Plant chimeric Ca2+/Calmodulin-dependent protein kinase. Role of the neural visinin-like domain in regulating autophosphorylation and calmodulin affinity

Chimeric Ca(2+)/calmodulin-dependent protein kinase (CCaMK) is characterized by a serine-threonine kinase domain, an autoinhibitory domain, a calmodulin-binding domain and a neural visinin-like domain with three EF-hands. The neural visinin-like Ca(2+)-binding domain at the C-terminal end of the CaM-binding domain makes CCaMK unique among all the known calmodulin-dependent kinases. Biological functions of the plant visinin-like proteins or visinin-like domains in plant proteins are not well known. Using EF-hand deletions in the visinin-like domain, we found that the visinin-like domain regulated Ca(2+)-stimulated autophosphorylation of CCaMK. To investigate the effects of Ca(2+)-stimulated autophosphorylation on the interaction with calmodulin, the equilibrium binding constants of CCaMK were measured by fluorescence emission anisotropy using dansylated calmodulin. Binding was 8-fold tighter after Ca(2+)-stimulated autophosphorylation. This shift in affinity did not occur in CCaMK deletion mutants lacking Ca(2+)-stimulated autophosphorylation. A variable calmodulin affinity regulated by Ca(2+)-stimulated autophosphorylation mediated through the visinin-like domain is a new regulatory mechanism for CCaMK activation and calmodulin-dependent protein kinases. Our experiments demonstrate the existence of two functional molecular switches in a protein kinase regulating the kinase activity, namely a visinin-like domain acting as a Ca(2+)-triggered switch and a CaM-binding domain acting as an autophosphorylation-triggered molecular switch.     

Autophosphorylation-dependent inactivation of plant chimeric calcium/calmodulin-dependent protein kinase.

Chimeric calcium/calmodulin dependent protein kinase (CCaMK) is characterized by the presence of a visinin-like Ca(2+)-binding domain unlike other known calmodulin- dependent kinases. Ca(2+)-Binding to the visinin-like domain leads to autophosphorylation and changes in the affinity for calmodulin [Sathyanarayanan P.V., Cremo C.R. & Poovaiah B.W. (2000) J. Biol. Chem. 275, 30417-30422]. Here, we report that the Ca(2+)-stimulated autophosphorylation of CCaMK results in time-dependent loss of enzyme activity. This time-dependent loss of activity or self-inactivation due to autophosphorylation is also dependent on reaction pH and ATP concentration. Inactivation of the enzyme resulted in the formation of a sedimentable enzyme due to self-association. Specifically, autophosphorylation in the presence of 200 microm ATP at pH 7.5 resulted in the formation of a sedimentable enzyme with a 33% loss in enzyme activity. Under similar conditions at pH 6.5, the enzyme lost 67% of its activity and at pH 8.5, 84% enzyme activity was lost. Furthermore, autophosphorylation at either acidic or alkaline reaction pH lead to the formation of a sedimentable enzyme. Transmission electron microscopic studies on autophosphorylated kinase revealed particles that clustered into branched complexes. The autophosphorylation of wild-type kinase in the presence of AMP-PNP (an unhydrolyzable ATP analog) or the autophosphorylation-site mutant, T267A, did not show formation of branched complexes under the electron microscope. Autophosphorylation- dependent self-inactivation may be a mechanism of modulating the signal transduction pathway mediated by CCaMK.     

Inhibitory autophosphorylation of multifunctional Ca2+/calmodulin-dependent protein kinase analyzed by site-directed mutagenesis.

Initial autophosphorylation of multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase) occurs at Thr286 (the "autonomy" site) and converts the kinase from a Ca(2+)-dependent to a partially Ca(2+)-independent or autonomous enzyme. After removal of Ca2+/calmodulin, the autonomous kinase undergoes a "burst" of inhibitory autophosphorylation at sites distinct from the autonomy site which may be masked in the presence of bound calmodulin. This burst of Ca(2+)-independent autophosphorylation blocks the ability of calmodulin to activate the kinase. We have used site-directed mutagenesis to replace putative inhibitory autophosphorylation sites within the calmodulin binding domain of recombinant alpha-CaM kinase with nonphosphorylatable alanines and examined the effects on autophosphorylation, kinase activity, and calmodulin binding. Although prominent Ca(2+)-independent autophosphorylation occurs within the calmodulin binding domain at Thr305, Thr306, and Ser314 in wild-type alpha-CaM kinase, the inhibitory effect on kinase activity and calmodulin binding is retained in mutants lacking any one of these three sites. However, when both Thr305 and Thr306 are converted to alanines the kinase does not display inhibition of either activity or calmodulin binding. Autophosphorylation at either Thr305 or Thr306 is therefore sufficient to block both binding and activation of the kinase by Ca2+/calmodulin. Thr306 is also slowly autophosphorylated in a basal reaction in the continuous absence of Ca2+/calmodulin. Autophosphorylation of Thr306 by the kinase in either its basal or autonomous state suggests that in the absence of bound calmodulin, the region of the autoregulatory domain surrounding Thr306, rather than the region near the autonomy site, lies nearest the peptide substrate binding site of the kinase.     

A model for molecular mechanisms of synaptic competition for a finite resource

Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) undergoes Ca(2+)/calmodulin-dependent autophosphorylation of threonine-286/287 (Thr(286/287)). Extremely high concentration of CaMKII in the postsynaptic spine indicates that increase in the Ca(2+) concentration in the spine induced by synaptic activation results in Thr(286/287) autophosphorylation of this enzyme. It has recently been shown that the K(d) value of CaMKII for Ca(2+)/calmodulin (Ca(2+)/CaM) drastically decreases after Thr(286/287) autophosphorylation. Therefore, Ca(2+)/CaM associated with CaMKII becomes tightly bound to this kinase after Thr(286/287) autophosphorylation. This has been called 'Ca(2+)/CaM trapping'. We discussed the functional significance of Ca(2+)/CaM trapping in the neuronal system by a mathematical-modelling approach. We considered neighbouring synapses formed on the same dendrite and different increase in the Ca(2+) concentration in each spine. CaMKII undergoing Thr(286/287) autophosphorylation in each spine is eager to recruit nearby calmodulin in the dendrite for Ca(2+)/CaM trapping. Since the amount of calmodulin is limited, recruiting calmodulin to each spine causes competition among synapses for this finite resource. The results of our computer simulation show that this competition leads to 'winner-take-all': almost all calmodulin is taken by a few synapses to which relatively large increases in the Ca(2+) concentration are assigned. These results suggest a novel form of synaptic encoding of information.     

Differential modulation of Ca2+/calmodulin-dependent protein kinase II activity by regulated interactions with N-methyl-D-aspartate receptor NR2B subunits and alpha-actinin

Neuronal Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) interacts with several prominent dendritic spine proteins, which have been termed CaMKII-associated proteins. The NR2B subunit of N-methyl-d-aspartate (NMDA)-type glutamate receptor, densin-180, and alpha-actinin bind comparable, approximately stoichiometric amounts of Thr(286)-autophosphorylated CaMKIIalpha, forming a ternary complex (Robison, A. J., Bass, M. A., Jiao, Y., Macmillan, L. B., Carmody, L. C., Bartlett, R. K., and Colbran, R. J. (2005) J. Biol. Chem. 280, 35329-35336), but their impacts on CaMKII function are poorly understood. Here we show that these interactions are differentially regulated and exert distinct effects on CaMKII activity. Nonphosphorylated and Thr(286)-autophosphorylated CaMKII bind to alpha-actinin with similar efficacy, but autophosphorylation at Thr(305/306) or Ca(2+)/calmodulin binding significantly reduce this binding. Moreover, alpha-actinin antagonizes CaMKII activation by Ca(2+)/calmodulin, as assessed by autophosphorylation and phosphorylation of a peptide substrate. CaMKII binding to densin (1247-1542) is partially independent of Thr(286) autophosphorylation and is unaffected by Ca(2+)-independent autophosphorylation or Ca(2+)/calmodulin. In addition, the CaMKII binding domain of densin-180 has little effect on CaMKII activity. In contrast, the interaction of CaMKIIalpha with NR2B requires either Thr(286) autophosphorylation or the binding of both Ca(2+)/calmodulin and adenine nucleotides. NR2B inhibits both the Ca(2+)/calmodulin-dependent and autonomous activities of CaMKII by a mechanism that is competitive with autocamtide-2 substrate, non-competitive with syntide-2 substrate, and uncompetitive with respect to ATP. In combination, these data suggest that dynamically regulated interactions with CaMKII-associated proteins could play pleiotropic roles in finetuning CaMKII signaling in defined subcellular compartments.     

Substrate-selective and calcium-independent activation of CaMKII by α-actinin

Protein-protein interactions are thought to modulate the efficiency and specificity of Ca(2+)/calmodulin (CaM)-dependent protein kinase II (CaMKII) signaling in specific subcellular compartments. Here we show that the F-actin-binding protein α-actinin targets CaMKIIα to F-actin in cells by binding to the CaMKII regulatory domain, mimicking CaM. The interaction with α-actinin is blocked by CaMKII autophosphorylation at Thr-306, but not by autophosphorylation at Thr-305, whereas autophosphorylation at either site blocks Ca(2+)/CaM binding. The binding of α-actinin to CaMKII is Ca(2+)-independent and activates the phosphorylation of a subset of substrates in vitro. In intact cells, α-actinin selectively stabilizes CaMKII association with GluN2B-containing glutamate receptors and enhances phosphorylation of Ser-1303 in GluN2B, but inhibits CaMKII phosphorylation of Ser-831 in glutamate receptor GluA1 subunits by competing for activation by Ca(2+)/CaM. These data show that Ca(2+)-independent binding of α-actinin to CaMKII differentially modulates the phosphorylation of physiological targets that play key roles in long-term synaptic plasticity.     

Isolation and characterization of a novel calcium/calmodulin-dependent protein kinase, AtCK, from arabidopsis

Protein phosphorylation is one of the major mechanisms by which eukaryotic cells transduce extracellular signals into intracellular responses. Calcium/calmodulin (Ca(2+)/CaM)-dependent protein phosphorylation has been implicated in various cellular processes, yet little is known about Ca(2+)/CaM-dependent protein kinases (CaMKs) in plants. From an Arabidopsis expression library screen using a horseradish peroxidase-conjugated soybean calmodulin isoform (SCaM-1) as a probe, we isolated a full-length cDNA clone that encodes AtCK (Arabidopsis thaliana calcium/calmodulin-dependent protein kinase). The predicted structure of AtCK contains a serine/threonine protein kinase catalytic domain followed by a putative calmodulin-binding domain and a putative Ca(2+)-binding domain. Recombinant AtCK was expressed in E. coli and bound to calmodulin in a Ca(2+)-dependent manner. The ability of CaM to bind to AtCK was confirmed by gel mobility shift and competition assays. AtCK exhibited its highest levels of autophosphorylation in the presence of 3 mM Mn(2+). The phosphorylation of myelin basic protein (MBP) by AtCK was enhanced when AtCK was under the control of calcium-bound CaM, as previously observed for other Ca(2+)/CaM-dependent protein kinases. In contrast to maize and tobacco CCaMKs (calcium and Ca(2+)/CaM-dependent protein kinase), increasing the concentration of calmodulin to more than 3 microgram suppressed the phosphorylation activity of AtCK. Taken together our results indicate that AtCK is a novel Arabidopsis Ca(2+)/CaM-dependent protein kinase which is presumably involved in CaM-mediated signaling.     

Chimeric calcium/calmodulin-dependent protein kinase in tobacco: differential regulation by calmodulin isoforms

cDNA clones of chimeric Ca2+/calmodulin-dependent protein kinase (CCaMK) from tobacco (TCCaMK-1 and TCCaMK-2) were isolated and characterized. The polypeptides encoded by TCCaMK-1 and TCCaMK-2 have 15 different amino acid substitutions, yet they both contain a total of 517 amino acids. Northern analysis revealed that CCaMK is expressed in a stage-specific manner during anther development. Messenger RNA was detected when tobacco bud sizes were between 0.5 cm and 1.0 cm. The appearance of mRNA coincided with meiosis and became undetectable at later stages of anther development. The reverse polymerase chain reaction (RT-PCR) amplification assay using isoform-specific primers showed that both of the CCaMK mRNAs were expressed in anther with similar expression patterns. The CCaMK protein expressed in Escherichia coli showed Ca2+-dependent autophosphorylation and Ca2+/calmodulin-dependent substrate phosphorylation. Calmodulin isoforms (PCM1 and PCM6) had differential effects on the regulation of autophosphorylation and substrate phosphorylation of tobacco CCaMK, but not lily CCaMK. The evolutionary tree of plant serine/threonine protein kinases revealed that calmodulin-dependent kinases form one subgroup that is distinctly different from Ca2+-dependent protein kinases (CDPKs) and other serine/threonine kinases in plants.     

Regulation of Ca(2+)/calmodulin-dependent protein kinase kinase alpha by cAMP-dependent protein kinase: I. Biochemical analysis

Ca(2+)/calmodulin-dependent protein kinases (CaM-kinases) I and IV are activated upon phosphorylation of their Thr(177) and Thr(196), respectively, by the upstream Ca(2+)/calmodulin-dependent protein kinases CaM-kinase kinase alpha and beta, and deactivated upon dephosphorylation by protein phosphatases such as CaM-kinase phosphatase. Recent studies demonstrated that the activity of CaM-kinase kinase alpha is decreased upon phosphorylation by cAMP-dependent protein kinase (PKA), and the relationship between the inhibition and phosphorylation of CaM-kinase kinase alpha by PKA has been studied. In the present study, we demonstrate that the activity of CaM-kinase kinase alpha toward PKIV peptide, which contains the sequence surrounding Thr(196) of CaM-kinase IV, is increased by incubation with PKA in the presence of Ca(2+)/calmodulin but decreased in its absence, while the activity toward CaM-kinase IV is decreased by incubation with PKA in both the presence and absence of Ca(2+)/calmodulin. Six phosphorylation sites on CaM-kinase kinase alpha, Ser(24) for autophosphorylation, and Ser(52), Ser(74), Thr(108), Ser(458), and Ser(475) for phosphorylation by PKA, were identified by amino acid sequence analysis of the phosphopeptides purified from the tryptic digest of the phosphorylated enzymes. The presence of Ca(2+)/calmodulin suppresses phosphorylation on Ser(52), Ser(74), Thr(108), and Ser(458) by PKA, but accelerates phosphorylation on Ser(475). The changes in the activity of the enzyme upon phosphorylation appear to occur as a result of conformational changes induced by phosphorylation on several sites.     

Interaction of plant chimeric calcium/calmodulin-dependent protein kinase with a homolog of eukaryotic elongation factor-1alpha

A chimeric Ca2+/calmodulin-dependent protein kinase (CCaMK) was previously cloned and characterized in this laboratory. To investigate the biological functions of CCaMK, the yeast two-hybrid system was used to isolate genes encoding proteins that interact with CCaMK. One of the cDNA clones obtained from the screening (LlEF-1alpha1) has high similarity with the eukaryotic elongation factor-1alpha (EF-1alpha). CCaMK phosphorylated LlEF-1alpha1 in a Ca2+/calmodulin-dependent manner. The phosphorylation site for CCaMK (Thr-257) was identified by site-directed mutagenesis. Interestingly, Thr-257 is located in the putative tRNA-binding region of LlEF-1alpha1. An isoform of Ca2+-dependent protein kinase (CDPK) phosphorylated multiple sites of LlEF-1alpha1 in a Ca2+-dependent but calmodulin-independent manner. Unlike CDPK, CCaMK phosphorylated only one site, and this site is different from CDPK phosphorylation sites. This suggests that the phosphorylation of EF-1alpha by these two kinases may have different functional significance. Although the phosphorylation of LlEF-1alpha1 by CCaMK is Ca2+/calmodulin-dependent, in vitro binding assays revealed that CCaMK binds to LlEF-1alpha1 in a Ca2+-independent manner. This was further substantiated by coimmunoprecipitation of CCaMK and EF-1alpha using the protein extract from lily anthers. Dissociation of CCaMK from EF-1alpha by Ca2+ and phosphorylation of EF-1alpha by CCaMK in a Ca2+/calmodulin-dependent manner suggests that these interactions may play a role in regulating the biological functions of EF-1alpha.     

Chemical quenched flow kinetic studies indicate an intraholoenzyme autophosphorylation mechanism for Ca2+/calmodulin-dependent protein kinase II

Autophosphorylation of alpha-Ca(2+)/calmodulin-dependent protein kinase II (CaM kinase II) at Thr-286 generates Ca(2+)-independent activity that outlasts the initial Ca(2+) stimulus. Previous studies suggested that this autophosphorylation occurs between subunits within each CaM kinase II holoenzyme. However, electron microscopy studies have questioned this mechanism because a large distance separates a kinase domain from its neighboring subunit. Moreover, the recently discovered ability of CaM kinase II holoenzymes to self-associate has raised questions about data interpretation in previous investigations of autophosphorylation. In this work, we characterize the mechanism of CaM kinase II autophosphorylation. To eliminate ambiguity arising from kinase aggregation, we used dynamic light scattering to establish the monodispersity of all enzyme solutions. We then found using chemical quenched flow kinetics that the autophosphorylation rate was independent of the CaM kinase II concentration, results corroborating intraholoenzyme activation. Experiments with a monomeric CaM kinase II showed that phosphorylation of this construct is intermolecular, supporting intersubunit phosphorylation within a holoenzyme. The autophosphorylation rate at 30 degrees C was approximately 12 s(-1), more than 10-fold faster than past estimates. The ability of CaM kinase II to autophosphorylate through an intraholoenzyme, intersubunit mechanism is likely central to its functions of decoding Ca(2+) spike frequency and providing a sustained response to Ca(2+) signals.     

A brain-specific Ca2+/calmodulin-dependent protein kinase (CaM kinase-Gr) is regulated by autophosphorylation. Relevance to neuronal Ca2+ signaling.

A neuronal Ca2+/calmodulin-dependent protein kinase (CaM kinase-Gr) undergoes autophosphorylation on a serine residue(s) in response to Ca2+ and calmodulin. Phosphate incorporation leads to the formation of a Ca(2+)-independent (autonomous) activity state, as well as potentiation of the Ca2+/calmodulin-dependent response. The autonomous enzyme activity of the phosphorylated enzyme approximately equals the Ca2+/calmodulin-stimulated activity of the unphosphorylated enzyme, but displays diminished affinity toward ATP and the synthetic substrate, syntide-2. The Km(app) for ATP and syntide-2 increased 4.3- and 1.7-fold, respectively. Further activation of the autonomous enzyme by Ca2+/calmodulin yields a marked increase in the affinity for ATP and peptide substrate such that the Km(app) for ATP and syntide-2 decreased by 14- and 8-fold, respectively. Both autophosphorylation and the addition of Ca2+/calmodulin are required to produce the maximum level of enzyme activation and to increase substrate affinity. Unlike Ca2+/calmodulin-dependent protein kinase type II that is dephosphorylated by the Mg(2+)-independent phosphoprotein phosphatases 1 and 2A, CaM kinase-Gr is dephosphorylated by a Mg(2+)-dependent phosphoprotein phosphatase that may be related to the type 2C enzyme. Dephosphorylation of CaM kinase-Gr reverses the effects of autophosphorylation on enzyme activity. A comparison between the autophosphorylation and dephosphorylation reactions of CaM kinase-Gr and Ca2+/calmodulin-dependent protein kinase type II provides useful insights into the operation of Ca(2+)-sensitive molecular switches.     

Cloning of a Ca(2+)/calmodulin-dependent protein kinase gene from the filamentous fungus Arthrobotrys dactyloides

A Ca(2+)/calmodulin-dependent protein kinase (CaMK) gene was cloned and characterized from Arthrobotrys dactyloides, a nematode-trapping fungus. The resulting 373-amino-acid protein, FCaMK, has significant homology to mammalian CaMKs. FCaMK contains a serine/threonine kinase domain followed by a calmodulin-binding domain. The activation loop in FCaMK (amino acids 184-199) contains a phosphorylation site at threonine-188, which could be the target of a kinase activator. Truncated FCaMK mutants revealed that amino acids 296-324 are essential for calmodulin binding. An oligopeptide designed from residues 297-324 formed a stable peptide-calmodulin complex of 1:1 stoichiometry. Southern blot analysis detected a single copy of the fcamk gene, suggesting that FCaMK plays an important role in Ca(2+)/calmodulin signaling in A. dactyloides.     

Activity-dependent gating of CaMKII autonomous activity by Drosophila CASK

The ability of CaMKII to act as a molecular switch, becoming Ca(2+) independent after activation and autophosphorylation at T287, is critical for experience-dependent plasticity. Here, we show that the Drosophila homolog of CASK, also known as Camguk, can act as a gain controller on the transition to calcium-independence in vivo. Genetic loss of dCASK significantly increases synapse-specific, activity-dependent autophosphorylation of CaMKII T287. In wild-type adult animals, simple and complex sensory stimuli cause region-specific increases in pT287. dCASK-deficient adults have a reduced dynamic range for activity-dependent T287 phosphorylation and have circuit-level defects that result in inappropriate activation of the kinase. dCASK control of the CaMKII switch occurs via its ability to induce autophosphorylation of T306 in the kinase's CaM binding domain. Phosphorylation of T306 blocks Ca(2+)/CaM binding, lowering the probability of intersubunit T287 phosphorylation, which requires CaM binding to both the substrate and catalytic subunits. dCASK is the first CaMKII-interacting protein other than CaM found to regulate this plasticity-controlling molecular switch.     

Calcium/calmodulin stimulates the autophosphorylation of elongation factor 2 kinase on Thr-348 and Ser-500 to regulate its activity and calcium dependence

Eukaryotic elongation factor 2 kinase (eEF-2K) is an atypical protein kinase regulated by Ca(2+) and calmodulin (CaM). Its only known substrate is eukaryotic elongation factor 2 (eEF-2), whose phosphorylation by eEF-2K impedes global protein synthesis. To date, the mechanism of eEF-2K autophosphorylation has not been fully elucidated. To investigate the mechanism of autophosphorylation, human eEF-2K was coexpressed with λ-phosphatase and purified from bacteria in a three-step protocol using a CaM affinity column. Purified eEF-2K was induced to autophosphorylate by incubation with Ca(2+)/CaM in the presence of MgATP. Analyzing tryptic or chymotryptic peptides by mass spectrometry monitored the autophosphorylation over 0-180 min. The following five major autophosphorylation sites were identified: Thr-348, Thr-353, Ser-445, Ser-474, and Ser-500. In the presence of Ca(2+)/CaM, robust phosphorylation of Thr-348 occurs within seconds of addition of MgATP. Mutagenesis studies suggest that phosphorylation of Thr-348 is required for substrate (eEF-2 or a peptide substrate) phosphorylation, but not self-phosphorylation. Phosphorylation of Ser-500 lags behind the phosphorylation of Thr-348 and is associated with the Ca(2+)-independent activity of eEF-2K. Mutation of Ser-500 to Asp, but not Ala, renders eEF-2K Ca(2+)-independent. Surprisingly, this Ca(2+)-independent activity requires the presence of CaM.     

Mapping of calmodulin-binding domain of Ca2+/calmodulin-dependent protein kinase II from rat brain

Recent molecular cloning experiments have identified a 25 amino-acid region as the calmodulin-binding domain of the alpha-subunit of rat brain Ca2+/calmodulin-dependent multifunctional protein kinase II (CaM-K II). Synthetic peptides, derived from the deduced amino-acid sequence encompassing this region, were examined for their ability to bind calmodulin in a calcium dependent manner and to inhibit the Ca2+/calmodulin-dependent autophosphorylation of CaM-K II. Comparison of these structure-function relationships highlighted a region of 5 amino-acids, which was essential for calmodulin interaction and inhibition of kinase activity. This region demonstrated some homology with other calmodulin-binding peptides, and may represent a key site of interaction of the kinase with calmodulin. These analyses provide additional insight into the molecular mechanism underlying the Ca2+ regulation of CaM-K II.     

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.     

Generation of autonomous activity of Ca(2+)/calmodulin-dependent protein kinase kinase β by autophosphorylation

Ca(2+)/calmodulin-dependent protein kinase kinases (CaMKKs) phosphorylate and activate specific downstream protein kinases, including CaMKI, CaMKIV, and 5'-AMP-activated protein kinase, which mediates a variety of Ca(2+) signaling cascades. CaMKKs have been shown to undergo autophosphorylation, although their role in enzymatic regulation remains unclear. Here, we found that CaMKKα and β isoforms expressed in nonstimulated transfected COS-7 cells, as well as recombinant CaMKKs expressed in and purified from Escherichia coli, were phosphorylated at Thr residues. Introduction of a kinase-dead mutation completely impaired the Thr phosphorylation of these recombinant CaMKK isoforms. In addition, wild-type recombinant CaMKKs were unable to transphosphorylate the kinase-dead mutants, suggesting that CaMKK isoforms undergo Ca(2+)/CaM-independent autophosphorylation in an intramolecular manner. Liquid chromatography-tandem mass spectrometry analysis identified Thr(482) in the autoinhibitory domain as one of the autophosphorylation sites in CaMKKβ, but phosphorylation of the equivalent Thr residue (Thr(446)) in the α isoform was not observed. Unlike CaMKKα that has high Ca(2+)/CaM-dependent activity, wild-type CaMKKβ displays enhanced autonomous activity (Ca(2+)/CaM-independent activity, 71% of total activity). This activity was significantly reduced (to 37%) by substitution of Thr(482) with a nonphosphorylatable Ala, without significant changes in Ca(2+)/CaM binding. In addition, a CaMKKα mutant containing the CaMKKβ regulatory domain was shown to be partially phosphorylated at Thr(446), resulting in a modest elevation of its autonomous activity. The combined results indicate that, in contrast to the α isoform, CaMKKβ exhibited increased autonomous activity, which was caused, at least in part, by autophosphorylation at Thr(482), resulting in partial disruption of the autoinhibitory mechanism.