of vol. 268, no. 35, of 15, pp. 26171-26176,1993 1993 by ... · the journal of biological chemistry...

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1993 by The American Society for Biochemistry and Molecula~ ’ Biology, Inc.

Vol. 268, No. 35, Issue of December 15, pp. 26171-26176,1993 Printed in U.S.A.

Biochemical Characterization of the Multifunctional Ca2+/Calmodulin- dependent Protein Kinase Type IV Expressed in Insect Cells*

(Received for publication, April 12, 1993, and in revised form, August 13, 1993)

Francisco H. CruzaleguiS and Anthony R. Means$ From the Department of Pharmacology, Duke University Medical Center, Durham North Carolina 27710

We have expressed the rat brain Caz+/calmodulin (CaM)-dependent protein kinase type IV in insect cells. The recombinant enzyme is produced as a single polypeptide that migrates on SDS-polyacrylamide gel electrophoresis at 61 kDa. Recombinant CaM kinase IV undergoes slow CaM-dependent autophosphorylation. The autophosphorylation of CaM kinase IV occurs on serine residues but is not accompanied by the generation of a CaM-independent activity, as previously reported for the cerebellar enzyme. Comparison of peptide and protein phosphorylation by the recombinant CaM kinase IV and the cerebellar enzyme showed differences in their catalytic activities. The deduced primary sequence of CaM kinase IV contained a domain, 316Phe-Asn-Ala-Arg-Arg-Lys-Leu-Lys323, also found in the regulatory domain of CaM kinase IIa (residues 293-300). Truncation of CaM kinase IV at Leu313 (at a position analogous to Leuzs0 in CaM kinase IIa) generated a fully active, CaM-independent enzyme. This truncated enzyme no longer bound CaM. These data confirm that CaM kinase IV demonstrates intrasteric regulation by an autoinhibitory domain and provides insight into a potentially common mechanism for the regulation of the CaM-dependent multifunctional protein kinases. A number of synthetic peptides were examined for their phosphorylation by both CaM kinase I1 and IV. These studies showed that several peptides derived from phospholamban were preferential substrates for CaM kinase I1 whereas a peptide derived from S6 ribosomal protein was selectively phosphorylated by CaM kinase IV. Kinetic analysis of several peptide substrates suggests that while both CaM kinase I1 and IV recognize the sequence motif represented by R-X-X-T/S, other structural features are also involved in defining the unique substrate specificity of CaM kinase IV.

~~

The multifunctional Ca2+/CaM1-dependent protein kinase

* This work was supported by National Institutes of Health Grant HD 07503. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Present address: Transcription Laboratory, Imperial Cancer Re- search Fund, Lincoln’s Inn Fields, London WC2A 3PX, UK.

8 To whom all correspondence should be addressed Dept. of Phar- macology, Box 3813, Duke University Medical Center, Durham, NC 27710. Tel.: 919-681-6209; Fax: 919-681-8461.

’ The abbreviations used are: CaM, calmodulin; CaM kinase I1 or IV, the Ca*+/calmodulin-dependent protein kinase types I1 or IV, respectively; MLC, myosin light chain; cAPK, cyclic AMP dependent protein kinase; CREB, cyclic AMP response element binding protein; C/EBP, CAAT/enhancer binding protein; SRF, serum response factor; ERK2, extracellular regulated protein kinase type 2; MAP, mitogen-activated protein kinase; smMLCK, smooth muscle myosin light chain kinase.

was first identified in the rat brain and is frequently referred to as Ca2+/CaM-dependent protein kinase I1 (CaM kinase 11) (1). Multiple isoforms of this enzyme have been cloned and are the products of distinct genes (2). lsoforms of CaM kinase I1 are present in many vertebrate tissues. By analogy to the intracellular actions of the second messenger, CAMP, which are mediated predominantly if not exclusively by the ubiqui- tous CAMP-dependent protein kinase, it has been suggested that many Ca2+-dependent events may be regulated by CaM kinase I1 (1). However, a second multifunctional Ca2+/CaM- dependent protein kinase has been identified in rat brain that shares approximately 40% identity with the CaM kinase I1 isoforms in its catalytic domain (3, 4). This enzyme, first discovered in the granule cell layer of the rat cerebellum and called CaM kinase-Gr (5), has been renamed CaM kinase IV following the demonstration that it was expressed in many other cells (3,4, 6, 7).

The amino acid sequence of CaM kinase IV predicted from the rat and mouse brain cDNAs (3,4,8) reveals an organiza- tion of domains very similar to the CaM kinase I1 subunits. The NH2-terminal half of CaM kinase IV contains the protein kinase domain (9). This is followed by a putative regulatory region which, by analogy with CaM kinase 11, may be involved in CaM binding and autoinhibition. Eight residues (Phe3I6- L Y S ~ * ~ ) in this region are identical to the sequence PheZg3- Lys3O0 in CaM kinase IT previously shown to be important for intrasteric inhibition of this enzyme (10). The COOH terminus of CaM kinase IV is only 15% identical to that of CaM kinase IIa and contains stretches of acidic residues that may be involved in its association with other proteins.

CaM kinase IV has also been purified from rat cerebellum and shows a sedimentation coefficient consistent with a monomeric protein of apparent Mr 67,000 (5 , 11). However, SDS- polyacrylamide gel electrophoresis of the purified enzyme showed two polypeptides of 61 and 64 kDa respectively. How- ever, only the smaller species is observed in forebrain preparations (11) or by in vitro translation of a full-length cDNA for CaM kinase IV (3). Since the known enzymatic properties of CaM kinase IV correspond to those of the cerebellar enzyme, the biochemical properties of the single protein product represented by the cDNA cloned from rat brain required investigation. In order to characterize this gene product, we produced CaM kinase IV in insect cells using the baculovirus system. Here, we report biochemical properties of the purified recombinant enzyme, including its subunit structure, modes of regulation, and substrate recognition. By a truncation which removed a regulatory domain also found to produce a constitutive CaM kinase IIa (lo), we have generated a constitutively active CaM kinase IV. These studies show that the intrasteric mode of regulation has been conserved in both CaM kinase I1 and CaM kinase IV and involves a similar autoinhibitory domain. Our results demonstrate that CaM kinase IV also shows many differences from CaM kinase I1

26171

26172 Recombinant Ca2+/CaM-dependent CaM Kinase IV

including its tertiary structure, regulation by autophosphorylation and the determinants for substrate recognition. These studies set the stage for future experiments which will inves- tigate the unique role of the two multifunctional kinases in calcium signaling in mammalian cells.

EXPERIMENTAL PROCEDURES

The plasmid vector pVL1393 for expression in insect cells was a gift from Dr. Ming-Jer Tsai (Baylor College of Medicine, Houston, TX) with permission from Dr. Max Summers (Texas A & M Univer- sity, College Station, TX). BlueBac I1 and transfection module were obtained from Invitrogen (Sorrento, CA). Site-directed mutagenesis kits and radioisotopes were purchased from Amersham. Chicken calmodulin was produced by bacterial expression as described before (12). GSlO peptide and a panel of peptide substrates were a kind gift from Dr. Bruce Kemp (St. Vincent's Institute for Medical Research, Melbourne, Australia). Autocamptide was synthesized at Research Genetics, Inc. (Huntsville, AL). Syntide-2 was purchased from Sigma. Grace's medium and fetal bovine serum were obtained from Life Technologies, Inc.

DNA Constructions and Mutagenesis-The cDNA encoding the rat brain CaM kinase IV (3) was introduced into the SmaI site of pVL1393 as a ApaII/SalI fragment resulting in pVLCaM kIV. The cDNA for the CaM kinase I1 a subunit was introduced in BlueBac I1 by ligation of a XbaI/BarnHI fragment into the NheIIBamHI sites. The BarnHIIPstI fragment was inserted into M13mp8 to produce single stranded DNA and mutagenesis was carried out according to Olsen and Eckstein (13) to generate a truncation mutant. The oligo- nucleotide 5' GCG TCC AGC ATT GAA TTT CTA GAG CTT TTT CTG AGC GGT ATC CAT GTG 3' was used for the generation of the truncation mutant. The mutant cDNA was transferred into pVLCaMkIV and transfected into insect Sf9 cells.

Cell Culture and Selection of Recombinant Baculouirus-Sf9 cells were maintained as monolayers and suspension cultures at 27 "C in supplemented Grace's medium containing 10% fetal bovine serum. Co-transfection of cells with pVLCaMkIV and wild-type baculovirus was carried out by the calcium phosphate method. Selection of recombinant virus was accomplished using the agarose overlay assay (14). To obtain recombinant virus encoding mutant CaM kinase IV, co-transfection was performed using cationic liposomes (Invitrogen). Selection of BlueBac I1 recombinants required the addition of 150 pg/ml X-Gal to the agarose.

Expression and Purification of CUM Kinase ZV-For large scale enzyme preparations, 20 X 10' cells in suspension culture were infected at a multiplicity of 10 pfu/cell. After 72 h, cells were har- vested, washed once with 1 X phosphate-buffered saline, resuspended in 10 ml of buffer A (25 mM Hepes, pH 7.5, 1 mM dithiothreitol, 2 mM EDTA, 2 mM EGTA, 50 mM NaCl, and 10% glycerol), and homogenized by sonication for 10 s using a Branson Sonifier 250. The suspension was clarified by centrifugation at 12,000 X g. The supernatant fluid was applied to a DE52 column equilibrated with buffer A. After washing the column with buffer A + 150 mM NaCl, CaM kinase IV was eluted with buffer A + 250 mM NaCl. Fractions were assayed for CaM kinase IV. The peak of kinase activity was pooled, made 2 mM CaC12 and loaded on a CaM-Sepharose column. After washing the column extensively with 2 mM CaC12, the kinase was eluted with a buffer containing 4 mM EGTA. All results presented are directly comparable as they were obtained with a single preparation of recombinant CaM kinase IV; in most cases a second preparation was used to confirm the values obtained.

CaM kinase IIa was similarly obtained from infected cells using the baculovirus system. The cell extract, prepared as described above, was loaded on a phosphocellulose column, washed with buffer A containing 250 mM NaCl and CaM kinase I1 and eluted at 450 mM NaCl. Gel filtration was carried out by fast pressure liquid chromatography on Superdex 200 HiLoad (Pharmacia LKB Biotechnology Inc.) in a buffer containing 25 mM Hepes, pH 7.5,0.5 mM EDTA, 0.5 mM dithiothreitol, 150 mM NaC1, and 10% glycerol at a flow rate of 1 ml/min. Thyroglobulin (669,000), ferritin (440,000), catalase (232,000), aldolase (158,000), bovine serum albumin (67,000), oval- bumin (43,000). and chymotrypsinogen A (25,000) were used as molecular weight standards. The fractions (2 ml) were assayed for protein kinase activity using GSlO peptide as a substrate.

Protein concentrations were determined by the method of Bradford (15).

Protein Kinase Assays-Phosphorylation assays (50 pl) were car-

ried out in 50 mM Hepes, pH 8.0, 0.5 mM dithiothreitol, 10 mM magnesium acetate, 100 p M ATP, 1 pM CaM, and either 1 mM CaC12 or 5 mM EGTA containing 100 p~ GSlO peptide (Pro-Leu-Arg-Arg- Thr-Leu-Ser-Val-Ala-Ala) at 30 "C (16). The reactions were initiated by addition of kinase. Specific activity of [y3'P]ATP ranged between 100 and 5000 cpm/pmol. Aliquots (10 pl) were removed, applied to P81 phosphocellulose discs (Whatman), and washed in 75 mM phos- phoric acid according to Roskoski (17). Unless stated otherwise the time used for CaM kinase IV was 6 min and for CaM kinase IIa, 30 s. Phosphorylation of protein substrates was carried out under similar conditions but reactions were stopped by addition of 4 X Laemmli sample buffer (18) and analyzed on 10% SDS-polyacrylamide gels. Autophosphorylation of CaM kinase IV was monitored in this assay in the absence of substrate. Proteins were visualized by staining with Coomassie Brilliant Blue. The gels were dried and subjected to autoradiography. Incorporation of 32P was determined by counting the excised bands as Cerenkov radiation or by analysis of on a Betascope 603 PhosphorImager (Betagen, Waltham, MA). The CURVE-FIT program designed by M. Smialek and obtained through Dr. A. Sobieszek (Austrian Academy of Sciences, Salzburg) was used to determine the kinetic constants (19).

Phosphoamino Acid Analysis-The purified CaM kinase IV (1-2 pg of protein) was subjected to autophosphorylation for 30 min, separated by SDS-PAGE and transferred to a poly(viny1idene fluo- ride) membrane. The membrane containing the kinase was hydro- lyzed in 6 M HCl at 110 "C overnight. The supernatant fluid was dried, resuspended in a buffer containing phosphoamino acid standards (Ser(P), Thr(P), and Tyr(P)), and subjected to two-dimensional electrophoresis on cellulose acetate thin layer plates. The first dimension was run at pH 1.9 and 700 V for 2-3 h and second dimension at pH 3.5 and 700 V for 1 h.

RESULTS

Expression of CaM Kinase I V in Insect Cells-Infection of Sf9 insect cells for 72 h with recombinant virus containing CaM kinase IV cDNA led to a 20-fold increase in the Ca2+/ CaM-dependent kinase activity of crude cell lysates, when compared to cells infected with the wild-type baculovirus. Recombinant CaM kinase IV was purified from cell extracts by ion exchange chromatography on DEAE-cellulose and CaM-affinity chromatography. As shown in Fig. lA, CaM kinase IV was purified to near homogeneity and was seen as a single 61-kDa polypeptide, by SDS-PAGE. Fig. 1B shows that the purified CaM kinase IV rapidly phosphorylated GS10, a peptide substrate mimicking the site-2 phosphorylated on glycogen synthase, in a Ca2+/CaM-dependent manner. GSlO was maximally phosphorylated after 10 min at 30 "C.

The recombinant CaM kinase IV eluted at a M, greater than 67,000, on gel filtration chromatography (Fig. IC) at a position compatible with its calculated stokes radius of 39 A (data no shown). This elution profile could indicate a dimer but as elution from gel filtration is independent of M, (33), it is more likely to be the result of the elongated shape of the molecule as determined previously by sedimentation equilib- rium analysis of the cerebellar enzyme (11, 20). The baculovirus system was also used to express the a subunit of rat brain CaM kinase I1 (CaM kinase IIa). The purified recombinant CaM kinase IIa (Fig. L4) eluted from the gel filtration column with an approximate M, of 600,000 (Fig. IC) consistent with the multimeric nature of the enzyme purified from many tissues.

The catalytic properties of the purified recombinant CaM kinase IV were compared to those reported for CaM kinase IV purified from cerebellum (Table I). Both enzymes dem- onstrated similar apparent K, values for ATP. However, the recombinant enzyme showed a significantly higher K, for both peptide and protein substrates (syntide-2 and synapsin I). However, the recombinant enzyme showed 2-fold higher maximal rate of phosphorylation for both of these substrates than reported for the cerebellar enzyme. The recombinant enzyme also showed a higher for activation by CaM.

Recombinant Ca2+/CaM-dependent CaM Kinase IV 26173

A 97.4-

66.2-

45 -

31 -

21.5-

20 30 40 50 60 70 80 90 100 110 120 ml

FIG. 1. Characterization of CaM kinase IV produced in insect cells. A, 12% SDS-polyacrylamide gel showing purified recombinant CaM kinase IV and CaM kinase 110 (silver-stained). B, time course of phosphorylation of GSlO peptide by recombinant CaM kinase IV in the presence of CaM and Ca2' ( sqwres) or EGTA (circles). C, elution profiles of CaM kinase IV (circles) and CaM kinase IIa ( sqwres) from a Superdex 200 gel filtration column; M , standards are shown with arrows.

TABLE I Kinetic parameters of recombinant CaM kinase IV

Apparent K,,, and V,. values for the phosphorylation of syntide-2 and synapsin I by the purified baculovirus-expressed CaM kinase IV were compared to values reported for the enzyme isolated from cerebellum. Phosphorylation of syntide-2 and synapsin I was carried out using 100 p~ ATP. K&aM was obtained using 100 p~ ATP and 100 p M GSlO peptide substrate. All assays were done in duplicate and differences in values were approximately 10%. The assays for K,,, for ATP and K, for CaM were also replicated using a different preparation of CaM kinase IV. Error determinations were as follows: K,,, for ATP (+2 p ~ ) ; K,,, and V,. values for syntide-2 and synapsin 1 (less than 10%); K&aM (k42 nM). Numbers in parentheses are reference citations.

Parameter Cerebellar CaMkIV Recombinant MkIV K,,, for ATP 19-48 FM (11,20) 27 pM K,,, for syntide-2 2.6-12 p~ (11,20) 187 p~ V,. for syntide-2 152 nmol/min/mg (11) 356 nmol/min/mg K,,, for synapsin I 1.2 p~ (5) 22.2 pM V,. for synapsin I 300 nmol/min/mg (5) 507 nmol/min/mg K , CaM 32 nM (11) 158 nM

~~

Autophosphoryhtion-Cerebellar CaM kinase IV has been reported to undergo stoichiometric CaM-dependent autophosphorylation, leading to a CaM-independent activity that was higher than the maximal activity stimulated by CaM prior to autophosphorylation (20). As shown in Fig. 2, A and B, a slow autophosphorylation of recombinant CaM kinase IV was also seen, reaching a maximum after 3-4 h of incu-

hours

B

C

I ' I ' I ' I 0 1 2 3 4 5 6 7

hours

!pH 1.9

& pH 3.5

FIG. 2. Autophosphorylation of CaM kinase IV. A , SDS-polyacrylamide gel showing a time course of autophosphorylation using 0.56 pg (10.6 pmol) of purified baculovirus expressed enzyme. B, quantitation of the autophosphorylation of CaM kinase IV kinase shown in A. C, phosphoamino acid analysis of CaM kinase IV autophosphorylated for 90 min, showing phosphorylation of serine only.

bation. The maximal autophosphorylation of CaM kinase IV was approximately 0.1 mol of phosphate/mol of enzyme. As reported for the cerebellar enzyme (20) autophosphorylation of CaM kinase IV occurred only on serine residues (Fig. 2C). Varying the concentration of the recombinant CaM kinase IV in the assay from 13 to 260 nM did not affect the rate of autophosphorylation (data not shown) nor was autophosphorylation increased by carrying out the reaction at room temperature (as described in Ref. 20). As seen in Fig. lB, phosphorylation of exogenous substrates, such as the GSlO synthetic peptide, reached a maximum after 10 min, when little autophosphorylation could be observed. This suggests that autophosphorylation of CaM kinase IV was not essential for activity. Effects of autophosphorylation on enzyme activity measured in GSlO kinase assays following preincubation with ATP, CaM, and Ca2+ or EGTA were monitored. Even after 90 min of the Ca2+/CaM-dependent autophosphorylation reaction, no significant increase in CaM-independent activity was observed (Fig. 3). However, the maximal Ca2+/ CaM-dependent activity was increased 1.5-fold by autophosphorylation, as also reported for the cerebellar CaM kinase IV (20). The higher CaM kinase IV activity following autophosphorylation may not be significant and may not be the result of a change in affinity for CaM as we only observed a slight decrease in the ECso for CaM from 158 to 126 nM. Unlike CaM kinase 11, which is unstable to prolonged incu-

26174 Recombinant Ca2+/CaM-

120 I4Ol T

60

40

20

I CONTROL ' AUTOPH. I

FIG. 3. Effect of autophosphorylation of CaM kinase IV on CaM-dependent and CaM-independent activity. Autophosphor- ylation was carried out for 90 min at 30 "C in the presence of CaM and CaZ+ (AUTOPH.) or EGTA (CONTROL). GSlO kinase activity was then measured in the presence of CaM and Ca2+ (filled bars) or EGTA (hutched bars). Error bars indicate the variation between values obtained in two separate experiments each done in duplicate. The two experiments each utilized a different preparation of recombinant CaM kinase IV.

bations at temperatures above 4 "C, no significant decrease in enzyme activity was seen with similar incubations of CaM kinase IV at 30 "C (Fig. 3).

Identification of an Autoinhibitory Domain in CaM Kinase IV-We and others have shown that removal of the COOH- terminal half of CaM kinase 11, including the autoinhibitory domain by proteolysis or mutagenesis generates a constitutively active form of the enzyme. Whereas truncation of CaM kinase IIa at Leuzgo produced a constitutive enzyme, extension by only four residues, 291Lys-Lys-Phe-Asn294, inhibited activity completely and were therefore suggested to be involved in intrasteric inhibition (10). As shown in Fig. 44, alignment of the amino acid sequences of CaM kinases IIa and IV reveals that they have conserved 8 residues in the regulatory domain, beginning with Phe316 of CaM kinase IV and Phem3 of CaM kinase IIa. Based on this conservation, we generated a truncation of CaM kinase IV at a site equivalent to Leum0 of CaM kinase 11. To accomplish this truncation Gln314 in CaM kinase IV was replaced by a termination codon. The mutant polypeptide, expressed in insect cells, eluted from anion-exchange chromatography on DEAE-cellulose at lower salt concentrations than the wild-type protein (150 mM as opposed to 200 mM), presumably due to the loss of the highly acidic COOH terminus. As shown in Fig. 4B, the purified mutant kinase showed a lower apparent M, compared to wild-type CaM kinase IV on SDS-polyacrylamide gels and also did not bind CaM in an overlay assay (data not shown). These data indicated that the truncation at Leu313 also removed the CaM- binding domain. In contrast to the wild-type enzyme (stokes radius 39 A), the truncated CaM kinase IV eluted on gel filtration between the 25- and 43-kDa markers at a stokes radius value of 27 A (data not shown).

truncation mutant in 5 mM EGTA was similar to that ob- Using synapsin I as substrate, the activity of the

-dependent CaM Kinase IV

served in the presence of the wild-type enzyme assayed in the presence of Ca2+/CaM (Fig. 4C). As shown in Fig. 40, even after 90 min of incubation in the presence or absence of Ca"/ CaM, autophosphorylation of the truncated purified protein was not detected, suggesting that the major autophosphorylation sites on CaM kinase IV are located within the 181 amino acids deleted from the COOH-terminal region.

Substrate Specificity-Although several proteins are substrates for both CaM kinase I1 and IV, their relative rates of phosphorylation by CaM kinase IV were orders of magnitude lower than by CaM kinase I1 (11). To date, only the synthetic peptide derived from the y subunit of CaM kinase I1 (Lys- Ser-Asp-Gly-Gly-Val-Lys-Lys-Arg-Lys-Ser-Ser-Ser-Ser) was better phosphorylated by CaM kinase IV (11). We have analyzed 28 synthetic peptides, which represented phosphorylation sites present in many different substrates of protein Ser/ Thr kinases, as substrates (at 300 p~ peptide concentration) for CaM kinase IV. The peptides were also examined for their phosphorylation by CaM kinase IIa. The values obtained with each peptide were compared with the standard GSlO peptide substrate (Table 11).

We found that several synthetic peptides derived from the yeast transcription factor ADRl(21) and which are substrates for CAMP-dependent protein kinase (cAPK), were also excellent substrates for CaM kinases IV and 11. In general, the best peptide substrates for CaM kinase IV contained an arginine at position -3 from the phosphorylated serine. This was most strikingly shown in the ADRl peptide 43, where a replacement of the arginine at position -3 with leucine greatly reduced its phosphorylation by both CaM kinase IV and 11. These data have been used to generate a consensus motif for CaM kinase IV of R-X-X-S/T-X. This consensus is also recognized by CaM kinase I1 and indicates that both CaM kinases share overlaping structural requirements for substrate recognition. In contrast to most of the ADRl peptides, the synthetic peptides derived from myosin light chain (MLC) or glycogen synthase (GS10 and GS(1-12)) did not contain a second basic residue at position -2 but were still substrates for CaM kinase IV. This contrasts with cAPK, which has been shown to require two basic residues at positions -3 and -2 for optimal substrate phosphorylation. Introduction of a hydrophobic residue, isoleucine, at position +2 in the ADRl peptide 48, decreased its phosphorylation by CaM kinase IIa and IV. Yet other substitutions, both NH2- and COOH-terminal to the phosphorylated serine in the ADRl as well as in the MLC and S6 peptides, differentially inhibited peptide phosphorylation by CaM kinase IV and CaM kinase 11. This suggests that the precise sequence surrounding the phosphorylation site may be an important discriminator of substrate recognition by these CaM kinases. Some of the peptides derived from MLC (nos. 22, 23, and 25) phospholamban (nos. 40 and 50) and phosphorylase kinase (no. 59) were significantly better substrates for CaM kinase IIa emphasizing that, although the multifunctional CaM kinases show overlaping substrate spec- ificities, the primary sequence in the vicinity of the phosphorylation site defines the ability of the peptides to act as effective substrates for these kinases.

A peptide derived from ribosomal protein S6 (no. 18) with the sequence Ala-Lys-Arg-Arg-Leu-Ser-Ser-Leu-Ala-Ala, was significantly better phosphorylated by CaM kinase IV than by CaM kinase 11. More detailed kinetic analysis of S6 peptide 18 phosphorylation by CaM kinase IV showed an apparent K,,, = 65.8 ,AM and a V,, = 120 nmol/min/mg. Although this was overall a poorer substrate for CaM kinase IV than the GSlO peptide, its virtual inability to act as a substrate for CaM kinase I1 may make this peptide useful in developing a

Recombinant Ca2+/CaM-dependent CUM Kinase IV

A "" Truncation Catalytic domain

284 290 t

' 3 0 0 I I Y O 340

CaMk IV --I? L T T F Q ~ H F W VT. .GKAANFV HMDTAQKKLQEFNARRKLKAAVKAW ASSRL GSAS S SHT -- CaMk Ila -7 I TAAEALKHT ISHRSTVASYMHRQET VDC\KKFNARRKLFGAI LT TM LATTN FSGG KSGV--

259 2 i o

W L313 t

kDa

97.4-

66.2-

45-

31 -

21.5-

WT L313

@ + - + - "

kDa

92.5 - 69 - -+- 0.

46 -

30 -

i l o 3 i 0 3 m

WT L313

@ + - + - "

kDa

92.5 - 69 - -0

46 .

30 -

26175

B C D FIG. 4. Truncation of CaM kinase IV at LeuS*' generates a constitutively active enzyme. A , alignment of the regulatory domains

of CaM kinase IV and CaM kinase IIa, indicating the site of truncation. TrpZg5 is the last residue conserved in the catalytic domain. B, silver- stained gel showing the purified wild-type and truncation mutant. C, phosphorylation of the approximately 85-kDa substrate, synapsin I (arrow) by the wild-type enzyme and the constitutively active truncation mutant shown by autoradiography. D, 90-min autophosphorylation reaction using the purified wild-type and truncation mutant enzymes.

TABLE I1 Phosphorylation of synthetic peptides by recombinant CUM kinases IV and I I

Peptides were assayed in duplicate reactions at a concentration of 300 PM. Variation between duplicate values was less than 20%. Level of phosphorylation is expressed as a percentage of the GSlO phosphorylation seen in parallel assays. The time used to assay CaM kinase IV was 6 min and that for CaM kinase IIa was 30 s.

CaMkIV ac- CaMkII activity (com-

pared to tivity (com-

GSIO) pared to GS10)

Peptide (serial no.) Sequence

GSlO

Kemptide GS[l-l2]K11,12 (32)

ADR1[225-2411 (30) [222-2341 (36)

(43) (46)

(48) (41) (47)

(42)

MLC [ll-231 (7) (8)

(11) (12) (9)

(62)

(22) (25)

(23)

Phospholamban [8-211 (40) Phospholamban [l-311 (50) Phospholamban kinase A [1014-10231 (59) cGMP-dependent kinase [72-831 (45) HMG CoA reductase [861-8761 (60) S6 [229-2391 A235 (17) S6 [229-2391 A238 (18) Autocamtide

PLRTLSVAA PLSRTLSVAAKK

LRRASLG LTRRASFSAQSASSYAL

LKKLTRRASFSAQ LKKLTLRASFSAQ LKKLTRKASFSAQ LKKLTRRASFRAQ LKKLTRRASFIAQ LKKLTRRASFSGQ LKKLTRRASSSAQ AKRPQRATSNVFA KARPQRATSNVFA KKAPQRATSNVFA KKRAARATSNVFA KKRPRAATSNVFA KKRWQRATSNVFA KKRPQRAVSNVFA KKRPQRAGSNVFA KKRPGRALSNVFA

TRSAIRRASTIEMP MDKVQYLTRSAIRRASTIEMPQQARQNLQNL

FRRLSISTES PRTKRQAISAEP

HLVKSHMINHNRSKINL AKRRRLASLRA AKRRRLSSLAA

KKALRROETVDAL

100 76 2

121 92

5 113 53 5

165 147 24 31 22 23 6 3 6 4

14 19 12 5 2 1 4

26 20

100 129

9 57 72

5 57 27 6

76 138 29 48 23 36 9 2

23 15 46

120 66 27 3 2 4 6

75

26176 Recombinant Ca2+/CaM-dependent CaM Kinase IV

more selective assay for CaM kinase IV. Selected peptides derived from ADR1, which were identified

as reasonable substrates for CaM kinase IV, were further analyzed for their kinetics of phosphorylation by this kinase under defined conditions. As shown in Table 111, many of these peptides were better phosphorylated by CaM kinase IV than the standard GSlO peptide substrate. ADRl (no. 36) and ADRl (no. 41) were five to six times better substrates than GS10. The maximal rate of phosphorylation of the ADRl peptide 41 by the recombinant CaM kinase IV was also significantly higher than that reported for the cerebellar CaM kinase IV using either syntide-2 or the y-peptide of CaM kinase I1 as substrates (11). Based on the V,, to K,,, ratio, peptide 36 was the best substrate. However, peptide 41 showed the highest rate of phosphorylation reported to date for any substrate of CaM kinase IV.

Phosphorylation of Transcription Factors-The analysis described above indicated that peptides derived from ADR1, a yeast transcription factor, were excellent substrates for CaM kinase IV. Therefore, we examined the phosphorylation of three mammalian transcription factors by the recombinant CaM kinase IV. The transcription factors CREB (CAMP response element binding protein), C/EBP 0 (a CAATlen- hancer binding protein) and SRF (the serum response factor) were chosen since each require phosphorylation for activity and at least two of these, CREB and C/EBP@ had been previously reported to be phosphorylated in a Ca2+-dependent reaction (22-27). Fig. 5 shows that recombinant CaM kinase IV will phosphorylate CREB and SRF in a Ca2+ dependent manner, but fails to phosphorylate C/EBPB. On the other hand, both CREB and C/EBPB can be phosphorylated by recombinant CaM kinase IIa consistent with previous reports (22-24). It can be seen in Fig. 5 that CaM kinase IV undergoes autophosphorylation in the presence of Ca2+ under the reaction conditions used (see the + lanes of AutoP, CIEBPB, and CREB). It is also apparent that SRF and CaM kinase IV comigrate in these gels so that a small portion of the radio- activity shown in the SRF (+ Cd+) lane in Fig. 5B is due to autophosphorylation of the kinase.

DISCUSSION

We have expressed Ca2+/CaM-dependent protein kinase type IV in insect cells and have characterized the biochemical properties of the purified enzyme. In contrast to CaM kinase IIa expressed in the same system, CaM kinase IV appears to be a monomeric enzyme and does not show stoichiometric autophosphorylation or the generation of a CaM-independent

TABLE I11 Kinetic analysis of phosphorylation of peptide substrates by

recombinant CUM kinase IV Assays for each concentration of peptide substrate were performed

once in duplicate except for peptide ADRl (30) which was assayed twice in duplicate. In all cases duplicate values varied from 10 to 20%. Kinetic parameters were derived by entering all values into the CURVEFIT program which makes use of every data point to calculate K, and V-.

Peptide Sequence Km V- V-lK, p~ nmolfminlmg

Syntide-2 PLART LSVA GLPGKK 187 356 GSlO

1.9 PLRRT LSVAA 50 293 5.9

ADRl(30) LTRR ASFS AQSASSYAL 12.2 259 21.2 ADRl(36) LKKLTRR ASFS AQ 12.4 445 35.8 ADRl(41) LKKLTRR ASFS GQ 25.5 798 31.3 ADRl(42) LKKLTRR ASFRAQ 39.5 280 7.1 ADRl(46) LKKLTRK ASFSAQ 28 608 21.7 ADRl(47) LKKLTRRASSSAQ 28.5 457 16.0

CaM kinase IV CaM kinase II CaM kinase IV AutoP C/EBPP CREB ClEBPP CREB SRF AutoP

Ca2’+ - + - + - + - + - Ca2’ + - + - kDa92.5 - kDa92.5 -

69 - SRF/ 69- CaMk IV .+ -

46 . 46 -

30 . 30 - .

A B FIG. 5. Phosphorylation of transcription factors by recom-

binant CaM kinases IV and I1 The kinase reactions were carried out in the presence (+) or absence (-) of Ca2+ as described under “Experimental Procedures”. A, AutoP repre- sents the autophosphorylation of CaM kinase IV without added substrate; in parallel experiments, C/EBPB (0.65 p ~ , M, 36,000) and CREB (1.76 p ~ , M, 40,000) were examined as substrates for CaM kinase IV and CaM kinase 11. B, SRF (0.58 p ~ , M , 67,000) was evaluated as a substrate for CaM kinase IV. Note that the autophosphorylated CaM kinase IV and SRF co-migrate in this gel system.

activity. On the other hand, the slow, substoichiometric (about 0.1 mol/mol of enzyme) autophosphorylation of CaM kinase IV resulted in an increase in the rate of CaM-dependent phosphorylation of exogenous substrates by this kinase. While the physiological relevance of this autophosphorylation remains to be established, it is interesting to note that the mitogen-activated-kinase ERK2 expressed in Escherichia coli also autophosphorylated at a very slow rate (0.001 mol/h) and was accompanied by an 3-fold increase in the overall kinase activity. This can be compared with a 2000-fold increase in enzyme activity following its phosphorylation by the MAP kinase activator or MAP kinase-kinase (28). By analogy, it is possible that the autophosphorylation of CaM kinase IV observed in our studies hints at a similar regulatory mechanism involving the phosphorylation of CaM kinase IV by unknown cellular protein kinases. It is noteworthy that phosphorylation of all known substrates by CaM kinase IV (either recombinant or purified from cerebellum) was 1 or 2 orders of magnitude slower than that seen with CaM kinase 11, an enzyme which is thought to be regulated by autophosphorylation (11). The cAPK has been shown to phosphorylate CaM kinase IV but this phosphorylation leads to a 50% decrease in CaM kinase IV activity (29). Thus, cAPK is unlikely to represent a CaM kinase IV activator. Based on the structural requirements for substrate recognition defined in our studies, CaM kinase IV contains a potential autophosphorylation site at Ser337. If this putative site is also phosphorylated by the putative CaM kinase IV activator, then its proximity to the CaM-binding domain might predict that CaM-binding would prevent access to the activator kinase. Phosphorylation of a serine at a similar position in smooth muscle myosin light chain kinase (smMLCK) by cAPK prevents CaM binding but this site is not phosphorylated in the presence of Ca2+/CaM (30). This then raises the possibility that other site(s) are phosphorylated during the activation of CaM kinase IV that requires Ca2+/CaM binding.

To interpret the differences in enzymatic properties of the baculovirus-expressed and the cerebellar CaM kinase IV, including the inability to generate autonomy by autophosphorylation and apparent K,,, and VmaX values for selected substrates, we must consider that the cerebellar preparations of

Recombinant Ca2+/CaM-dependent CaM Kinase IV 26177

CaM kinase IV (11,20) contained more than one polypeptide. In most tissues (forebrain, thymus, spleen, testis), CaM kinase IV activity correlates with a single 60-65-kDa polypeptide (i.e. similar to the enzyme expressed using the baculovirus system or translated in uitro in reticulocyte lysates), although the biochemical properties of the enzymes from forebrain, thymus, or spleen have not been extensively characterized. Furthermore, the additional polypeptide(s) are found only in the adult cerebellum and are absent at all stages up to 7 days after birth. These data suggest that the enzymes characterized by others (11, 20) contained polypeptides whose relationship to the CaM kinase IV gene product remains unclear. Regard- less of their origin, one or more of the additional polypeptides may also possess protein kinase activity and undergo autophosphorylation (11). Thus, the unique enzymatic properties of the recombinant CaM kinase IV may reflect the elimination of other protein kinases present in the cerebellar preparations.

Further understanding of the physiological fbnctions of CaM kinase IV in the brain and other tissues will result from the identification of its cellular substrates. The apparent similarities in the requirements for substrate recognition by CaM kinase I1 and IV may be consistent with the conservation of structural elements in the catalytic domain of these two enzymes. This is supported by the phosphorylation of a number of synthetic peptides. Although there is a significant overlap in the structural requirements for substrate recognition between CaM kinase IV and CaM kinase 11, the phosphorylation of many CaM kinase I1 substrates by CaM kinase IV are several orders of magnitude slower (11). Moreover, the Synthetic peptides poorly recognized by CaM kinase I1 (S6 peptide and CaMkII y peptide (11) suggest that CaM kinase IV may also phosphorylate a unique set of protein substrates. The higher rate of phosphorylation observed with peptides derived from ADRl by CaM kinase IV and the comparison of the ADRl sequences with the S6(18) peptide suggests an arrangement of basic amino acids on the NHz-terminal side, followed by groups of hydrophobic residues COOH-terminal to the phosphorylated serine. This array of amino acids is also found in the y peptide of CaMkII (11). Finally, the efficient phosphorylation of ADRl raises the possibility that CaM kinase IV could regulate the function of a putative mammalian homolog of ADRl or other transcription factors such as CREB or SRF.

Our studies indicate that CREB and SRF can be phosphorylated in uitro by CaM kinase IV, whereas C/EBPB cannot. Whereas we did not have sufficient CREB or SRF to determine the kinetic constants, both appear to be reasonable substrates as the extent of phosphorylation of CREB or SRF at 1.76 is similar to that observed for synapsin at the same substrate concentration (data not shown). Under our assay conditions both CREB and SRF incorporate 1 mol of phosphate/mol. In contrast, CREB and C/EBPP were phosphorylated by CaM kinase I1 in uitro, whereas SRF has been shown to be phosphorylated by casein kinase I1 (25-27). The CREB protein is also phosphorylated by CaM kinase I1 on S e P which is the site recognized by cyclic AMP dependent protein kinase. These results led Sheng et al. (23) to suggest that both Caz+ and CAMP pathways could regulate transcriptional activation by CREB. On the other hand, phosphorylation of C/ EBPB by CaM kinase I1 on Ser276 seems specific for this enzyme since protein kinase A, protein kinase C, or CaM kinase IV will not modify this protein (24) (Fig. 5). Phos- phorylation of C/EBPB on S e P 6 which is located in the leucine zipper region is required for transcriptional activation in vitro and was shown by Wegner et al. (24) to also occur in intact cells. Phosphorylation of SRF by a CaM dependent

protein kinase has not previously been reported although phosphorylation of SRF has been reported to occur under conditions that alter intracellular Ca2+ (25). It remains to be conclusively shown which residues are phosphorylated on SRF and how the activity of SRF is affected by CaM kinase IV-mediated phosphorylation. Jensen et al. (31) used antibod- ies to CaM kinase IV to suggest that this enzyme is present in nuclei of cerebellar granule cells. These results raise the intriguing possibility that CaM kinase IV may be involved in regulation of nuclear events that are triggered by Ca2+/ca1- modulin dependent pathways.

Generation of a constitutively active form of CaM kinase IV was accomplished by truncation at Leu313. Being a Ca2+/ CaM-dependent kinase, we predicted that, as with the MLCKs or CaM kinase IIs, the mechanism of activation by CaM would involve a conformational change in the regulatory region which results in the removal of autoinhibition of the kinase involving an adjacent domain (10, 32). Previous mutagenesis studies of CaM kinase 1Ia revealed that removal of the autoinhibitory domain required the deletion of amino acids 291Lys-Lys-Phe-Asn-Ala-Arg-Arg-Lys-Leu-Lys3w (10). The conservation of the sequence 293Phe-Asn-Ala-Arg-Arg- L y s - L e u - L y ~ ~ ~ a t position 316-323 in CaM kinase IV suggested that similar autoinhibitory determinants could be involved in this enzyme. Indeed, our Leu313 mutant was constitutively active and is remarkably stable to thermal denatur- ation. The high activity of our truncation mutant suggests that the COOH-terminal acidic domain of CaM kinase IV is not required for catalytic activity or recognition of protein substrates. The absence of significant autophosphorylation of the mutant protein also suggested that the major autophosphorylation sites have been removed.

In summary, our results suggest a conservation of some of the structural components regulating enzyme activity, substrate specificity and CaM activation of CaM kinase I1 and IV. This is counteracted by differences in the regulation of these kinases by autophosphorylation. CaM kinase I1 can switch rapidly to a CaM-independent state, and has thus been postulated to maintain long term biological responses initiated by a transient Ca2+ signal. By comparison, CaM kinase IV shows a mechanism for regulation which is dependent on increases and decreases in Caz+ levels although additional modulation by phosphorylation by other kinases has not been discounted. The divergence of the COOH termini of these multifunctional CaM kinases may also suggest that unique associations with subcellular compartments could account for their distinct localization in the cell. The intracellular distri- bution of these enzymes may play an important role in tar- geting the unique cellular functions of these multifunctional Ca2+/CaM-regulated protein kinases.

Acknowledgments-We thank Dr. Bruce Kemp for providing us with the synthetic peptides used in the substrate specificity studies; Drs. Angus Nairn and Andrew Czernick for synapsin I, Dr. M. G. Rosenfeld for CDBPB, Dr. Mark Montiminny for CREB, and Dr. Michael Greenberg for SRF; as well as Dr. Shirish Shenolikar for his helpful suggestions and redaction of the manuscript. We are also appreciative of EIizabeth McDougall and Qi-Hui Huang for their valuable technical help as well as Elizabeth Fletcher for preparation of the typescript.

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