Expression and characterization of calmodulin-dependent protein kinase II from cloned cDNAs in Chinese hamster ovary cells

cDNAs containing the entire coding regions of the alpha and beta subunits of calmodulin-dependent protein kinase II (CaM kinase II) were isolated from a rat cerebrum cDNA library, ligated into an expression vector under the control of SV40 early promoter and introduced into Chinese hamster ovary (CHO) cells. To investigate the role of the alpha and beta subunits and their functional domains in CaM kinase II activity, the properties of the kinases expressed in the transfected cells were studied. CaM kinase II activity was detected in the transfected cells when the alpha and beta cDNAs were introduced into CHO cells simultaneously. RNA transfer blot and protein immunoblot analyses demonstrated the expression of the mRNAs and proteins of both alpha and beta subunits in the cloned cells. When alpha or beta cDNA was introduced into CHO cells separately, a significant level of the enzyme activity was also expressed, indicating that the alpha and beta subunits exhibited enzyme activity individually. The apparent Km values for ATP and MAP 2 were almost the same for the alpha subunit, beta subunit, alpha beta complex, and brain CaM kinase II. However, there was a slight difference in the affinity for calmodulin between the expressed proteins. The alpha and beta subunits expressed in the same cells polymerized to form alpha beta complex of a size similar to that of brain CaM kinase II. The alpha subunit also polymerized to form an oligomer, which showed almost the same S value as that of alpha beta complex and brain CaM kinase II. In contrast, the beta subunit did not polymerize. The alpha subunit, beta subunit, alpha beta complex, and brain CaM kinase II were autophosphorylated with [gamma-32P]ATP in the presence of Ca2+ and calmodulin, which resulted in the appearance of Ca2+-independent activity. The Ca2+-independent activity was 60-75% of the total activity as measured in the presence of Ca2+ plus calmodulin. To examine the functional relationship of peptide domains of the subunits of CaM kinase II, deleted cDNAs were introduced into CHO cells and the properties of the expressed proteins were studied. In cells transfected with alpha or beta cDNA from which the association domain was deleted, a significant level of kinase activity was expressed. However, the expressed proteins showed hardly any autophosphorylation and the appearance of Ca2+-independent enzyme activity was very low, indicating that the association domain was essential for the autophosphorylation and for the appearance of the Ca2+-independent activity.     

KN-62, 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazi ne, a specific inhibitor of Ca2+/calmodulin-dependent protein kinase II

1-[N,O-Bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpipera zine (KN-62), a selective inhibitor of rat brain Ca2+/calmodulin-dependent protein kinase II (Ca2+/CaM kinase II) was synthesized and its inhibitory properties in vitro and in vivo were investigated. KN-62 inhibited phosphorylation of exogenous substrate (chicken gizzard myosin 20-kDa light chain) by Ca2+/CaM kinase II with Ki value of 0.9 microM, but no significant effect up to 100 microM on activities of chicken gizzard myosin light chain kinase, rabbit brain protein kinase C, and bovine heart cAMP-dependent protein kinase type II. KN-62 also inhibited the Ca2+/calmodulin-dependent autophosphorylation of both alpha (50 kDa) and beta (60 kDa) subunits of Ca2+/CaM kinase II dose dependently in the presence or absence of exogenous substrate. Kinetic analysis indicated that this inhibitory effect of KN-62 was competitive with respect to calmodulin. However, KN-62 did not inhibit the activity of autophosphorylated Ca2+/CaM kinase II. Moreover, Ca2+/CaM kinase II bound to a KN-62-coupled Sepharose 4B column, but calmodulin did not. These results suggest that KN-62 affects the interaction between calmodulin and Ca2+/CaM kinase II following inhibition of this kinase activity by directly binding to the calmodulin binding site of the enzyme but does not affect the calmodulin-independent activity of already autophosphorylated (activated) enzyme. We examined the effect of KN-62 on cultured PC12 D pheochromocytoma cells. KN-62 suppressed the A23187 (0.5 microM)-induced autophosphorylation of the 53-kDa subunit of Ca2+/CaM kinase in PC12 D cells, which was immunoprecipitated with anti-rat forebrain Ca2+/CaM kinase II polypeptides antibodies coupled to Sepharose 4B, thereby suggesting that KN-62 could inhibit the Ca2+/CaM kinase II activity in vivo.     

CaM-kinase II dephosphorylates Thr(286) by a reversal of the autophosphorylation reaction

Autophosphorylation of CaM-kinase II produces a form of the enzyme not requiring Ca(2+)/calmodulin for sustained activity. We report that autophosphorylated CaM-kinase II dephosphorylates itself in the presence of ADP (termed autodephosphorylation). The dephosphorylation was unaffected by phosphatase inhibitors and was nucleotide specific, occurring with ADP but not with AMP, GTP, or GDP. (32)P-ATP was produced when ADP was added to (32)P-labeled CaM-kinase II, indicating that the enzyme was undergoing dephosphorylation through a reversal of the autophosphorylation reaction. ATP addition also produced loss of (32)P from the autophosphorylated enzyme while maintaining the kinase in a phosphorylated state. This indicates that the enzyme was undergoing cycles of autophosphorylation and dephosphorylation in the activated state. Autothiophosphorylated CaM-kinase II was resistant to autodephosphorylation. Site-directed mutants were used to show that Thr(286) was the predominant site dephosphorylated. Additionally, CaM-kinase II composed of beta subunits exhibited autodephosphorylation. Ca(2+)/CaM-independent activity expressed by the autophosphorylated alpha and beta holoenzymes was reversed following autodephosphorylation.     

Comparative analyses of the three-dimensional structures and enzymatic properties of alpha, beta, gamma and delta isoforms of Ca2+-calmodulin-dependent protein kinase II

Ca(2+)-calmodulin-dependent protein kinase II (CaM-kinase II) is a ubiquitous Ser/Thr-directed protein kinase that is expressed from a family of four genes (alpha, beta, gamma, and delta) in mammalian cells. We have documented the three-dimensional structures and the biophysical and enzymatic properties of the four gene products. Biophysical analyses showed that each isoform assembles into oligomeric forms and their three-dimensional structures at 21-25 A revealed that all four isoforms were dodecamers with similar but highly unusual architecture. A gear-shaped core comprising the association domain has the catalytic domains tethered on appendages, six of which extend from both ends of the core. At this level of resolution, we can discern no isoform-dependent differences in ultrastructure of the holoenzymes. Enzymatic analyses showed that the isoforms were similar in their K(m) for ATP and the peptide substrate syntide, but showed significant differences in their interactions with Ca(2+)-calmodulin as assessed by binding, substrate phosphorylation, and autophosphorylation. Interestingly, the rank order of CaM binding affinity (gamma > beta > delta > alpha) does not directly correlate with the rank order of their CaM dependence for autophosphorylation (beta > gamma > delta > alpha). Simulations utilizing this data revealed that the measured differences in CaM binding affinities play a minor role in the autophosphorylation of the enzyme, which is largely dictated by the rate of autophosphorylation for each isoform.     

Regulatory Properties of Calcium/Calmodulin-Dependent Protein Kinase II in Rat Brain Postsynaptic Densities

Calcium/calmodulin (CaM)-dependent protein kinase II (CaM-kinase II) contained within the postsynaptic density (PSD) was shown to become partially Ca2+-independent following initial activation by Ca2+/CaM. Generation of this Ca2+-independent species was dependent upon autophosphorylation of both subunits of the enzyme in the presence of Mg2+/ATP/Ca2+/CaM and attained a maximal value of 74 +/- 5% of the total activity within 1-2 min. Subsequent to the generation of this partially Ca2+-independent form of PSD CaM-kinase II, addition of EGTA to the autophosphorylation reaction resulted in further stimulation of 32PO4 incorporation into both kinase subunits and a loss of stimulation of the kinase by Ca2+/CaM. Examination of the sites of Ca2+-dependent autophosphorylation by phosphoamino acid analysis and peptide mapping of both kinase subunits suggested that phosphorylation of Thr286/287 of the alpha- and beta-subunits, respectively, may be responsible for the transition of PSD CaM-kinase II to the Ca2+-independent species. A synthetic peptide 281-309 corresponding to a portion of the regulatory domain (residues 281-314) of the soluble kinase inhibited syntide-2 phosphorylation by the Ca2+-independent form of PSD CaM-kinase II (IC50 = 3.6 +/- 0.8 microM). Binding of Ca2+/CaM to peptide 281-309 abolished its inhibitory property. Phosphorylation of Thr286 in peptide 281-309 also decreased its inhibitory potency. These data suggest that CaM-kinase II in the PSD possesses regulatory properties and mechanisms of activation similar to the cytosolic form of CaM-kinase II.     

Structural features of Ca2+/calmodulin-dependent protein kinase II revealed by electron microscopy

The molecular conformation of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) from the rat forebrain and cerebellum was studied by means of EM using a quick-freezing technique. Each molecule appeared to be composed of two kinds of particles, with one larger central particle and smaller peripheral particles and had shapes resembling that of a flower with 8 or 10 "petals". A favorable shadowing revealed that each peripheral particle had a thin link to the central particle. We predicted that the 8-petal molecules and 10-petal molecules were octamers and decamers of CaM kinase II subunits, respectively, each assembled with the association domains of subunits gathered in the center, and the catalytic domains in the peripheral particles. Binding of antibodies to the enzyme molecules suggested that molecules with 8 and 10 peripheral particles were homopolymers composed only of beta subunit and of alpha subunit, respectively, specifying that CaM kinase II consists of homopolymer of either alpha or beta subunits.     

Alternative splicing modulates the frequency-dependent response of CaMKII to Ca(2+) oscillations

Ca(2+) oscillations are required in various signal trans duction pathways, and contain information both in their amplitude and frequency. Remarkably, the Ca(2+)/calmodulin(CaM)-dependent protein kinase II (CaMKII) can decode such frequencies. A Ca(2+)/CaM-stimulated autophosphorylation leads to Ca(2+)/CaM-independent (autonomous) activity of the kinase that outlasts the initial stimulation. This autonomous activity increases exponentially with the frequency of Ca(2+) oscillations. Here we show that three beta-CaMKII splice variants (beta(M), beta and beta(e)') have very similar specific activity and maximal autonomy. However, their autonomy generated by Ca(2+) oscillations differs significantly. A mechanistic basis was found in alterations of the CaM activation constant and of the initial rate of autophosphorylation. Structurally, the splice variants differ only in a variable 'linker' region between the kinase and association domains. Therefore, we propose that differences in relative positioning of kinase domains within multimeric holoenzymes are responsible for the observed effects. Notably, the beta-CaMKII splice variants are differentially expressed, even among individual hippocampal neurons. Taken together, our results suggest that alternative splicing provides cells with a mechanism to modulate their sensitivity to Ca(2+) oscillations.     

Global Forebrain Ischemia Induces a Posttranslational Modification of Multifunctional Calcium- and Calmodulin-Dependent Kinase II

The activity of multifunctional calcium/calmodulin-dependent protein kinase II (CaM kinase II) has recently been shown to be inhibited by transient global ischemia. To investigate the nature of ischemia-induced inhibition of the enzyme, CaM kinase II was purified to greater than 1,000-fold from brains of control and ischemic gerbils. The characteristics of CaM kinase II from control and ischemic preparations were compared by numerous parameters. Kinetic analysis of purified control and ischemic CaM kinase II was performed for autophosphorylation properties, ATP, magnesium, calcium, and calmodulin affinity, immunoreactivity, and substrate recognition. Ischemia induced a reproducible inhibition of CaM kinase II activity, which could not be overcome by increasing the concentration of any of the reaction parameters. Ischemic CaM kinase II was not different from control enzyme in affinity for calmodulin, Ca2+, Mg2+, or exogenously added substrate or rate of autophosphorylation. CaM kinase II isolated from ischemic gerbils displayed decreased immunoreactivity with a monoclonal antibody (immunoglobulin G3) directed toward the beta subunit of the enzyme. In addition, ischemia caused a significant decrease in affinity of CaM kinase II for ATP when measured by extent of autophosphorylation. To characterize further the decrease in ATP affinity of CaM kinase II, the covalent-binding ATP analog 8-azido-adenosine-5'-[alpha-32P]triphosphate was used. Covalent binding of 25 microM azido-ATP was decreased 40.4 +/-12.3% in ischemic CaM kinase II when compared with control enzyme (n = 5; p less than 0.01 by paired Student's t test). Thus, CaM kinase II levels for ischemia and control fractions were equivalent by protein staining, percent recovery, and calmodulin binding but were significantly different by immunoreactivity and ATP binding. The data are consistent with the hypothesis that ischemia induces a posttranslational modification that alters ATP binding in CaM kinase II and that results in an apparent decrease in enzymatic activity.     

Autophosphorylation and activation of Ca2+/calmodulin-dependent protein kinase II in intact nerve terminals

The autophosphorylation of purified Ca2+/calmodulin-dependent protein kinase II (Ca2+/CaM kinase II) on a threonine-containing phosphopeptide common to both the alpha and beta subunits was previously shown to convert this enzyme into a catalytically active Ca2+-independent species. We now have examined the phosphorylation and activation of Ca2+/CaM kinase II in synaptosomes, a Ca2+-dependent neurosecretory system consisting of isolated nerve terminals. Synaptosomes were prelabeled with 32Pi and the alpha subunit of Ca2+/CaM kinase II was immunoprecipitated. Under basal incubation conditions the alpha subunit was phosphorylated. Depolarization of synaptosomes produced a rapid (2-5 s) Ca2+-dependent increase of about 50% in the state of phosphorylation of the alpha subunit. This was followed by a slower increase in the 32P content of the alpha subunit over the next 5 min of depolarization. The enhanced phosphorylation was characterized by an initial rise (2 s) and subsequent decrease (30 s) in the phosphothreonine content of the alpha subunit. In contrast, the phosphoserine content of the alpha subunit slowly increased during the course of depolarization. Thermolytic two-dimensional phosphopeptide maps of the alpha subunit demonstrated that depolarization stimulated the labeling of a phosphopeptide associated with autoactivation. In parallel experiments, unlabeled synaptosomes were depolarized, and lysates of these synaptosomes were assayed for Ca2+/CaM kinase II activity. Depolarization produced a rapid (less than or equal to 2 s) increase in Ca2+-independent Ca2+/CaM kinase II activity. This activity returned to basal levels by 60 s. Thus, depolarization of intact synaptosomes is associated with the transient phosphorylation of Ca2+/CaM kinase II on threonine residues, presumably involving an autophosphorylation mechanism and concomitantly the transient generation of the Ca2+-independent form of Ca2+/CaM kinase II.     

A Retinal Calmodulin-Dependent Kinase: Calcium/ Calmodulin-Stimulated and -Inhibited States

Abstract: A calcium/calmodulin-dependent protein kinase was isolated from retina. The retinal enzyme is composed exclusively of 50-kilodalton (kD) subunits and has a molecular mass of approximately 275 kD, in contrast to forebrain calmodulin kinase II, which is composed of 50-kD and 60-kD subunits in a 3:1 ratio and has a molecular mass of approximately 520 kD. Similar substrate specificities, kinetic properties, capacity to bind calmodulin, and immunoreactivity suggest that the retinal kinase is an isoenzyme of forebrain calmodulin kinase II. Both kinases autophosphorylate in an intramolecular manner; however, auto-phosphorylation has different effects on the activities of the two enzymes. Autophosphorylation of retinal calmodulin kinase converts the enzyme from a calcium/calmodulin-dependent to a calcium/calmodulin-inhibited kinase, with high activity in the absence of calcium, whereas autophosphorylation of the forebrain kinase results in a less active, calcium/calmodulin-independent enzyme. These properties of calmodulin kinase may play an important role in retinal function.     

Staurosporine: An Effective Inhibitor for Ca2+/Calmodulin-Dependent Protein Kinase II

We investigated the effect of staurosporine on Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) purified from rat brain. (a) Staurosporine (10-100 nM) inhibited the activity of CaM kinase II. The half-maximal and maximal inhibitory concentrations were 20 and 100 nM, respectively. (b) The inhibition with staurosporine was of the noncompetitive type with respect to ATP, calmodulin, and phosphate acceptor (beta-casein). (c) Staurosporine suppressed the auto-phosphorylation of alpha- and beta-subunits of CaM kinase II at concentrations similar to those at which the enzyme activity was inhibited. (d) Staurosporine also attenuated the Ca2+/calmodulin-independent activity of the autophosphorylated CaM kinase II. These results suggest that staurosporine inhibits CaM kinase II by interacting with the catalytic domain, distinct from the ATP-binding site or substrate-binding site, of the enzyme and that staurosporine is an effective inhibitor for CaM kinase II in the cell system.     

Autophosphorylation of the type II calmodulin-dependent protein kinase is essential for formation of a proteolytic fragment with catalytic activity. Implications for long-term synaptic potentiation

Autophosphorylation plays an essential role in proteolytic activation of the type II calmodulin-dependent protein kinase (CaM kinase II). Limited proteolysis of CaM kinase II by trypsin, alpha-chymotrypsin, and Ca2+-stimulated neutral protease (calpain) yielded a catalytically active kinase fragment only when the holoenzyme was autophosphorylated prior to proteolysis. Slightly larger, inactive fragments were obtained from nonphosphorylated CaM kinase II, regardless of whether Ca2+/calmodulin or Mg2+/ATP were present or absent. The active fragment exhibited Ca2+/calmodulin-dependent kinase activity with kinetic parameters identical with those of the activated holoenzyme. The key autophosphorylation site of CaM kinase II was absent from the active fragment which indicates that proteolysis can effectively uncouple the activation state and Ca2+/calmodulin independence of the kinase from the action of phosphoprotein phosphatases. Because autophosphorylation exerts such a tight control over this irreversible process, proteolytic activation of CaM kinase II by intracellular proteases offers an attractive mechanism for prolonging the effects of Ca2+ at the synapse.     

Functional analysis of Ca2+/calmodulin-dependent protein kinase II expressed in bacteria

The cDNA encoding the 50-kDa subunit of Ca2+/calmodulin (CaM)-dependent protein kinase II from adult rat brain was cloned into the bacterial expression vector pK223-2 and produced in bacteria. Extensive modification of the cDNA was required to express detectable levels of enzyme. The activity of the bacterially expressed kinase was stringently dependent on Ca2+/CaM but did not exhibit cooperative activation kinetics characteristic of the forebrain enzyme and required 10-fold greater amounts of CaM for half-maximal activation. The bacterially expressed enzyme displayed an apparent Km for a synthetic peptide substrate similar to that of the forebrain enzyme (12 and 10 microM, respectively). Limited proteolysis maps of autophosphorylated peptides, and Western blot analysis demonstrated that the bacterially expressed enzyme was structurally and immunologically indistinguishable from the 50-kDa subunit of the rat forebrain holoenzyme. The bacterially expressed enzyme became Ca2+/CaM-independent after Ca2+/CaM-dependent autophosphorylation in a fashion identical to the forebrain enzyme.     

Reversible generation of a Ca2+-independent form of Ca2+(calmodulin)-dependent protein kinase II by an autophosphorylation mechanism

The Ca2+(calmodulin (CaM))-dependent protein kinase II, purified from either rabbit liver or rat brain, was preincubated under conditions that are known to promote its autophosphorylation. When kinase activity was assayed after this preincubation, it was observed that excess EGTA could block no more than 40-60% of the total Ca2+- and CaM-dependent activity compared to 95% inhibition by EGTA prior to preincubation. In the EGTA assay, free Ca2+ was calculated to be less than 1 nM; therefore, this activity was designated Ca2+-independent activity. Formation of this Ca2+-independent form of the kinase was shown to be associated with autophosphorylation based on the following observations: (a) it required the presence of Ca2+, CaM, and ATP; (b) the ATP analogs adenylyl imidodiphosphate and adenylyl methylenediphosphate could not substitute for ATP; (c) generation of the independent form was associated with incorporation of phosphate into the kinase; and (d) addition of protein phosphatase partially dephosphorylated the kinase and restored its Ca2+ dependence. This phenomenon may be of physiological importance because it would prolong the effects of extracellular signals that only transiently increase the intracellular Ca2+ level.     

Differential autophosphorylation of CaM kinase II from phasic and tonic smooth muscle tissues

Ca+/calmodulin-dependent protein kinase II (CaM kinase II) is regulated by calcium oscillations, autophosphorylation, and its subunit composition. All four subunit isoforms were detected in gastric fundus and proximal colon smooth muscles by RT-PCR, but only the gamma and delta isoforms are expressed in myocytes. Relative gamma and delta message levels were quantitated by real-time PCR. CaM kinase II protein and Ca2+/calmodulin-stimulated (total) activity levels are higher in proximal colon smooth muscle lysates than in fundus lysates, but Ca2+/calmodulin-independent (autonomous) activity is higher in fundus lysates. CaM kinase II in fundus lysates is relatively unresponsive to Ca2+/calmodulin. Alkaline phosphatase decreased CaM kinase II autonomous activity in fundus lysates and restored its responsiveness to Ca2+/calmodulin. Acetylcholine (ACh) increased autonomous CaM kinase II activity in fundus and proximal colon smooth muscles in a time- and dose-dependent manner. KN-93 enhanced ACh-induced fundus contractions but inhibited proximal colon contractions. The different properties of CaM kinase II from fundus and proximal colon smooth muscles suggest differential regulation of its autophosphorylation and activity in tonic and phasic gastrointestinal smooth muscles.     

A mechanism for synaptic frequency detection through autophosphorylation of CaM kinase II.

A model for the regulation of CaM kinase II is presented based on the following reported properties of the molecule: 1) The holoenzyme is composed of 8-12 subunits, each with the same set of autophosphorylation sites; 2) Autophosphorylation at one group of sites (A sites) requires the presence of Ca2+ and causes a subunit to remain active following the removal of Ca2+; 3) Autophosphorylation at another group of sites (B sites) occurs only after the removal of Ca2+ but requires prior phosphorylation of a threshold number of A sites within the holoenzyme. Because B-site phosphorylation inhibits Ca2+/calmodulin binding, we propose that, for a given subunit, phosphorylation of a B site before an A site prevents subsequent phosphorylation at the A site and thereby locks that subunit in an inactive state. The model predicts that a threshold activation by Ca2+ will initiate an "autophosphorylation phase." Once started, intra-holoenzyme autophosphorylation will proceed, on A sites during periods of high [Ca2+] and on B sites during periods of low [Ca2+]. At "saturation," that is when every subunit has been phosphorylated on a B site, the number of phosphorylated A sites and, therefore, the kinase activity will reflect the relative durations of periods of high [Ca2+] to periods of low [Ca2+] that occurred during the autophosphorylation phase. Using a computer program designed to simulate the above mechanism, we show that the ultimate state of phosphorylation of an array of CaM kinase II molecules could be sensitive to the temporal pattern of Ca2+ pulses. We speculate that such a mechanism may allow arrays of CaM kinase II molecules in postsynaptic densities to act as synaptic frequency detectors involved in setting the direction and level of synaptic modification.     

Sequences of autophosphorylation sites in neuronal type II CaM kinase that control Ca2(+)-independent activity

After initial activation by Ca2+, the catalytic activity of type II Ca2+/calmodulin-dependent protein kinase rapidly becomes partially independent of Ca2+. The transition is caused by autophosphorylation of a few subunits in the dodecameric holoenzyme, which is composed of varying proportions of two homologous types of subunits, alpha (50 kd) and beta (58-60 kd). We have identified one site in the alpha subunit (Thr286) and two in the beta subunit (Thr287 and Thr382) that are rapidly autophosphorylated. We show that phosphorylation of alpha-Thr286 and beta-Thr287, which are located immediately adjacent to the calmodulin binding domain, controls Ca2(+)-independent activity. In contrast, phosphorylation of beta-Thr382 is not required to maintain Ca2+ independence. It is absent in the alpha subunit and is selectively removed from the minor beta' subunit, apparently by alternative splicing. Regulation of the presence of beta-Thr382 in the holoenzyme by both differential gene expression and alternative splicing suggests that it may have an important but highly specialized function.     

Studies of the regulatory mechanism of Ca2+/calmodulin-dependent protein kinase II. Mutation of threonine 286 to alanine and aspartate

A cDNA clone for the alpha subunit of mouse brain Ca2+/CaM-dependent protein kinase II (CaM-kinase II) was transcribed in vitro and translated in a rabbit reticulocyte lysate system. Inclusion of [35S]methionine in the translation system yielded a single 35S-polypeptide of about 50 kDa. When the translation system was assayed for CaM-kinase II activity, there was a 5-10-fold enrichment of kinase activity which was totally dependent on Ca2+/calmodulin (CaM). Both the 50-kDa 35S-polypeptide and the Ca2+/CaM-dependent protein kinase activity were quantitatively immunoprecipitated by rat brain CaM-kinase II antibody. When the translated wild-type kinase was subjected to autophosphorylation conditions in the presence of Ca2+, CaM, Mg2+, and ATP, the Ca2+-independent activity (assayed in the presence of [ethylenebis(oxyethylenenitrilo)]tetraacetic acid) increased from 5.8 +/- 0.7 to 26.5 +/- 2.1% of total activity (assayed in the presence of Ca2+/CaM). These properties confirm the identity of the kinase translated in vitro as CaM-kinase II. The role of Thr-286 autophosphorylation in formation of the Ca2+-independent activity was investigated by site-directed mutation of Thr-286 to Ala (Ala-286 kinase) and to Asp (Asp-286 kinase). The Ala-286 kinase was completely dependent on Ca2+/CaM for activity prior and subsequent to autophosphorylation. The Asp-286 kinase exhibited 21.9 +/- 0.8% Ca2+-independent activity, and this was not increased by autophosphorylation. These results establish that introduction of negative charge(s) at residue 286, either by autophosphorylation of Thr or by mutation to Asp, is sufficient and necessary to generate the partially Ca2+-independent form of CaM-kinase II.     

Regulation of Ca2+/calmodulin-dependent protein kinase II by Ca2+/calmodulin-independent autophosphorylation

The autophosphorylation of Ca2+/calmodulin-dependent protein kinase II (CaM-KII) results in the generation of kinase activity that is largely Ca2+/CaM-independent. We report that continued Ca2+/CaM-independent autophosphorylation of CaM-KII results in the generation of distinct phosphopeptides as identified by high performance liquid chromatography and enzymatic properties that are different than those observed for Ca2+/CaM-dependent autophosphorylation. These Ca2+/CaM-independent properties include (a) increased catalytic activity, (b) higher substrate affinity for the phosphorylation of synapsin I, and (c) decreased CaM-binding to both CaM-KII subunits as analyzed by gel overlays. Our results indicate that the autophosphorylation of only one subunit per holoenzyme is required to generate the Ca2+/CaM-independent CaM-KII. We suggest a two-step process by which autophosphorylation regulates CaM-KII. Step I requires Ca2+/CaM and underlies initial kinase activation. Step II involves continued autophosphorylation of the Ca2+/CaM-independent kinase and results in increased affinity for its substrate synapsin I and decreased affinity for calmodulin. These results indicate a complex mechanism through which autophosphorylation of CaM-KII may regulate its activity in response to transient fluctuations in intracellular calcium.