CaM kinase II


Table of contents

CaM kinase interaction with Calmodulin and CaM kinase activation

The transduction of many cellular stimuli results in oscillations in the intracellular concentration of calcium ions (Ca2+). Although information is thought to be encoded in the frequency of such oscillations, no frequency decoder has been identified. Rapid superfusion of immobilized Ca2+- and calmodulin-dependent protein kinase II (CaM kinase II) in vitro shows that the enzyme can decode the frequency of Ca2+ spikes into distinct amounts of kinase activity. The frequency response of CaM kinase II is modulated by several factors, including the amplitude and duration of individual spikes as well as the subunit composition and previous state of activation of the kinase. These features should provide specificity in the activation of this multifunctional enzyme by distinct cellular stimuli and may underlie its pivotal role in activity-dependent forms of synaptic plasticity (De Koninck, 1998).

Autophosphorylation of multifunctional Ca2+/calmodulin-dependent protein kinase makes it Ca2+ independent by trapping bound calmodulin and by enabling the kinase to remain partially active even after calmodulin dissociates. Autophosphorylation is an intersubunit reaction between neighbors in the multimeric kinase that requires two molecules of calmodulin. Ca2+/calmodulin acts not only to activate the "kinase" subunit, but also to effectively present the "substrate" subunit for autophosphorylation. Conversion of the kinase to the potentiated or trapped state is a cooperative process, inefficient at low Ca2++ occupancy of calmodulin. Simulations show that repetitive Ca2+ pulses at limiting levels of calmodulin lead to the recruitment of calmodulin to the holoenzyme, which further stimulates autophosphorylation and trapping. This cooperative, positive feedback loop will potentiate the responseof the kinase to sequential Ca2+ transients and establish a threshold frequency at which the enzyme becomes highly active (Hanson, 1994).

Calmodulin dependent-kinase II is inactive in the absence of Ca2+ Calmodulin due to interaction of the CaM kinase II autoinhibitory domain with its own catalytic domain. Synthetic autoinhibitory domain peptides (residues 281-302) identify several residues that are important for inhibitory potency and suggested that His282 may interact with the ATP-binding motif of the catalytic domain. Purified mutant proteins have many biochemical properties identical to wild-type kinase, but certain single residue mutants have 10-20% constitutive Ca(2+)-independent activities, indicating that these residues are involved in the autoinhibitory interaction. The Ca(2+)-independent activities of the some of these mutants exhibit 10-fold lower Km values for ATP than the wild-type kinase. Wild-type and certain mutant kinases, generate Ca2+ independence upon autophosphorylation in the presence of Ca2+/CaM; those mutants having constitutive Ca2+ independence also exhibit enhanced Ca2+/CaM-independent autophosphorylation. This Ca(2+)-independent autophosphorylation results in a decrease in total kinase activity, but there is little increase in Ca(2+)-independent activity, consistent with autophosphorylation of predominantly Thr306 rather than Thr286. These results are consistent with an inhibitory interaction of His282 and possibly Arg283 with the ATP-binding motif of the catalytic domain, and they indicate that constitutively active CaM-kinase II cannot autophosphorylate on Thr286 in the absence of bound Ca2+/CaM. Based on these and other biochemical characterizations, a molecular model is proposed for the interaction of a bisubstrate autoinhibitory domain with the catalytic domain of CaM-kinase II (Brinkey, 1994).

Ca2+/calmodulin-dependent protein kinase kinase (CaM-KK) activates both CaM-kinase IV (CaM-K IV) and CaM-K I. Activation of CaM-K IV through phosphorylation of Thr196 by CaM-KK is triggered by elevated intracellular Ca2+ in intact cells and requires binding of Ca2+/Calmodulin to both enzymes. An expressed fragment of CaM-K IV (CaM-K IV178-246), which contains the activating phosphorylation site (Thr196) but not the autoinhibitory domain or the CaM-binding domain, still requires Ca2+/Calmodulin for phosphorylation by wild-type CaM-KK. A truncated mutant of CaM-KK (CaM-KK1-434) phosphorylates CaM-K IV178-246 in a Ca2+/Calmodulin-independent manner, but this constitutively active CaM-KK1 434 requires Ca2+/Calmodulin for phosphorylation and activation of wild-type CaM-K IV. These results demonstrate that binding of Ca2+/Calmodulin to both CaM-K IV and CaM-KK is required for the CaM-kinase cascade. Both CaM-KK and CaM-K IV appear to have similar Ca2+/Calmodulin requirements with EC50 values of approximately 100 nM. CaM-KK rapidly activates both total and Ca2+/Calmodulin-independent activities of wild-type CaM-K IV, but not the Thr196 --> Ala mutant, upon ionomycin stimulation (Tokumitsu, 1996).

The calmodulin-dependent kinase (CaM-K) cascade, a Ca2+-triggered system involving phosphorylation and activation of CaM-KI and CaM-KIV by CaM kinase kinase (CaM-KK), regulates transcription through direct phosphorylation of transcription factors such as cAMP response element-binding protein. Activated CaM-KIV can activate the mitogen-activated protein kinases. The present paper describes a novel regulatory cross-talk between cAMP kinase (PKA) and CaM-KK. PKA gives rapid phosphorylation in vitro and in cells of recombinant CaM-KK, resulting in 50-75% inhibition of CaM-KK activity, part of which is due to suppression of CaM-binding by phosphorylation of Ser458 in the CaM-binding domain. However, the Ser458 --> Ala mutant, or a truncation mutant in which the CaM-binding and autoinhibitory domains are deleted, is still partially suppressed by PKA-mediated phosphorylation. The second inhibitory site is identified as Thr108 by site-specific mutagenesis. Treatments of COS-7, PC12, hippocampal, or Jurkat cells with the PKA activators give 30-90% inhibition of either endogenous or transfected CaM-KK and/or CaM-KIV activities. These results demonstrate that the CaM kinase cascade is negatively regulated in cells by the cAMP/PKA pathway (Wayman, 1997).

Brain-derived neurotrophic factor (BDNF) stimulates both the phosphorylation of the Ca2+/calmodulin-dependent protein kinase 2 (CaMK2) and its kinase activity in rat hippocampal slices. It has been found that (1) the time course of BDNF action is not accompanied by a change in the spectrum of either the alpha- or beta-subunits of CaMK2 detected by immunoblotting; (2) both treatment of solubilized CaMK2 with alkaline phosphatase and treatment of immunoprecipitated CaMK2 with protein phosphatase 1 reverse phosphorylation and activation of the kinase, and (3) phospholipase C inhibitor D609 and intracellular Ca2+ chelation abolish the stimulatory effect of BDNF on phosphorylation and activation of CaMK2. These results strongly suggest that the conversion of CaMK2 into its active, autophosphorylated form, but not its concentration, is increased by BDNF via stimulation of phospholipase C and subsequent intracellular Ca2+ mobilization (Blanquet, 1997).

Many cells express more than one integrin receptor for extracellular matrix, and in vivo these receptors may be simultaneously engaged. Ligation of one integrin may influence the behavior of others on the cell, a phenomenon termed integrin crosstalk. Ligation of the integrin alphavbeta3 inhibits both phagocytosis and migration mediated by alpha5beta1 on the same cell, and the beta3 cytoplasmic tail is necessary and sufficient for this regulation of alpha5beta1. Ligation of alpha5beta1 activates the calcium- and calmodulin-dependent protein kinase II (CamKII). This activation is required for alpha5beta1-mediated phagocytosis and migration. Simultaneous ligation of alphavbeta3 or expression of a chimeric molecule with a free beta3 cytoplasmic tail prevents alpha5beta1-mediated activation of CamKII. Expression of a constitutively active CamKII restores alpha5beta1 functions blocked by alphavbeta3-initiated integrin crosstalk. Thus, alphavbeta3 inhibition of alpha5beta1 activation of CamKII is required for its role in integrin crosstalk. Structure-function analysis of the beta3 cytoplasmic tail demonstrates a requirement for Ser752 in beta3-mediated suppression of CamKII activation, while crosstalk is independent of Tyr747 and Tyr759, implicating Ser752, but not beta3 tyrosine phosphorylation in initiation of the alphavbeta3 signal for integrin crosstalk (Blystone, 1999).

Changes in synaptic strength that underlie memory formation in the CNS are initiated by pulses of Ca2+ flowing through NMDA-type glutamate receptors into postsynaptic spines. Differences in the duration and size of the pulses determine whether a synapse is potentiated or depressed after repetitive synaptic activity. Calmodulin (CaM) is a major Ca2+ effector protein that binds up to four Ca2+ ions. CaM with bound Ca2+ can activate at least six signaling enzymes in the spine. In fluctuating cytosolic Ca2+, a large fraction of free CaM is bound to fewer than four Ca2+ ions. Binding to targets increases the affinity of CaM's remaining Ca2+-binding sites. Thus, initial binding of CaM to a target may depend on the target's affinity for CaM with only one or two bound Ca2+ ions. To study CaM-dependent signaling in the spine, mutant CaMs were designed that bind Ca2+ only at the two N-terminal or two C-terminal sites by using computationally designed mutations to stabilize the inactivated Ca2+-binding domains in the 'closed' Ca2+-free conformation. Their interactions with CaMKII, a major Ca2+/CaM target that mediates initiation of long-term potentiation, were measured. CaM with two Ca2+ ions bound in its C-terminal lobe not only binds to CaMKII with low micromolar affinity but also partially activates kinase activity. These results support the idea that competition for binding of CaM with two bound Ca2+ ions may influence significantly the outcome of local Ca2+ signaling in spines and, perhaps, in other signaling pathways (Shifman, 2006).

Alternative forms of CaM kinases

The delta B-CaM kinase isoform is targeted to the nucleus in transfected cells while the delta A- and delta C-CaM kinase isoforms are cytosolic/cytoskeletal. A chimeric construct of alpha-CaM kinase containing the delta B-CaM kinase variable domain is rerouted to the nucleus while the native alpha-CaM kinase and chimeras of alpha-CaM kinase that contain either the delta A- or delta C-CaM kinase variable domains are retained in the cytoplasm. A nuclear localization signal (NLS) has been found within an 11-amino acid sequence, likely inserted by alternative splicing, in the variable domain of delta B-CaM kinase. Isoform-specific nuclear targeting of CaM kinase is probably a key mechanism in the selective regulation of nuclear functions by CaM kinase. CaM kinase is a multimer that can be composed of several isoforms. When cells express two different isoforms of CaM kinase, cellular targeting is determined by the ratio of the isoforms to each other. When an excess of the cytoplasmic isoform of CaM kinase is coexpressed along with the nuclear isoform, both isoforms are localized in the cytoplasm. Conversely an excess of the nuclear isoform can reroute the cytoplasmic isoform to the nucleus. The nuclear isoform likely coassembles with the cytosolic isoform, to form a heteromultimeric holoenzyme that is transported into the nucleus. These experiments demonstrate isoform-specific targeting of CaM kinase and indicate that such targeting can be modified by the expression of multiple isoforms of the enzyme (Srinivasan, 1994).

In rat brain, the alpha- and beta-CaM kinase isoforms of multifunctional Ca2+/calmodulin-dependent protein kinase are both neuron-specific and highly abundant. The variable domain of CaM kinase is a potential site for the generation of isoform diversity by alternative spicing of its N- and/or C-terminal segments. Three new isoforms have been isolated from rat brain, namely alpha B-, beta e- and beta'e-CaM kinase. alpha beta-CaM kinase contains 11 amino acids, likely inserted by alternative splicing, at the C-terminal segment of the variable domain. This insertion introduces a nuclear localization signal (NLS) that targets alpha B-CaM kinase to the nucleus of transfected cells; alpha-CaM kinase is excluded from the nucleus. The mRNA and the protein corresponding to this isoform are detected only in the diencephalon/midbrain regions. Two alternatively spliced isoforms of beta-CaM kinase have been identified that lack the 24 amino acid sequence at the N-terminal segment of the variable domain. Alternative splicing of these two isoforms occurs with a three base pair shift of the 3'-splice site. These new beta-CaM kinase isoforms are expressed primarily in early developmental stages, and are therefore term beta e - (embryonic) and beta' e-CaM kinase. Recombinant alpha B-, beta e and beta' e-CaM kinase expressed in COS-7 cells exhibit characteristic Ca2+/calmodulin-dependent protein kinase activity and autophosphorylation (Brocke, 1995).

Two novel CaM kinase II isoforms have been discovered in rat heart. The presence of these additional subtypes makes the heart the organ which possesses the greatest number of different and unusual CaM kinase II isoforms throughout the body except for the brain. The importance of this finding is underscored by the fact that calcium is involved in the regulation of many crucial cardial parameters. The amino acid sequence indicates a molecular organization that could make the design of subtype-specific inhibitory drugs for CaM kinase II possible. Such compounds would act in a similar manner to digitalis glycosides, but be more selective and would likely produce fewer side effects (Mayer, 1995).

The gene for the alpha isoform of Ca2+/calmodulin-dependent kinase II (alpha CaMKII) codes for a multifunctional protein kinase that is found exclusively in the brain. In skeletal muscle, an alternative nonkinase product, alpha CaMKII association protein (alpha KAP), is expressed from the same gene. alpha KAP consists of a C-terminal region that is identical to the association domain of alpha CaMKII, with the exception of 11 amino acids inserted in the variable region. The N-terminal sequence of alpha KAP is highly hydrophobic and not present in any known CaMKII protein. The catalytic and regulatory domains of alpha CaMKII are missing in alpha KAP. Analysis of the exon-intron structure revealed that the alpha KAP transcript is derived from the alpha CaMKII gene by alternative promoter usage and RNA splicing. The transcriptional start site of alpha KAP mRNA is located within an intron of the alpha CaMKII gene. Therefore, the relationship between alpha KAP and alpha CaMKII is that of a gene within a gene. Immunostaining using anti-alpha KAP antibodies suggests that alpha KAP is associated with sarcomeres of skeletal muscle fibers. On the basis of its primary structure and specific location, one possible function for alpha KAP is as an anchoring protein for CaMKII (Bayer, 1996).

Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) is present in a membrane-bound form that phosphorylates synapsin I on neuronal synaptic vesicles and the ryanodine receptor at skeletal muscle sarcoplasmic reticulum (SR), but it is unclear how this soluble enzyme is targeted to membranes. alphaKAP, a non-kinase protein encoded by a gene within the gene of alpha-CaM kinase II, is shown to target the CaM kinase II holoenzyme to the SR membrane. alphaKAP is anchored to the membrane via its N-terminal hydrophobic domain and can co-assemble with catalytically competent CaM kinase II isoforms and target them to the membrane regardless of their state of activation. Alpha Kap is co-localized and associated with rat skeletal muscle CaM kinase II in vivo. alphaKAP is therefore the first demonstrated anchoring protein for CaM kinase II. CaM kinase II assembled with alphaKAP retains normal enzymatic activity and the ability to become Ca2+-independent following autophosphorylation. A new variant of beta-CaM kinase II, termed betaM-CaM kinase II, is one of the predominant CaM kinase II isoforms associated with alphaKAP in skeletal muscle SR (Bayer, 1998).

Calmodulin kinase IV is induced at a very early stage of brain differentiation by the thyroid hormone T3 in a time- and concentration-dependent manner. The induction of the enzyme observed, both at the mRNA and the protein levels, is T3-specific; that is, it cannot be induced by retinoic acid or reverse T3, and can be inhibited on both the transcriptional and the translational level by adding to the culture medium actinomycin D or cycloheximide, respectively. The earliest detection of calmodulin kinase IV in the fetal rat brain tissue occursat days E16/E17, at both the mRNA and protein levels. This is the first report in which a second messenger-dependent kinase involved in the control of cell regulatory processes is itself controlled by a primary messenger, the thyroid hormone (Krebs, 1996).

In situ hybridization histochemistry and immunocytochemistry were used to study localization and activity-dependent regulation of alpha, beta, gamma, and delta isoforms of type II calcium/calmodulin-dependent protein kinase (CaMKII) and their mRNAs in areas 17 and 18 of normal and monocularly deprived adult macaques. CaMKII-alpha is expressed overall at levels three to four times higher than that of CaMKII-beta and at least 15 times higher than that of CaMKII-gamma and -delta. All isoforms are expressed primarily in pyramidal cells of both areas, especially those of layers II-III, IVA (in area 17), and VI, but isoforms are also expressed in nonpyramidal, non-GABAergic cells of layer IV of both areas and in interstitial neurons of the white matter. CaMKII-alpha and -beta are colocalized, suggesting the formation of heteromers. There was no evidence of expression in neuroglial cells. Each isoform has a unique pattern of laminar and sublaminar distribution, but cortical layers or sublayers enriched for one isoform do not correlate with layers receiving inputs only from isoform-specific layers of the lateral geniculate nucleus. CaMKII-alpha and -beta mRNA and protein levels in layer IVC of area 17 are subject to activity-dependent regulation, with brief periods of monocular deprivation caused by intraocular injections of tetrodotoxin leading to a 30% increase in CaMKII-alpha mRNA and a comparable decrease in CaMKII-beta mRNA in deprived ocular dominance columns, especially of layer IVCbeta. Expression in other layers and expression of CaMKII-gamma and delta are unaffected. Changes occurring in layer IVC may influence the formation of heteromers and protect supragranular layers from CaMKII-dependent plasticity in the adult (Tighilet, 1998).

Ca2+ oscillations are required in various signal transduction pathways, and contain information both in their amplitude and frequency. Remarkably, the Ca2+/calmodulin(CaM)-dependent protein kinase II (CaMKII) can decode such frequencies. A Ca2+/CaM-stimulated autophosphorylation leads to Ca2+/CaM-independent (autonomous) activity of the kinase that outlasts the initial stimulation. This autonomous activity increases exponentially with the frequency of Ca2+ oscillations. Three ß-CaMKII splice variants (ßM, ß and ße') have very similar specific activity and maximal autonomy. However, their autonomy generated by Ca2+ 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, it is proposed that differences in relative positioning of kinase domains within multimeric holoenzymes are responsible for the observed effects. Notably, the ß-CaMKII splice variants are differentially expressed, even among individual hippocampal neurons. Taken together, these results suggest that alternative splicing provides cells with a mechanism to modulate their sensitivity to Ca2+ oscillations (Bayer, 2002).

What are the functional implications of the differential Ca2+ oscillation response detected for the different CaMKII isoforms? In skeletal muscle, ßM-CaMKII is one of the most prominent CaMKII isoforms and the only ß variant expressed. There, ßM is targeted by a CaMKII-related anchoring protein, aKAP, to the membrane of the sarcoplasmic reticulum, where CaMKII controls Ca2+ release from intracellular stores in a negative feedback loop by phosphorylating the ryanodine receptor (the Ca2+ release channel), phospholamban (a regulator of the Ca2+ pump protein) and the Ca2+ pump protein itself. Thus, CaMKII can control Ca2+ oscillations, and its own sensitivity to such oscillations, modulated by kinase isoform and splice variant expression, would influence this feedback regulation. Expression of both ß- and ße'-CaMKII is restricted to the central nervous system. However, a- and ß-CaMKII are by far the most abundant isoforms in the mature brain. In contrast, ße- and ße'-CaMKII are the predominant ß variants during early postnatal development, a time when there is virtually no a- or ß-CaMKII present. Like ße'-CaMKII, the ße variant has reduced CaM affinity. Thus, neonatal neurons mostly express CaMKII isoforms that can, in contrast to a-CaMKII, associate with the actin cytoskeleton, but have a high frequency threshold, more similar to the a isoform. A requirement for distinct frequencies of spontaneous Ca2+ spikes and waves in neural development has been demonstrated, and Ca2+ and CaMKII have actin-based morphological effects on dendritic arbors and spines, probably with distinct functions in differentiating versus mature neurons. High frequencies of neuronal stimulation can trigger long-term potentiation of synaptic strength, whereas low-frequency stimulation leads to long-term depression. The autophosphorylation state of CaMKII is thought to be involved in setting the frequency threshold for the induction of long-term potentiation. Thus, this form of metaplasticity would be modulated by differential expression of CaMKII isoforms with distinct Ca2+ oscillation sensitivities for autophosphorylation. Indeed, such metaplasticity is known to change during development (Bayer, 2002).

Remarkably, ßM-, ß- and ße'-CaMKII are differentially expressed even among individual mature hippocampal neurons. Thus, regulated alternative splicing may tune the sensitivity to Ca2+ oscillations not only during development and for different tissues, but also for individual cells of the same type. Thereby, differential expression of CaMKII variants can provide a novel level of neuronal plasticity, and it will be interesting to elucidate the mechanisms and cellular stimuli that regulate the alternative splice events. The ubiquitous gamma and delta isoforms of CaMKII show a high degree of structural homology to a- and ß-CaMKII and are subject to even more extensive alternative splicing. Thus, alternative splicing and co-assembly into heteromeric holoenzymes with mixed properties can give rise to finely tuned arrays of molecular decoders of Ca2+ oscillations in a large variety of different cell types (Bayer, 2002).

CaM kinase subcellular localization

Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a serine/threonine protein kinase that regulates long-term potentiation and other forms of neuronal plasticity. Functional differences between the neuronal CaMKIIalpha and CaMKIIbeta isoforms are not yet known. Green fluorescent protein-tagged (GFP-tagged) CaMKII isoforms were used, and show that CaMKIIbeta is bound to F-actin in dendritic spines and cell cortex while CaMKIIalpha is largely a cytosolic enzyme. When expressed together, the two isoforms form large heterooligomers; a small fraction of CaMKIIbeta is sufficient to dock the predominant CaMKIIalpha to the actin cytoskeleton. Thus, CaMKIIbeta functions as a targeting module that localizes a much larger number of CaMKIIalpha isozymes to synaptic and cytoskeletal sites of action (Shen, 1998).

What is the functional advantage of a subcellular localization of CaMKII? The main advantages of localizing enzymes and signaling proteins are (1) increased efficiency and (2) increased specificity. By targeting CaMKII to dendritic spines, its local concentration is elevated and the efficiency of substrate phosphorylation is increased. Important dendritic substrates of CaMKII include AMPA receptors, NMDA receptors, SynGAP, and MAP2. Hence, by localizing CaMKII to dendritic spines, the kinase can more effectively exert its functions in synaptic plasticity. The postsynaptically localized AMPA receptor has been shown to be an important substrate of CaMKII, and phosphorylation by CaMKII increases the AMPA receptor conductivity 3-fold. This upregulation of the AMPA receptor is thought to be one of the critical functions of CaMKII in synaptic plasticity. The postsynaptically localized NMDA receptor has also been shown to be a substrate for CaMKII, although the function of the NMDA receptor phosphorylation by CaMKII is not well understood. Furthermore, the NMDA receptor has been shown to bind to alpha-actinin, an actin binding protein present in PSDs. A recent study has also suggested that CaMKII has an important postsynaptic role in inhibiting the activity of the PSD protein SynGAP, a Ras-GTPase activating protein that will likely prove important in regulating the dendritic MAP kinase pathway (Shen, 1998 and references).

The dendritic tubulin binding protein MAP2 is a major CaMKII substrate and is known to form a bridge between actin and tubulin. Phosphorylation by CaMKII has been shown to break its binding interaction with actin, which is potentially important for a reorganization of the dendritic morphology. An important role of CaMKII in stabilizing dendritic branches has been suggested in recent studies of neurons using expressed catalytically active CaMKII. In addition to its postsynaptic functions, CaMKII may also be relevant for regulation of the presynaptic actin binding protein synapsin 1. It is likely that the direct association of CaMKII with F-actin is important for the enhancement of synapsin 1 phosphorylation by CaMKII. Functionally, phosphorylation by CaMKII has been shown to disable the synapsin-actin binding interaction and thereby enables synaptic vesicles to become available for secretion. Biochemical evidence suggests that a significant fraction of CaMKII is enriched in postsynaptic densities (PSDs). These structures have been shown to contain a marked amount of CaMKII (and a lesser amount of actin and CaMKII) in hippocampal neurons. Interestingly, CaMKII and actin are present in PSDs at about the same molar concentration, making it possible that CaMKII localization to dendritic spines is mediated by a direct CaMKII-actin interaction. Furthermore, CaMKII, once autophosphorylated, can bind to at least two postsynaptic density proteins of 140 and 190 kDa. In light of the data in this study, it is conceivable that CaMKII oligomers are initially prelocalized to the PSD by CaMKII reversible actin binding interaction and then either switch their PSD binding partner or further translocate to PSDs following CaMKII autophosphorylation (Shen, 1998 and references).

Calcium-calmodulin-dependent protein kinase II (CaMKII) is thought to increase synaptic strength by phosphorylating postsynaptic density (PSD) ion channels and signaling proteins. It has been shown that N-methyl-D-aspartate (NMDA) receptor stimulation reversibly translocates green fluorescent protein-tagged CaMKII from an F-actin-bound to a PSD-bound state. The translocation time is controlled by the ratio of expressed beta-CaMKII to alpha-CaMKII isoforms. Although F-actin dissociation into the cytosol requires either autophosphorylation of beta-CaMKII or calcium-calmodulin binding to beta-CaMKII, PSD translocation requires binding of calcium-calmodulin to either the alpha- or beta-CaMKII subunits. Autophosphorylation of CaMKII indirectly prolongs its PSD localization by increasing the calmodulin-binding affinity (Shen, 1999).

The synapse contains densely localized and interacting proteins that enable it to adapt to changing inputs. A Ca2+-sensitive protein complex is described involved in the regulation of AMPA receptor synaptic plasticity. The complex is comprised of (1) MUPPI, a multi-PDZ domain-containing protein, (2) SynGAP, a synaptic GTPase-activating protein, and (3) the Ca2+/calmodulin-dependent kinase CaMKII. In synapses of hippocampal neurons, SynGAP and CaMKII are brought together by direct physical interaction with the PDZ domains of MUPP1, and in this complex, SynGAP is phosphorylated. Ca2+CaM binding to CaMKII dissociates it from the MUPP1 complex, and Ca2+, entering the cell via the NMDAR, drives the dephosphorylation of SynGAP. Specific peptide-induced SynGAP dissociation from the MUPP1-CaMKII complex results in SynGAP dephosphorylation accompanied by P38 MAPK inactivation, potentiation of synaptic AMPA responses, and an increase in the number of AMPAR-containing clusters in hippocampal neuron synapses. siRNA-mediated SynGAP knockdown confirms these results. These data implicate SynGAP in NMDAR- and CaMKII-dependent regulation of AMPAR trafficking (Krapivinsky, 2004).

Activating the PARP-1 sensor component of the Groucho/ TLE1 corepressor complex mediates a CaMKinase II-dependent neurogenic gene activation pathway

Switching specific patterns of gene repression and activation in response to precise temporal/spatial signals is critical for normal development. This study reports a pathway in which induction of CaMKII triggers an unexpected switch in the function of the HES1 transcription factor from a TLE-dependent repressor to an activator required for neuronal differentiation. These events are based on activation of the poly(ADP-ribose) polymerase1 (PARP-1) sensor component of the groucho/TLE-corepressor complex mediating dismissal of the corepressor complex from HES1-regulated promoters. In parallel, CaMKII mediates a required phosphorylation of HES1 to permit neurogenic gene activation, revealing the ability of a specific signaling pathway to modulate both the derepression and the subsequent coactivator recruitment events required for transcriptional activation of a neurogenic program. The identification of PARP-1 as a regulated promoter-specific exchange factor required for activation of specific neurogenic gene programs is likely to be prototypic of similar molecular mechanisms (Ju, 2004).

A covalent modification recently linked to transcription is poly(ADP-ribosyl)ation of proteins mediated by the poly(ADP-ribose) polymerase1 (PARP-1) enzyme. PARP-1 catalyzes the transfer of ADP-ribose chains onto glutamic acid residues of acceptor proteins, including itself (automodification), histones, transcription factors, and DNA repair proteins using NAD+ as a substrate involved in chromatin decondensation, DNA replication, and DNA repair. Therefore, poly(ADP-ribosyl)ation by PARP-1 affects cellular processes such as apoptosis, necrosis, cellular differentiation, malignant transformation, and modulations activities of transcription factors. While it has been recently reported that PARP influences both the expression and silencing at diverse times during Drosophila development (Tulin, 2002), it has been demonstrated that high PARP enzymatic activity is observed in areas of high transcriptional activity and chromatin decondensation on the polytene chromatin (Tulin, 2003). Together these observations suggest that PARP-1 may exert its function in transcription through direct binding to the gene-regulating sequences and through modification of transcription factors by poly(ADP-ribosyl)ation (Ju, 2004).

This study finds that HES1-dependent repression of MASH1 is dependent upon the actions of the TLE1 corepressor complex. Not only are additional insights provided into the molecular mechanisms of TLE1-mediated repression but also the molecular mechanism of the switch to activation function has been uncovered. The composition of this TLE1 complex is distinct from those of other reported corepressor complexes such as N-CoR/SMRT and CtBP. Interestingly, roles for transcriptional regulation and chromatin remodeling activities have been described for most of the components of the TLE1 complex identified, but no component alone is indispensable for at least some level of TLE1-mediated repression (Ju, 2004).

Consistent with the observation that the enzymatic activity of PARP-1 is not required for HES1-mediated MASH1 repression, these data favors a model predicting that in the TLE1 holorepressor complex, the enzymatic activity of PARP-1 is inhibited. Exposure to a signal inducing neuronal differentiation causes activation of CaMKIIdelta, which is proven to be required for activation of neurogenic genes. Once sufficient levels of CaMKIIdelta are achieved (5-7 hr), it will, directly or indirectly, mediate phosphorylation and activation of PARP-1, which then catalyzes poly(ADP-ribosyl)ation of TLE1 and most of the other components to the corepressor complex. This is consistent with the observation that calcium signaling evoked by extrinsic and intrinsic cues can induce auto-poly(ADP-ribosyl)ation of PARP-1; however, CaMKIIdelta may also be activated in a calcium-independent fashion. This covalent modification is suggested to result in their dismissal from the biochemical complex and derepression of the MASH1 gene. The role of PARP-1 in derepression of MASH1 and its retention on the activated MASH1 promoter is quite consistent with reports that poly(ADP-ribosyl)ation of chromatin-associated proteins induce major changes in chromosomal architecture. However, in the case of MASH1, it was found that derepression alone is insufficient for induction. This is in accord with the findings for other regulated transcription factors. For example, the loss of the N-CoR corepressor is not alone sufficient to activate most AP-1-regulated genes, and only in a subset of RAR target genes does derepression result in a signal-independent 'default' activation of gene targets.

These data also indicate that, in addition to Ca2+-CaMKII-dependent dissociation of the TLE1 corepressor from HES1, a covalent modification of HES1 itself is required to permit activation of MASH1. Thus, activation of MASH1 is linked to sequential CaMKIIdelta-dependent activation of PARP-1 enzymatic activity, which was previously inhibited in the TLE1 holocorepressor complex, permitting dismissal of the TLE1 complex, derepression and phosphorylation of HES1, and recruitment of specific coactivators and thus causing and maintaining derepression of genes mediating neuronal differentiation. The actions of PARP-1 in the TLE1-mediated events are thus analogous to the effects of covalent modifications by phosphorylation and acetylation, as mediators of switches from repressor to activator function (Ju, 2004).

While HES1 is recognized to regulate tissue morphogenesis by maintaining undifferentiated cells and preventing differentiation, continued occupancy of the MASH1 promoter by HES1 in the differentiating neural stem cells has surprisingly proven to be required to initiate MASH1 activation events. Indeed, previous, seemingly contradictory reports of transient transfection assays in which HES1 can inhibit acid beta-glucosidase genes in HepG2 cells but cause activation in fibroblasts are likely to be explained by findings of signal-dependent HES1 switching events (Ju, 2004).

This cortical progenitor culture system has permitted identification of a regulatory pathway that may be, at least in part, partially compensated in vivo because many nestin-positive neural stem cells in the subventricular zone proliferate without losing the multipotentiality to differentiate into neurons in HES1 mutant or even HES1/HES5 double mutant mice. It is suggested that there may be additional HES1-like repressors or unidentified protein partners, including HERP and other E box binding proteins, that are also potentially involved in MASH1 gene activation. The identification of this unexpected mechanism of HES1 action in cortical progenitor cell cultures suggest that, in this system, the other molecules that could assume a similar function were either not expressed or required. The finding of a requirement of HES1 in activation of neurogenic genes is consistent with suggestions that HES1 might promote differentiation, in addition to its role in maintenance of the undifferentiated state, at multiple steps of neural stem cell development (Ju, 2004).

In summary, a pathway is suggested in which a PARP-1-containing TLE1 complex is recruited by the Notch-induced bHLH factor, HES1, initially mediating repression of MASH1 in the proliferating neural stem cells. The data suggest that signals that induce neuronal differentiation, such as PDGF in neural stem cells, act to induce the CaMKIIdelta isoform, which, in turn, is required for HES1-mediated MASH1 activation . The temporal aspects of CaMKIIdelta induction appear to account for the delay in derepression and activation of MASH1 expression following critical PDGF signaling. CaMKIIdelta-induced phosphorylation of a specific serine residue in the orange domain of HES1 permits it to recruit coactivators, including CBP, and has proven to be required for activation of the MASH1. The conserved relationship of the HES1 orange domain with the HLH domain raises the possibility of an additional role in protein interactions that include dimerization (Ju, 2004).

In a sense, the observation that a component of the TLE1-mediated repression complex, PARP-1, is also required for derepression events and maintenance of activation, parallels the requirement of TBL1/TBLR1 complex for ligand-dependent exchange of N-CoR corepressor complexes for coactivators in the switch of nuclear receptor function from repression to activation. TBLR1 is required for recruitment of the ubiquitylation/19S proteosome complex to prevent N-CoR/SMRT-dependent maintenance of a repression checkpoint. It is suggested that PARP-1 may achieve the same effects by a distinct modification strategy and serves as a regulated sensor of neuron-inducing signals based on the actions of CaMKIIdelta induced by the initial stimulus to neuronal differentiation. Therefore, it is tempting to speculate that PARP-1 and TBLR1 may be critical for the exchange events required to overcome the repression checkpoint for TLE- and N-CoR-regulated repressors, respectively (Ju, 2004).

The dual functions of PARP-1 and HES1 in the progression of neural stem cells along a neuronal pathway indicate that while the initial Notch signal causes repression of neurogenic genes by induction of HES1, it simultaneously arms the response to subsequent Ca2+-CaMKII signals that permit MASH1 gene activation events. The data illustrate how a single signaling pathway can mediate a sequential, two-step derepression/activation process required for development in gene activation. Induction of a Ca2+/CaMKII-dependent program initiates both PARP-1 activation, which is required for dismissal of the TLE1 corepressor complex, and a second event, covalent modification of HES1, which is required for target gene activation. The requirement for both a derepression and independently mediated activation event is likely to be prototypic of many similar functions of PARP-1 factors in development. The sequential calcium-regulated PARP-1-dependent switch from repression to derepression to activation function of HES1 is clearly an effective strategy to maximize the amplitude of the transcriptional response of neurogenic gene expression to signals and to permit temporally precise patterns of cellular response (Ju, 2004).

Calcium-independent activation of CaMKII by methionine oxidation

Calcium/calmodulin (Ca2+/CaM)-dependent protein kinase II (CaMKII) couples increases in cellular Ca2+ to fundamental responses in excitable cells. CaMKII was identified over 20 years ago by activation dependence on Ca2+/CaM, but recent evidence shows that CaMKII activity is also enhanced by pro-oxidant conditions. This study shows that oxidation of paired regulatory domain methionine residues sustains CaMKII activity in the absence of Ca2+/CaM. CaMKII is activated by angiotensin II (AngII)-induced oxidation, leading to apoptosis in cardiomyocytes both in vitro and in vivo. CaMKII oxidation is reversed by methionine sulfoxide reductase A (MsrA), and MsrA-/- mice show exaggerated CaMKII oxidation and myocardial apoptosis, impaired cardiac function, and increased mortality after myocardial infarction. These data demonstrate a dynamic mechanism for CaMKII activation by oxidation and highlight the critical importance of oxidation-dependent CaMKII activation to AngII and ischemic myocardial apoptosis (Erickson, 2008).

Table of contents

CaM kinase II: Biological Overview | Evolutionary Homologs part 2/2 | Regulation | Developmental Biology | Effects of Mutation | References
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