obtained with the purified brain CaM-kinase kinase(10) , indicated that expressed CaM-kinase kinase not only bound Ca/CaM (Fig. 3A) but also required it for activity.

Activation of CaM-kinases I and II

A 52-kDa activator of CaM-kinase I has been purified from rat brain(14) , so it was of interest to determine whether our expressed 68-kDa CaM-kinase kinase could also activate CaM-kinase I. CaM-kinase I phosphorylates in vitro synapsin I (20) and the cystic fibrosis transmembrane conductance regulator(21) . Fig. 4A shows that CaM-kinase kinase catalyzed a 10-fold increase in the total activity of recombinant CaM-kinase I; unlike with CaM-kinase IV, there was no effect on Ca-independent activity of CaM-kinase I. CaM-kinase II is known to be regulated by autophosphorylation, which only increases its Ca-independent activity(22) . There is no strong evidence that its total activity can be increased by phosphorylation, and it was not activated by CaM-kinase kinase (Fig. 4B).


Figure 4: The effect of CaM-kinase kinase on CaM-kinase I and II activities. Purified Sf9 cell-expressed wild-type CaM-kinase I (2.0 µM, panelA) and alpha CaM-kinase II (2.0 µM, panelB) were incubated with either the lysate buffer (Buffer) or COS-7 cell lysate (180 ng) transfected with plasmid (Mock) or plasmid-expressing CaM-kinase kinase (CaMKK) in the presence of 1 mM CaCl(2) plus 10 µM CaM under the same conditions as described in Fig. 3B. CaM-kinase I and II activities were measured using 40 µM syntide-2 in the presence of either 1 mM EGTA (openbar) or 1 mM CaCl(2) plus 1 µM CaM (closedbar) under standard assay conditions(10) . Syntide-2 phosphorylation activity of the lysate from COS-7 cells transfected with pMECaMKK was also measured under the same conditions (righttwolanes in each panel). The results indicate the mean ± S.E. of three experiments using three independent transfections.



Enhanced Gene Expression by Co-expression of CaM-kinase Kinase and CaM-kinase IV

Considerable evidence exists that CaM-kinase IV is involved in transcriptional regulation through the phosphorylation of CREB(5, 6, 7) , but CaM-kinase IV is less potent than protein kinase A and may need to be activated by CaM-kinase kinase(5) . We further tested the CaM-kinase cascade hypothesis by determining whether co-transfection of COS cells with CaM-kinase kinase and CaM-kinase IV would enhance gene transcription compared with CaM-kinase IV alone. COS-7 cells transfected with either CaM-kinase IV or CaM-kinase kinase exhibited little CREB-dependent luciferase gene expression under these conditions (Fig. 5). The weak effect of transfection by CaM-kinase IV alone was consistent with the apparent lack of CaM-kinase kinase in COS-7 cells (Fig. 3). Co-transfection of CaM-kinase IV with CaM-kinase kinase gave a 14-fold enhancement of luciferase expression compared with transfection with either kinase alone (Fig. 5). However, the dependence on ionomycin for the transcriptional enhancement by CaM-kinase kinase was only about 2-fold (not shown). This small Ca effect could be due to Ca-independent activity of the overexpressed CaM-kinase kinase, or it could indicate that factors in addition to Ca are required for full activation of CaM-kinase kinase (see below). These possibilities are being explored.


Figure 5: Demonstration of the CaM-kinase cascade in CREB-mediated gene transcription. COS-7 cells were transfected with plasmid alone (Mock) or plasmid(s) encoding CaM-kinase IV (CaMK IV) and/or CaM-kinase kinase (CaMKK) as indicated with plasmids encoding a fusion protein for GAL4-CREB and GAL4-luciferase as described under ``Experimental Procedures.'' After 2 h in 5 µM ionomycin and 10 mM CaCl(2), luciferase activity was determined. Results are the mean ± S.E. for three dishes per condition.



Conclusions

The results presented in this paper are consistent with a signal transduction cascade involving CaM-kinase kinase and CaM-kinase IV, which regulates gene expression. Protein kinase cascades, such as the protein kinase A/phosphorylase kinase cascade (23) and the mitogen-activated protein kinase cascade(24) , are well established physiological pathways. Agonists that trigger the CaM-kinase cascade are currently being defined. Activation of the CD3 receptor in Jurkat cells, which stimulates tyrosine and Ser/Thr protein kinases and Ca mobilization via inositol trisphosphate formation(25) , gives a 10-20-fold activation of CaM-kinase IV (26) and also initiates Ca-dependent gene transcription(27) . This CD3-mediated activation of CaM-kinase IV is presumably due to CaM-kinase kinase since the CaM-kinase IV activation is reversed by treatment in vitro with protein phosphatase 2A but not phosphatase 1. (^2)We are currently investigating which agonists can activate CaM-kinase IV in various cells and whether Ca mobilization alone is sufficient to maximally activate CaM-kinase kinase or whether other second messengers are also required.


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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank®/EMBL Data Bank with accession number(s) L42810[GenBank].

§
To whom correspondence should be addressed: Vollum Institute L-474, 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 used are: CaM-kinase, Ca/CaM-dependent protein kinase; CaM, calmodulin; PAGE, polyacrylamide gel electrophoresis; CREB, cAMP-response element-binding protein; PVDF, polyvinylidene difluoride; PCR, polymerase chain reaction; bp, base pair(s); kbp, kilobase pair(s); RACE, rapid amplification of cDNA ends.

(^2)
A. Park and T. R. Soderling, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank D. McMillen (University of Oregon) for protein sequencing, Dr. A. Yamakawa (Dana Farber Cancer Institute) for providing the rat brain cDNA libraries, Drs. P. Sun and R. A. Maurer for the gift of Sf9-expressed CaM-kinase I, and Drs. Richard Goodman and John Scott for critically reading the manuscript.


REFERENCES

  1. Schulman, H. (1993) Curr. Opin. Cell Biol. 5,247-253 [Medline] [Order article via Infotrieve]
  2. Ohmstede, C. A., Jensen, K. F., and Sahyoun, N. E. (1989) J. Biol. Chem. 264,5866-5875 [Abstract/Free Full Text]
  3. Miyano, O., Kameshita, I., and Fujisawa, H. (1992) J. Biol. Chem. 267,1198-1203 [Abstract/Free Full Text]
  4. Cruzalegui, F. H., and Means, A. R. (1993) J. Biol. Chem. 268,26171-26178 [Abstract/Free Full Text]
  5. Enslen, H., Sun, P., Brickey, D., Soderling, S. H., Klamo, E., and Soderling, T. R. (1994) J. Biol. Chem. 269,15520-15527 [Abstract/Free Full Text]
  6. Sun, P., Enslen, H., Myung, P. S., and Maurer, R. A. (1994) Genes & Dev. 8,2527-2539
  7. Matthews, R. P., Guthrie, C. R., Wailes, L. M., Zhao, X., Means, A. R., and McKnight, G. S. (1994) Mol. Cell. Biol. 14,6107-6116 [Abstract]
  8. Jensen, K. F., Ohmstede, C. A., Fisher, R. S., and Sahyoun, N. (1991) Proc. Natl. Acad. Sci. U. S. A. 88,2850-2853 [Abstract]
  9. Frangakis, M. V., Ohmstede, C. A., and Sahyoun, N. (1991) J. Biol. Chem. 266,11309-11316 [Abstract/Free Full Text]
  10. Tokumitsu, H., Brickey, D. A., Gold, J., Hidaka, H., Sikela, J., and Soderling, T. R. (1994) J. Biol. Chem. 269,28640-28647 [Abstract/Free Full Text]
  11. Okuno, S., and Fujisawa, H. (1993) J. Biochem. (Tokyo) 114,167-170 [Abstract]
  12. Okuno, S., Kitani, T., and Fujisawa, H. (1994) J. Biochem. (Tokyo) 116,923-930 [Abstract]
  13. Enslen, H., Tokumitsu, H., and Soderling, T. R. (1995) Biochem. Biophys. Res. Commun. 207,1038-1043 [CrossRef][Medline] [Order article via Infotrieve]
  14. Lee, J. C., and Edelman, A. M. (1994) J. Biol. Chem. 269,2158-2164 [Abstract/Free Full Text]
  15. Brickey, D. A., Colbran, R. J., Fong, Y. L., and Soderling, T. R. (1990) Biochem. Biophys. Res. Commun. 173,578-584 [Medline] [Order article via Infotrieve]
  16. Sun, P., and Maurer, R. A. (1995) J. Biol. Chem. 270,7041-7044 [Abstract/Free Full Text]
  17. Kozak, M. (1991) J. Cell Biol. 115,887-903 [Abstract]
  18. Frangakis, M. V., Chatila, T., Wood, E. R., and Sahyoun, N. (1991) J. Biol. Chem. 266,17592-17596 [Abstract/Free Full Text]
  19. Rhodes, N., Connell, L., and Errede, B. (1990) Genes & Dev. 4,1862-1874
  20. Nairn, A. C., and Greengard, P. (1987) J. Biol. Chem. 262,7273-7281 [Abstract/Free Full Text]
  21. Picciotto, M. R., Cohn, J. A., Bertuzzi, G., Greengard, P., and Nairn, A. C. (1992) J. Biol. Chem. 267,12742-12752 [Abstract/Free Full Text]
  22. Colbran, R. J., and Soderling, T. R. (1990) Curr. Top. Cell Regul. 31,181-221 [Medline] [Order article via Infotrieve]
  23. Walsh, D. A., Perkins, J. P., Brostrom, C. O., Ho, E. S., and Krebs, E. G. (1971) J. Biol. Chem. 246,1968-1976 [Abstract/Free Full Text]
  24. Nakielny, S., Cohen, P., Wu, J., and Sturgill, T. (1992) EMBO J. 11,2123-2129 [Abstract]
  25. Howe, L. R., and Weiss, A. (1995) Trends Biochem. Sci. 20,59-64 [CrossRef][Medline] [Order article via Infotrieve]
  26. Hanissian, S. H., Frangakis, M., Bland, M. M., Jawahar, S., and Chatila, T. A. (1993) J. Biol. Chem. 268,20055-20063 [Abstract/Free Full Text]
  27. Irving, S. G., June, C. H., Zipfel, P. F., Siebenlist, U., and Kelly, K. (1989) Mol. Cell. Biol. 9,1034-1040 [Medline] [Order article via Infotrieve]
  28. Lin, C. R., Kapiloff, M. S., Durgerian, K., Tatemoto, K., Russo, A. F., Hanson, H., Schulman, H., and Rosenfeld, M. G. (1987) Proc. Natl. Acad. Sci. U. S. A. 84,5962-5966 [Abstract]
  29. Means, A. R., Cruzalegui, F., LeMagueresse, B., Needleman, D. S., Slaughter, G. R., and Ono, T. (1991) Mol. Cell. Biol. 11,3960-3971 [Medline] [Order article via Infotrieve]
  30. Picciotto, M. R., Czernik, A. J., and Nairn, A. C. (1993) J. Biol. Chem. 268,26512-26521 [Abstract/Free Full Text]
  31. Wiemann, S., Voss, H., Kinzel, V., and Pyerin, W. (1991) Biochim. Biophys. Acta 1089,254-256 [Medline] [Order article via Infotrieve]
  32. Miranti, C. K., Ginty, D. D., Huang, G., Chatila, T., and Greenberg, M. E. (1995) Mol. Cell. Biol. 15,3672-3684 [Abstract]

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