©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Mutational Analysis of Ca-independent Autophosphorylation of Calcium/Calmodulin-dependent Protein Kinase II (*)

Sucheta Mukherji , Thomas R. Soderling (§)

From the (1) Vollum Institute, Oregon Health Sciences University, Portland, Oregon 97201

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Previous studies with synthetic peptides indicate that residues 290-309, corresponding to the calmodulin (CaM)-binding domain of Ca/CaM-dependent protein kinase II interact with the catalytic core of the enzyme as a pseudosubstrate (Colbran, R. J., Smith, M. K., Schworer, C. M., Fong, Y. L., and Soderling, T. R.(1989) J. Biol. Chem. 264, 4800-4804). In the present study, we attempted to locate the pseudosubstrate motif by generation or removal of potential substrate recognition sequences (RXXS/T) at selected positions using site-directed mutagenesis. Based on previous results, Arg, Thr, and Ser were selected as key residues. Single mutations such as N294S, K300S, A302R, A309R, and R311A were expressed, purified, and characterized. Several of the mutants exhibited decreased binding of and activation by CaM, not surprising since the mutations were within the CaM-binding domain. None of the mutants exhibited enhanced Ca-independent kinase activity toward exogenous substrate, but the K300S and N294S mutants showed a significant enhancement in the rate and stoichiometry of P incorporation during Ca-independent autophosphorylation. Using two-dimensional peptide mapping and phosphoamino acid analyses, enhanced phosphorylation of the introduced Ser residue was demonstrated in the K300S mutant but not in the N294S mutant. This specific Ca-independent autophosphorylation of Ser is consistent with the hypothesis that Arg may occupy the P(-3) position in a pseudosubstrate autoinhibitory interaction with the catalytic core in the nonactivated state of the kinase.


INTRODUCTION

Ca/CaM-dependent protein kinase II (CaM kinase II)() is a multifunctional serine/threonine kinase known to be involved in a variety of cellular functions (reviewed in Refs. 1 and 2). The enzyme is present in most tissues but is particularly abundant in brain. The adult rat brain kinase is an oligomeric enzyme composed of 10-12 subunits (50-60 kDa each) of several isoforms in varying molar ratios. All isoforms share at their NH termini a highly conserved catalytic domain characteristic of serine/threonine kinases in general. The central portion of each subunit contains the regulatory domain comprised of an autoinhibitory sequence overlapping a CaM-binding region, and the COOH terminus contains an association domain, which is important in holoenzyme formation.

Like many other protein kinases, CaM kinase II is regulated by an autoinhibitory mechanism. In the absence of Ca/CaM, low basal kinase activity is due to interaction of amino acid residues within the regulatory domain with the catalytic region, thereby blocking access of exogenous protein substrates and ATP. Binding of Ca/CaM to the CaM-binding motif (residues 293-310 in the subunit) removes the regulatory domain from the catalytic domain, which converts the kinase to its fully active form. Several recent studies (3, 4) have addressed the mechanisms of autoinhibition and regulation of CaM kinase II. For example, studies with synthetic peptides corresponding to the regulatory domain of CaM kinase II suggest that residues between 281 and 289 interact with the ATP-binding motif, whereas residues between 290 and 309 interact with the protein/peptide-binding elements of the catalytic domain (3) . Thus, a bisubstrate-directed autoinhibitory mechanism was indicated, and we have recently proposed a molecular model for CaM kinase II, illustrating a possible interaction of the bisubstrate autoinhibitory domain with the bilobal catalytic domain of the enzyme (4) .

For several protein kinases, the autoinhibitory mechanism has been suggested to be due to interaction of a pseudosubstrate sequence in the regulatory domain with the active site of the enzyme (5, 6, 7, 8) . A pseudosubstrate type of inhibition has also been proposed for CaM kinase II (3, 9) , and in our molecular model of CaM kinase II (4) , Arg was proposed to occupy the P(-3) position of a putative pseudosubstrate sequence. In the present study, we have investigated the pseudosubstrate mechanism by generating or removing substrate consensus sequences (RXXS/T) at several potential sites in CaM kinase II through site-specific mutagenesis. Since most of the mutations were introduced into the CaM-binding region of the regulatory domain, this study also revealed some important information about the structural basis of regulation by CaM in the context of the holoenzyme structure of CaM kinase II.


EXPERIMENTAL PROCEDURES

Oligonucleotide-directed Mutagenesis

The cDNA encoding the 50-kDa mouse brain CaM kinase II subunit was inserted into the EcoRI site of pVL1393 baculovirus transfer vector and digested with XbaI and PstI. A 680-base pair SphI-PstI fragment from the 3`-end of the mouse CaM kinase II cDNA (corresponding to the regulatory and subunit association domains of the kinase) was inserted into M13 mp19 to generate single-stranded DNA for mutagenesis. The following oligonucleotides were designed for carrying out the mutations mentioned (mutated bases underlined): N294S, 5`-TTCCTCCTGGCACTGAACTTCTTCAG-3`; K300S, 5`-GAGGATGGCTCCACTCAGTTTCCTCCT-3`; A302R, 5`-TGGTGAGGATTCTTCCCTTCAGTTTCC-3`; A309R, 5`-GAGAAGTTCCTGGTTCTCAGCATAGTGGT-3`; R311A, 5`-TCCGGAGAAGTTCGCGGTGGCCAGCATAGT-3`.

Mutagenesis was carried out by using Sculptorin vitro mutagenesis kit (Amersham Corp.). The mutations were confirmed by sequencing the entire 650-base pair fragment using the Sequenase kit from U. S. Biochemical Corp. After mutation, the SphI-PstI fragments were ligated to XbaI-SphI-digested 5`-fragment (corresponding to catalytic region) of mouse CaM kinase-II , and the total cDNA molecule corresponding to the 50-kDa subunit was inserted into XbaI- and PstI-digested pVL1393 for subsequent expression in baculovirus/Sf9 expression system.

Expression and Purification of Mutant CaM Kinase II Enzymes

The wild-type and mutant CaM kinase II enzymes were expressed in the baculovirus/Sf9 cell expression system as previously described (10) with slight modifications. Recombinant viruses were isolated via homologous recombination and plaque purification. Isolated recombinant viruses were titered by plaque assay method (11) . About 150 ml of Sf9 cell culture at log phase (1.2-1.6 10 cells/ml) were infected with recombinant viruses at a multiplicity of infection of 3 in Corning spinner flasks. Infected cells were harvested between 48-72 h, frozen in liquid N, and stored at -70 °C until purification. The wild-type and mutant CaM kinase II enzymes were purified by CaM-Sepharose affinity chromatography as described before (10) . Because of the poor affinity for CaM, mutants K300S and A302R were eluted in the salt wash step. All the enzyme preparations were dialyzed against 100 mM HEPES, pH 7.5, 10% ethylene glycol, 0.5 mM EDTA, 5 mM dithiothreitol, 50% glycerol and stored at -20 °C.

Kinase Assays

CaM kinase II activities were measured routinely at 30 °C for 1 min using standard assay conditions containing 40 µM syntide-2, 0.5 mM [-P]ATP (600-2000 cpm/pmol), and either 0.5 mM Ca, 3 µM CaM (total activity) or 1 mM EGTA (Ca-independent activity) in a final volume of 25 µl (10) . All assays were initiated by the addition of kinases diluted appropriately in dilution buffer (50 mM HEPES, pH 7.5, 2 mg/ml bovine serum albumin, and 10% ethylene glycol). P incorporation was determined by spotting 15-µl aliquots onto Whatman P-81 phosphocellulose paper followed by washing in 75 mM phosphoric acid as described (12) .

Immunodetection of CaM Kinase II Subunits in Western Blot

The purified proteins were separated by 10% SDS-PAGE and transferred to nitrocellulose membrane (Schleicher & Schuell). Blocking, washing, and antibody dilution were done with 50 mM Tris-HCl buffer, pH 8.0, 150 mM NaCl, and 0.2% Tween 20. The stock solution of 4-chloro-1-naphthol (3 mg/ml methanol) was diluted 6-fold in 50 mM Tris-HCl buffer, pH 8.0, containing 150 mM NaCl. Goat polyclonal anti-rat brain CaM kinase II was diluted 500-fold. Anti-CaM kinase II was prepared by Bethyl Laboratories (Montgomery, TX) and was purified by affinity chromatography using CaM kinase II linked to Affi-Gel 15 (Bio-Rad). The peroxidase-conjugated swine anti-goat IgG antibody (Boehringer Mannheim) was diluted 1000-fold.

CaM Overlay

Wild-type and mutant CaM kinase II enzymes were resolved on 10% SDS-PAGE and then electrophoretically transferred onto nitrocellulose membrane. The membrane was blocked with 150 mM NaCl, 50 mM Tris-HCl (pH 7.4), 1 mM CaCl 0.2% Tween 20, and 5% nonfat dry milk for 1 h at room temperature. Biotinylated CaM was added at a final concentration of 0.4 µg/ml in the above buffer without milk and incubated for 1 h. After washing with buffer alone, the membranes were incubated with 1:500-diluted avidin D-conjugated horseradish peroxidase (Vector Laboratories, Inc.) The membranes were washed extensively with 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 1 mM CaCl and developed with 4-chloro-1-naphthol and HO.

Two-dimensional Peptide Mapping

Mapping of the phosphorylation sites was performed as previously described (13) but with some modifications. The P-labeled proteins or peptides were transferred to nitrocellulose membranes after separation by 10% SDS-PAGE and 18% Tricine gel. Subsequent digestions with CNBr or trypsin were carried out on the corresponding membrane fragments containing the labeled protein or peptides as described earlier (13) .

Phosphoamino Acid Analysis

Aliquots of the P-labeled tryptic peptides for two-dimensional peptide mapping were dried by speed vac, resuspended in 100 µl of 6 N HCl at 110 °C for 1 h. Hydrolyzed samples were dried and resuspended in pH 1.9 electrophoresis buffer (formic acid:glacial acetic acid:water, 25:78:897, v/v/v). The P-labeled phosphoamino acids were separated by one-dimension electrophoresis in pH 1.9 buffer for 1 h at 1.5 kV and detected by autoradiography. For phosphoamino acid analysis of peptides from two-dimensional maps, the corresponding spot was scraped off the cellulose plate and washed with pH 1.9 buffer to elute the phosphopeptide (14) . The P-labeled phosphopeptide was hydrolyzed with 6 N HCl and subjected to phosphoamino acid analysis as described above.

Other Materials and Methods

CaM was purified from bovine brain according to the method of Gopalakrishna and Anderson (15). Syntide-2 was synthesized and purified as previously described (16). Protein concentration was determined by the Bio-Rad protein binding assay using bovine serum albumin (Pierce) as standard.


RESULTS

Mutagenesis in the Pseudosubstrate Region of CaM Kinase II

Previous studies with synthetic peptides showed that residues 290-309 in the autoinhibitory domain of CaM kinase II inhibited a catalytic fragment of CaM kinase II competitively with substrate. This result suggests that residues 290-309 interact as a pseudosubstrate with the catalytic core of the enzyme in the absence of Ca/CaM (3) . In the present study, we attempted to locate the pseudosubstrate sequence by generating or removing potential substrate consensus sequences (RXXS/T) through site-specific mutagenesis of selected residues as shown below.

On-line formulae not verified for accuracy

On-line formulae not verified for accuracy

On-line formulae not verified for accuracy

Arg, Thr, and Ser, indicated by the asterisks, were selected as key residues for analyses. On the basis of substitution studies in synthetic peptides (9) , Arg was suggested to be an important determinant for interaction of the autoinhibitory domain peptide of CaM kinase II with the protein-binding motif of the catalytic core, and it has been modeled by our laboratory to occupy the P(-3) position of a pseudosubstrate (4) . Thr and Ser have been shown to be the major autophosphorylation sites in the absence of Ca/CaM (17, 18) . We made single mutants by introducing Ser residues in place of Asn and Lys, which are at the P-0 positions on the NH- and COOH-terminal sides, respectively, of Arg because the relative orientation of the autoinhibitory region with respect to the active site of the enzyme is not established yet. Two other single mutants were made by introducing an Arg in place of Ala or Ala, which would place them three residues to the NH- and COOH-terminal sides of Thr, respectively. It is known that CaM kinase II recognizes phosphorylation sites that have an Arg three residues NH-terminal of the phosphorylated Ser or Thr. Since the orientation of the autoinhibitory polypeptide sequence in the catalytic site (see model in Ref. 4) may be opposite to that of the normal substrate, we wanted to also place an Arg three residues COOH-terminal of Thr (i.e. mutant A309R). The R311A mutant was constructed to test the effect of disruption of the substrate recognition sequence around Ser.

General Characteristics of the Mutants

The mutants were expressed using the baculovirus/Sf9 cell system and purified on CaM-Sepharose as described under ``Experimental Procedures.'' All the mutants were at least 90% pure and gave a major 50-kDa band on SDS-PAGE by Coomassie stain (Fig. 1A) and by Western analyses (Fig. 1B). Four of our mutations are in the CaM-binding region of the autoinhibitory domain, and these mutants showed lower affinity for CaM. Fig. 1C shows the CaM overlay assay for all the mutants. The relative CaM binding affinities were wild type = R311A > N294S > A309R > K300S > A302R. The abilities of the mutants to be activated by CaM are shown in Fig. 2 . Mutants N294S and R311A exhibited similar CaM activation profiles as the wild-type enzyme with A values of 0.1-0.15 µM in the presence of 0.5 mM CaCl The A309R and K300S mutants showed significant loss in CaM activation with A values of 0.6 and 1.6 µM, respectively. The A302R mutant showed a severe loss in CaM activation, and the A value could not be determined for this mutant.


Figure 1: Coomassie, Western blot, and CaM overlay analyses of purified CaM Kinase II mutants. Equal amounts (2 µg) of wild-type CaM kinase II and the indicated mutants were run on 10% polyacrylamide gel in the presence of SDS and analyzed by Coomassie Blue (A), Western blot with anti CaM kinase II (B), or reacted with biotinylated CaM in the presence of 1 mM CaCl (C) as described under ``Experimental Procedures.'' STDS, molecular mass standards ranging from 31 to 106 kDa.




Figure 2: Ca/CaM activation of wild-type and mutant CaM kinase II. Kinase assays were performed in the presence of 0.5 mM Ca and the indicated concentrations of CaM. Equivalent amounts (15 nM) of wild-type enzyme (), N294S (), K300S (), A302R (), A309R (⊞), and R311A () mutants were used. Maximal activation (100%) was the activity at 12 µM CaM (see Table I), where activation had reached a plateau for all the mutants except A302R.



Catalytic Properties of the Mutants

All of the mutants had activities in the absence of Ca/CaM similar to wild-type kinase (), and this indicates that the mutations, which are not in the catalytic domain, had no significant effect on the basic catalytic properties of the kinase. When assayed in the presence of saturating CaM (12 µM), most of the mutants had specific activities () between 14-22 µmol/min/mg, comparable to the wild-type enzyme. Mutant K300S displayed a 10-fold lower specific activity, probably a consequence of its altered interaction with CaM. It is known that mutations within CaM can not only affect the apparent K for activation of its target proteins but also the extent of activation at saturating CaM (19) . It is not surprising that mutation within the CaM-binding domain of a target protein may have a similar effect.

Ca-independent Autophosphorylation of the Mutants

Truncation of CaM kinase II at residue 316 produces a monomeric species of CaM kinase II that requires Ca/CaM for activation (20) . This indicates that interaction of the autoinhibitory domain with the catalytic domain is intrasubunit in the holoenzyme. Furthermore, CaM kinase II undergoes slow, intrasubunit autophosphorylation at 23 °C in the absence of Ca/CaM (i.e. 1 mM EGTA) (13). It has been demonstrated that this Ca-independent autophosphorylation occurs predominantly at Thr in the CaM-binding domain, and this blocks subsequent binding of CaM and prevents activation of the enzyme (17, 18) . Therefore, if any of our autoinhibitory domain mutations converted a pseudosubstrate motif into a substrate site, we would expect enhanced intrasubunit, Ca-independent autophosphorylation of that mutant.

When we subjected the mutant kinases to Ca-independent autophosphorylation conditions, the K300S and N294S mutants showed enhanced rates of P incorporation compared with wild-type kinase and the other mutants (Fig. 3). The stoichiometry of P incorporation per mole of enzyme subunit was also increased. For the K300S mutant, about 0.4 mol of P was incorporated per mol of enzyme subunit in 15 min at room temperature compared with about 0.1 mol for wild-type kinase (Fig. 3). The N294S mutant incorporated 0.2 mol of P under identical conditions. The A302R, A309R, and R311A mutants did not show any significant differences in either rate or stoichiometry of P incorporation compared with wild-type enzyme, and all showed a similar loss (50-70%) in Ca/CaM-stimulated activity after 15 min of incubation in the presence of 1 mM EGTA and 0.5 mM ATP at room temperature (data not shown). This loss of activation by Ca/CaM is consistent with the known Ca-independent autophosphorylation of Thr. Since the K300S and N294S mutants demonstrated enhanced Ca-independent autophosphorylation, we carefully examined the effects of this autophosphorylation on their kinase activities. The K300S mutant did exhibit a slightly higher rate of inactivation, whereas the N294S mutant showed a rate of inactivation similar to the wild-type kinase (Fig. 4).


Figure 3: Kinetics and stoichiometry of P incorporation for Ca-independent autophosphorylation of wild-type and mutant CaM kinase II enzymes. Wild-type and mutant enzymes (1 µM) were incubated for 15 min at 23 °C in 50 mM HEPES, pH 7.5, 10 mM magnesium acetate, 1 mM EGTA, and 0.5 mM [-P]ATP (12000-17000 cpm/pmol). At the indicated times, aliquots were removed, and EDTA was added to a final concentration of 28 mM to stop the autophosphorylation reaction. Aliquots from these solutions were spotted directly on P-81 paper for measurement of P incorporation. Symbols are the same as in Fig. 2.




Figure 4: Inactivation of N294S and K300S mutants due to Ca-independent autophosphorylation. Wild-type CaM kinase II, N294S, and K300S mutant enzymes were autophosphorylated in the absence of Ca/CaM as described in Fig. 3. At the indicated times, aliquots were removed, and autophosphorylation reactions were stopped by adding EDTA. An aliquot from these solutions was diluted 35-150-fold in dilution buffer, and total kinase activities (i.e. + Ca/CaM) were measured as described under ``Experimental Procedures.'' Assays were done in duplicate, and the experiment is representative of three similar experiments. Kinase activities of the wild-type (), N294S (), and K300S () mutant enzymes are expressed as a percentage of corresponding controls incubated under identical conditions except for the absence of ATP.



When autophosphorylation was performed in the presence of Ca/CaM, all of the mutants, except A302R, generated similar Ca-independent activity indicative of autophosphorylation of Thr (data not shown except for K300S mutant in ). Also, enhanced P incorporation was observed for autophosphorylation of the K300S mutant only in the absence (Fig. 3) and not in the presence of Ca/CaM ().

Two-dimensional Phosphopeptide Mapping and Phosphoamino Acid Analysis Studies

Finally, we analyzed the phosphorylation sites for the K300S and N294S mutants by two-dimensional P peptide mapping. To simplify the analysis, we restricted our analysis to the 3-kDa CNBr fragment, which contains the autophosphorylation sites of interest (13) . The patterns of the CNBr/tryptic phosphopeptide maps were the same for wild-type enzyme, N294S and K300S mutants, showing only one major radioactive P peptide with comparable mobility (Fig. 5). Since Ser and Thr are present in the same CNBr/tryptic phosphopeptide (LS*GAILTT*M), only one spot was expected to be generated due to phosphorylation of either Ser or Thr. The absence of any additional spots in the phosphopeptide map of the N294S mutant indicated that Ser was not phosphorylated. We also carried out phosphoamino acid analyses on the 3-kDa CNBr/tryptic peptide fragment from the wild-type enzyme, N294S and K300S mutants as described under ``Experimental Procedures.'' For wild-type enzyme and N294S mutant, only Thr residues were phosphorylated (Fig. 6A, lanes1 and 2). However, the K300S mutant gave phosphorylation of both Thr and Ser (Fig. 6A, lane3). Phosphoamino acid analysis of the phosphopeptide 1 (Fig. 5D) also showed the presence of both phosphoserine and phosphothreonine (Fig. 6B) in the same spot, thereby confirming the phosphorylation of Ser in the K300S mutant.


Figure 5: Two-dimensional P peptide mapping of N294S and K300S mutants after Ca-independent autophosphorylation. Wild-type, N294S, and K300S mutants (2 µM) were autophosphorylated in the presence of 50 mM HEPES, pH 7.5, 10 mM magnesium acetate, 1 mM EGTA, and [-P]ATP (9 Ci/mmol) at 23 °C for 15 min. The 50-kDa P kinase subunits were isolated by SDS-PAGE and cleaved with CNBr. The 3-kDa CNBr fragments, corresponding to residues 282-307, were isolated, digested with trypsin, and subjected to two-dimensional peptide mapping as described under ``Experimental Procedures.'' In panelA, synthetic peptide 290-309 was specifically P labeled on Thr, digested with CNBr and trypsin, and subjected to two-dimensional peptide mapping as described (13). PanelsB-D represent Ca-independent autophosphorylation of wild-type, N294S, and K300S mutants, respectively.




Figure 6: Phosphoamino acid analysis of P-labeled tryptic fragments from N294S and K300S mutants. A, aliquots of the P-labeled CNBr/tryptic peptides prepared for two-dimensional peptide maps as described in Fig. 5 were hydrolyzed in 6 N HCl, and the released P amino acids were separated by thin layer electrophoresis at pH 1.9 as described under ``Experimental Procedures.'' Lane1 represents wild-type enzyme; lane2, N294S mutant; lane3, K300S mutant, B, peptide 1 from panelD of Fig. 5.




DISCUSSION

CaM binding motifs of several Ca/CaM-dependent enzymes act as autoinhibitory domains of their respective enzyme functions (21, 22) . In some cases, autoinhibitory domains have been proposed to interact with the catalytic core of the enzyme as a pseudosubstrate (6, 7) . In CaM kinase II, residues 281-290 interact with the ATP-binding pocket of the catalytic domain, whereas residues 290-309, which also comprise the CaM-binding domain, have been proposed on the basis of synthetic peptide and mutagenesis studies (3, 9) to act as a pseudosubstrate by blocking the protein-substrate motif. The present study was aimed at exploring the pseudosubstrate sequence in the autoinhibitory domain by generating or removing possible substrate consensus sequences around three potential sites: Arg, Thr, or Ser.

The unaltered Ca-independent specific activities of the mutants () indicated there were no major changes in basic catalytic properties of the enzymes due to mutations. This conclusion was supported by the examination of other catalytic properties of the enzymes, such as autophosphorylation properties. After activation by Ca/CaM in the presence of Mg/ATP, CaM kinase II autophosphorylates itself on Thr in the autoinhibitory domain in an intersubunit manner (13, 23) , and the enzyme becomes partially Ca/CaM independent. All the mutant enzymes, except A302R, exhibited normal Ca/CaM-dependent autophosphorylation and generation of Ca-independent activity similar to the wild-type enzyme. Because the specific activity of the A302R mutant in the presence of Ca/CaM was so low, this mutant could not be tested for any Ca/CaM-dependent phenomena.

Changing a pseudosubstrate autoinhibitory sequence to a substrate consensus sequence would be expected to enhance intrasubunit autophosphorylation in the absence of Ca/CaM. The A302R, A309R, and R311A mutants exhibited the same rates and stoichiometries of P incorporation during Ca-independent autophosphorylation as the wild-type enzyme, thereby excluding Thr and Ser as possible pseudosubstrate sites. This is consistent with previous data indicating that the autoinhibitory domain resides within residues 281-302 (9) . However, both the K300S and the N294S mutants exhibited enhanced rates and stoichiometries of Ca-independent autophosphorylation (Fig. 3). When we measured the corresponding rates of inactivation for these two mutants due to Ca-independent autophosphorylation, only the K300S mutant showed a higher rate of inactivation compared with wild-type enzyme (Fig. 4). As discussed below, Lys of the CaM kinase II peptide interacts with glutamate residues in the NH-terminal lobe of Ca/CaM (24) , and the K300S mutation in CaM kinase II reduced CaM binding (Fig. 1C). It is likely that introduction of negative charge by phosphorylation of Ser in the K300S mutant would further disrupt the interaction with CaM, causing an enhanced rate of inactivation during Ca-independent autophosphorylation. Phosphorylation of Ser, but not Ser, was demonstrated by two-dimensional peptide mapping and phosphoamino acid analysis ( Fig. 5and 6). The enhanced autophosphorylation observed with the N294S mutant was due to phosphophorylation of an unknown site. Multiple sites are subject to autophosphorylation in the absence and presence of Ca/CaM (1, 2) , and we did not attempt to identify this site in the N294S mutant since its autophosphorylation did not produce any detectable changes in the kinase.

The mutations that we studied were also within the CaM-binding domain, and many of the mutants (Fig. 1C, Fig. 2, and ) were altered in their abilities to bind to and be activated by Ca/CaM. This is to be expected since the crystal structure of Ca/CaM bound to peptide 290-314 of CaM kinase II (24) has shown that residues 293-310 form electrostatic and hydrophobic interactions with CaM. The cluster of three basic residues (Arg-Arg-Lys and Lys in the NH-terminal half of CaM kinase II peptide form salt bridges with glutamate residues on both the NH- and COOH-terminal lobes of CaM.

Hydrophobic residues Leu and Ile on the NH-terminal half and Leu on the COOH-terminal half of the peptide are involved in hydrophobic interactions with the COOH- and NH-terminal lobes, respectively, of CaM. We found that insertion of charge at position 302 (A302R mutant), which should disrupt the hydrophobic interaction with the COOH-terminal lobe of CaM, caused the most severe loss in binding (Fig. 1C) and activation by CaM (Fig. 2, ). The hydrophobic interaction with the NH-terminal lobe of CaM, on the other hand, appeared to be more involved in CaM binding (Fig. 1C) than in kinase activation (Fig. 2, ), as observed with the A309R mutant. Similar but less pronounced effects were observed with the N294S mutant, probably because the charge was not altered. The K300S mutant showed a strong effect on binding to and activation by CaM, presumably by disrupting the electrostatic interaction of Lys with glutamate residues in the NH-terminal lobe of CaM. Although 12 µM CaM was not able to fully activate the K300S mutant (), autophosphorylation on Thr, which normally does not increase total kinase activity assayed in the presence of Ca/CaM, caused a 4-fold increase in total kinase activity of the K300S mutant (). This result is consistent with impaired binding to and activation by CaM for this mutant. Thus, our mutagenesis results suggest that although both lobes of CaM are involved in binding to CaM kinase II through electrostatic and hydrophobic interactions, the COOH-terminal lobe of CaM probably plays the major role in the binding and activation mechanism.

Although several of the mutants in this study had altered CaM binding properties, none of the mutants demonstrated enhanced Ca-independent activity toward exogenous substrate (i.e. syntide 2) in the absence of Ca/CaM compared with wild-type kinase (). This indicates that none of the mutations weakened the interaction of the autoinhibitory domain with the catalytic domain. Similar findings were obtained with another Ca/CaM-regulated enzyme, myosin light chain kinase (25, 26) . Mutation to acidic residues of the basic residues RRK on the NH-terminal side of the CaM-binding domain of myosin light chain kinase altered the CaM binding affinity but did not give rise to Ca-independent activity (26) . It appears, therefore, that the mechanisms of CaM activation and mechanism of autoinhibition involve different residues. This is contrary to a ``flip-flop model'' proposed earlier (27) , which assumed an autoinhibitory mechanism in which the CaM-binding domain interacted with a ``CaM-like binding region'' on the enzyme surface.

In summary, the observation that Ser is autophosphorylated in the absence of Ca/CaM is consistent with our previous synthetic peptide study (4) and our molecular model (4) , indicating that Arg may occupy the P(-3) position of a pseudosubstrate inhibitor. Our results also support some of the conclusions made from the crystal structure of CaM with the synthetic CaM-binding peptide complex (24) . In addition, this study shows in the context of the entire enzyme structure which residues are obligatory for high affinity CaM binding versus those that may be necessary for conformational changes and those that participate directly in modulation of enzyme activity.

  
Table: Specific activities of the wild-type and mutant kinases

Specific activities were measured for total kinase activities in the presence of 12 µM CaM, 0.5 mM CaCl, 10 mM magnesium acetate, 0.5 mM [-P]ATP, and 40 µM syntide 2 as described under ``Experimental Procedures.'' Ca-independent specific activities were measured in the absence of Ca/CaM under standard conditions (see ``Experimental Procedures''). All assays were done in duplicate. Values obtained from more than two independent assays are given as the mean ± S.D.


  
Table: Effect of Ca/CaM-dependent autophosphorylation on K300S mutant

Wild-type and K300S mutant enzymes (1 µM) were incubated for 15 min at 5 °C in 50 mM HEPES buffer, pH 7.5, 10 mM magnesium acetate, 0.5 mM CaCl, 27 µM CaM, with or without 0.5 mM [-P]ATP (6600 cpm/pmol). Autophosphorylation reactions were stopped by adding EDTA to a final concentration of 28 mM. Aliquots from these solutions were spotted directly on P-81 paper for measurement of P incorporation or assayed for CaM-kinase II activities (±Ca/CaM) as described under ``Experimental Procedures'' after appropriate dilution.



FOOTNOTES

*
Supported in part by National Institutes of Health Grant GM41292. 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, Oregon Health Sciences University, 3181 S. W. Sam Jackson Park Rd., Portland, OR 97201.

The abbreviations used are: CaM kinase II, Ca/calmodulin-dependent protein kinase II; PAGE, polyacrylamide gel electrophoresis.


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