Brain-derived neurotrophic factor activation of CaM-kinase kinase via transient receptor potential canonical channels induces the translation and synaptic incorporation of GluA1-containing calcium-permeable AMPA receptors.

Glutamatergic synapses in early postnatal development transiently express calcium-permeable AMPA receptors (CP-AMPARs). Although these GluA2-lacking receptors are essential and are elevated in response to brain-derived neurotrophic factor (BDNF), little is known regarding molecular mechanisms that govern their expression and synaptic insertion. Here we show that BDNF-induced GluA1 translation in rat primary hippocampal neurons requires the activation of mammalian target of rapamycin (mTOR) via calcium calmodulin-dependent protein kinase kinase (CaMKK). Specifically, BDNF-mediated phosphorylation of threonine 308 (T308) in AKT, a known substrate of CaMKK and an upstream activator of mTOR-dependent translation, was prevented by (1) pharmacological inhibition of CaMKK with STO-609, (2) overexpression of a dominant-negative CaMKK, or (3) short hairpin-mediated knockdown of CaMKK. GluA1 surface expression induced by BDNF, as assessed by immunocytochemistry using an extracellular N-terminal GluA1 antibody or by surface biotinylation, was impaired following knockdown of CaMKK or treatment with STO-609. Activation of CaMKK by BDNF requires transient receptor potential canonical (TRPC) channels as SKF-96365, but not the NMDA receptor antagonist d-APV, prevented BDNF-induced GluA1 surface expression as well as phosphorylation of CaMKI, AKT(T308), and mTOR. Using siRNA we confirmed the involvement of TRPC5 and TRPC6 subunits in BDNF-induced AKT(T308) phosphorylation. The BDNF-induced increase in mEPSC was blocked by IEM-1460, a selected antagonist of CP-AMPARs, as well as by the specific repression of acute GluA1 translation via siRNA to GluA1 but not GluA2. Together these data support the conclusion that newly synthesized GluA1 subunits, induced by BDNF, are readily incorporated into synapses where they enhance the expression of CP-AMPARs and synaptic strength.

Regulation of neuronal mRNA translation by CaM-kinase I phosphorylation of eIF4GII.

Ca²âº/calmodulin-dependent kinases (CaMKs) are essential for neuronal development and plasticity, processes requiring de novo protein synthesis. Roles for CaMKs in modulating gene transcription are well established, but their involvement in mRNA translation is evolving. Here we report that activity-dependent translational initiation in cultured rat hippocampal neurons is enhanced by CaMKI-mediated phosphorylation of Ser1156 in eukaryotic initiation factor eIF4GII (4GII). Treatment with bicuculline or gabazine to enhance neuronal activity promotes recruitment of wild-type 4GII, but not the 4GII S1156A mutant or 4GI, to the heterotrimeric eIF4F (4F) complex that assembles at the 5' cap structure (m7GTP) of mRNA to initiate ribosomal scanning. Recruitment of 4GII to 4F is suppressed by pharmacological inhibition (STO-609) of CaM kinase kinase, the upstream activator of CaMKI. Post hoc in vitro CaMKI phosphorylation assays confirm that activity promotes phosphorylation of S1156 in transfected 4GII in neurons. Changes in cap-dependent and cap-independent translation were assessed using a bicistronic luciferase reporter transfected into neurons. Activity upregulates cap-dependent translation, and RNAi knockdown of CaMKIß and γ isoforms, but not α or δ, led to its attenuation as did blockade of NMDA receptors. Furthermore, RNAi knockdown of 4GII attenuates cap-dependent translation and reduces density of dendritic filopodia and spine formation without effect on dendritic arborization. Together, our results provide a mechanistic link between Ca²âº influx due to neuronal activity and regulation of cap-dependent RNA translation via CaMKI activation and selective recruitment of phosphorylated 4GII to the 4F complex, which may function to regulate activity-dependent changes in spine density.

Structural modulation of dendritic spines during synaptic plasticity.

The majority of excitatory synaptic input in the brain is received by small bulbous actin-rich protrusions residing on the dendrites of glutamatergic neurons. These dendritic spines are the major sites of information processing in the brain. This conclusion is reinforced by the observation that many higher cognitive disorders, such as mental retardation, Rett syndrome, and autism, are associated with aberrant spine morphology. Mechanisms that regulate the maturation and plasticity of dendritic spines are therefore fundamental to understanding higher brain functions including learning and memory. It is well known that activity-driven changes in synaptic efficacy modulate spine morphology due to alterations in the underlying actin cytoskeleton. Recent studies have elucidated numerous molecular regulators that directly alter actin dynamics within dendritic spines. This review will emphasize activity-dependent changes in spine morphology and highlight likely roles of these actin-binding proteins.

Local application of neurotrophins specifies axons through inositol 1,4,5-trisphosphate, calcium, and Ca2+/calmodulin-dependent protein kinases.

Neurons are highly polarized cells that have structurally distinct processes-the axons and dendrites-that differentiate from common immature neurites. In cultured hippocampal neurons, one of these immature neurites stochastically initiates rapid extension and becomes an axon, whereas the others become dendrites. Various extracellular and intracellular signals contribute to axon specification; however, the specific intracellular pathways whereby particular extracellular stimuli lead to axon specification remain to be delineated. Here, we found that the neurotrophins brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) were required for axon specification in an autocrine or a paracrine fashion. Using local application with a micropipette to selectively stimulate individual neurites, we found that stimulation of a selected neurite by BDNF or NT-3 induced neurite outgrowth and subsequent axon formation. NT-3 induced a rapid increase in calcium ions (Ca(2+)) in an inositol 1,4,5-trisphosphate (IP(3))-dependent fashion as well as local activation of the Ca(2+) effector Ca(2+)/calmodulin-dependent protein kinase kinase (CaMKK) in the growth cone. Inhibition of neurotrophin receptors or CaMKK attenuated NT-3-induced axon specification in cultured neurons and axon formation in cortical neurons in vivo. These results identify a role for IP(3)-Ca(2+)-CaMKK signaling in axon specification.

Analysis of CaM-kinase signaling in cells.

A change in intracellular free calcium is a common signaling mechanism that modulates a wide array of physiological processes in most cells. Responses to increased intracellular Ca(2+) are often mediated by the ubiquitous protein calmodulin (CaM) that upon binding Ca(2+) can interact with and alter the functionality of numerous proteins including a family of protein kinases referred to as CaM-kinases (CaMKs). Of particular interest are multifunctional CaMKs, such as CaMKI, CaMKII, CaMKIV and CaMKK, that can phosphorylate multiple downstream targets. This review will outline several protocols we have used to identify which members and/or isoforms of this CaMK family mediate specific cellular responses with a focus on studies in neurons. Many previous studies have relied on a single approach such as pharmacological inhibitors or transfected dominant-negative kinase constructs. Since each of these protocols has its limitations, that will be discussed, we emphasize the necessity to use multiple, independent approaches in mapping out cellular signaling pathways.

Calmodulin-kinases regulate basal and estrogen stimulated medulloblastoma migration via Rac1.

Medulloblastoma is a highly prevalent pediatric central nervous system malignancy originating in the cerebellum, with a strong propensity for metastatic migration to the leptomeninges, which greatly increases mortality. While numerous investigations are focused on the molecular mechanisms of medulloblastoma histogenesis, the signaling pathways regulating migration are still poorly understood. Medulloblastoma likely arises from aberrant proliferative signaling in cerebellar granule precursor cells during development, and estrogen is a morphogen that promotes medulloblastoma cell migration. It has been previously shown that the calcium/calmodulin activated kinase kinase (CaMKK) pathway promotes cerebellar granule precursor migration and differentiation during normal cerebellar development via CaMKIV. Here we investigate the regulatory role of the CaMKK pathway in migration of the human medulloblastoma DAOY and cerebellar granule cells. Using pharmacological inhibitors and dominant negative approaches, we demonstrate that the CaMKK/CaMKI cascade regulates basal medulloblastoma cell migration via Rac1, in part by activation of the RacGEF, ßPIX. Additionally, pharmacological inhibition of CaMKK blocks both the estrogen induced Rac1 activation and medulloblastoma migration. The CaMKK signaling module described here is one of the first reported calcium regulated pathways that modulates medulloblastoma migration. Since tumor dissemination requires cell migration to ectopic sites, this CaMKK pathway may be a putative therapeutic target to limit medulloblastoma metastasis.

Long-term potentiation-dependent spine enlargement requires synaptic Ca2+-permeable AMPA receptors recruited by CaM-kinase I.

It is well established that long-term potentiation (LTP), a paradigm for learning and memory, results in a stable enlargement of potentiated spines associated with recruitment of additional GluA1-containing AMPA receptors (AMPARs). Although regulation of the actin cytoskeleton is involved, the detailed signaling mechanisms responsible for this spine expansion are unclear. Here, we used cultured mature hippocampal neurons stimulated with a glycine-induced, synapse-specific form of chemical LTP (GI-LTP). We report that the stable structural plasticity (i.e., spine head enlargement and spine length shortening) that accompanies GI-LTP was blocked by inhibitors of NMDA receptors (NMDARs; APV) or CaM-kinase kinase (STO-609), the upstream activator of CaM-kinase I (CaMKI), as well as by transfection with dominant-negative (dn) CaMKI but not dnCaMKIV. Recruitment of GluA1 to the spine surface occurred after GI-LTP and was mimicked by transfection with constitutively active CaMKI. Spine enlargement induced by transfection of GluA1 was associated with synaptic recruitment of Ca(2+)-permeable AMPARs (CP-AMPARs) as assessed by an increase in the rectification index of miniature EPSCs (mEPSCs) and their sensitivity to IEM-1460, a selective antagonist of CP-AMPARs. Furthermore, the increase in spine size and mEPSC amplitude resulting from GI-LTP itself was blocked by IEM-1460, demonstrating involvement of CP-AMPARs. Downstream signaling effectors of CP-AMPARs, identified by suppression of their activation by IEM-1460, included the Rac/PAK/LIM-kinase pathway that regulates spine actin dynamics. Together, our results suggest that synaptic recruitment of CP-AMPARs via CaMKI may provide a mechanistic link between NMDAR activation in LTP and regulation of a signaling pathway that drives spine enlargement via actin polymerization.

Regulation of spine and synapse formation by activity-dependent intracellular signaling pathways.

Formation of the human brain during embryonic and postnatal development is an extraordinarily complex process resulting at maturity in billions of neurons with trillions of specialized connections called synapses. These synapses, composed of a varicosity or bouton from a presynaptic neuron that communicates with a dendritic spine of the postsynaptic neuron, comprise the neural network that is essential for complex behavioral phenomena and cognition. Inappropriate synapse formation or structure is thought to underlie several developmental neuropathologies. Even in the mature CNS, alterations in synapse structure and function continues to be a very dynamic process that is foundational to learning and memory as well as other adaptive abilities of the brain. This synaptic plasticity in mature neurons, which is often triggered by certain patterns of neural activity, is again multifaceted and involves post-translational modifications (e.g. phosphorylation) and subcellular relocalization or trafficking (endocytosis/exocytosis) of existing synaptic proteins, initiation of protein synthesis from existing mRNAs localized in dendrites or spines, and triggering of new gene transcription in the nucleus. These various cellular processes support varying temporal components of synaptic plasticity that begin within 1-2 min but can persist for hours to days. This review will give a critical assessment of activity-dependent molecular modulations of synapses reported over the past couple years. Owing to space limitations, it will focus on mammalian excitatory (i.e. glutamatergic) synapses and will not consider several activity-independent signaling pathways (e.g. ephrinB receptor) that also modulate spine and synapse formation.

An activity-induced microRNA controls dendritic spine formation by regulating Rac1-PAK signaling.

Activity-regulated gene expression is believed to play a key role in the development and refinement of neuronal circuitry. Nevertheless, the transcriptional networks that regulate synaptic plasticity remain largely uncharacterized. We show here that the CREB- and activity-regulated microRNA, miR132, is induced during periods of active synaptogenesis. Moreover, miR132 is necessary and sufficient for hippocampal spine formation. Expression of the miR132 target, p250GAP, is inversely correlated with miR132 levels and spinogenesis. Furthermore, knockdown of p250GAP increases spine formation while introduction of a p250GAP mutant unresponsive to miR132 attenuates this activity. Inhibition of miR132 decreases both mEPSC frequency and the number of GluR1-positive spines, while knockdown of p250GAP has the opposite effect. Additionally, we show that the miR132/p250GAP circuit regulates Rac1 activity and spine formation by modulating synapse-specific Kalirin7-Rac1 signaling. These data suggest that neuronal activity regulates spine formation, in part, by increasing miR132 transcription, which in turn activates a Rac1-Pak actin remodeling pathway.

Transient receptor potential canonical 5 channels activate Ca2+/calmodulin kinase Igamma to promote axon formation in hippocampal neurons.

Functionality of neurons is dependent on their compartmentalized polarization of dendrites and an axon. The rapid and selective outgrowth of one neurite, relative to the others, to form the axon is critical in initiating neuronal polarity. Axonogenesis is regulated in part by an optimal intracellular calcium concentration. Our investigation of Ca(2+)-signaling pathways involved in axon formation using cultured hippocampal neurons demonstrates a role for Ca(2+)/calmodulin kinase kinase (CaMKK) and its downstream target Ca(2+)/calmodulin kinase I (CaMKI). Expression of constitutively active CaMKI induced formation of multiple axons, whereas blocking CaMKK or CaMKI activity with pharmacological, dominant-negative, or short hairpin RNA (shRNA) methods significantly inhibited axon formation. CaMKK signals via the gamma-isoform of CaMKI as shRNA to CaMKIgamma, but not the other CaMKI isoforms, inhibited axon formation. Furthermore, overexpression of wild-type CaMKIgamma, but not a mutant incapable of membrane association, accelerated the rate of axon formation. Pharmacological or small interfering RNA inhibition of transient receptor potential canonical 5 (TRPC5) channels, which are present in developing axonal growth cones, suppressed CaMKK-mediated activation of CaMKIgamma as well as axon formation. We demonstrate using biochemical fractionation and immunocytochemistry that CaMKIgamma and TRPC5 colocalize to lipid rafts. These results are consistent with a model in which highly localized calcium influx through the TRPC5 channels activates CaMKK and CaMKIgamma, which subsequently promote axon formation.

Calmodulin-kinases: modulators of neuronal development and plasticity.

In the nervous system, many intracellular responses to elevated calcium are mediated by CaM kinases (CaMKs), a family of protein kinases whose activities are initially modulated by binding Ca(2+)/calmodulin and subsequently by protein phosphorylation. One member of this family, CaMKII, is well-established for its effects on modulating synaptic plasticity and learning and memory. However, recent studies indicate that some actions on neuronal development and function attributed to CaMKII may instead or in addition be mediated by other members of the CaMK cascade, such as CaMKK, CaMKI, and CaMKIV. This review summarizes key neuronal functions of the CaMK cascade in signal transduction, gene transcription, synaptic development and plasticity, and behavior. The technical challenges of mapping cellular protein kinase signaling pathways are also discussed.

Recruitment of calcium-permeable AMPA receptors during synaptic potentiation is regulated by CaM-kinase I.

Ca(2+)-permeable AMPA receptors (CP-AMPARs) at central glutamatergic synapses are of special interest because of their unique biophysical and signaling properties that contribute to synaptic plasticity and their roles in multiple neuropathologies. However, intracellular signaling pathways that recruit synaptic CP-AMPARs are unknown, and involvement of CP-AMPARs in hippocampal region CA1 synaptic plasticity is controversial. Here, we report that intracellular infusion of active CaM-kinase I (CaMKI) into cultured hippocampal neurons enhances miniature EPSC amplitude because of recruitment of CP-AMPARs, likely from an extrasynaptic pool. The ability of CaMKI, which regulates the actin cytoskeleton, to recruit synaptic CP-AMPARs was blocked by inhibiting actin polymerization with latrunculin A. CaMK regulation of CP-AMPARs was also confirmed in hippocampal slices. CA1 long-term potentiation (LTP) after theta bursts, but not high-frequency tetani, produced a rapid, transient expression of synaptic CP-AMPARs that facilitated LTP. This component of TBS LTP was blocked by inhibition of CaM-kinase kinase (CaMKK), the upstream activator of CaMKI. Our calculations show that adding CP-AMPARs numbering <5% of existing synaptic AMPARs is sufficient to account for the potentiation observed in LTP. Thus, synaptic expression of CP-AMPARs is a very efficient mechanism for rapid enhancement of synaptic strength that depends on CaMKK/CaMKI signaling, actin dynamics, and the pattern of synaptic activity used to induce CA1 LTP.

An activity-regulated microRNA controls dendritic plasticity by down-regulating p250GAP.

Activity-regulated gene expression is believed to play a key role in the development and refinement of neuronal circuitry. Nevertheless, the transcriptional networks that regulate synapse growth and plasticity remain largely uncharacterized. Here, we show that microRNA 132 (miR132) is an activity-dependent rapid response gene regulated by the cAMP response element-binding (CREB) protein pathway. Introduction of miR132 into hippocampal neurons enhanced dendrite morphogenesis whereas inhibition of miR132 by 2'O-methyl RNA antagonists blocked these effects. Furthermore, neuronal activity inhibited translation of p250GAP, a miR132 target, and siRNA-mediated knockdown of p250GAP mimicked miR132-induced dendrite growth. Experiments using dominant-interfering mutants suggested that Rac signaling is downstream of miR132 and p250GAP. We propose that the miR132-p250GAP pathway plays a key role in activity-dependent structural and functional plasticity.

Activity-dependent synaptogenesis: regulation by a CaM-kinase kinase/CaM-kinase I/betaPIX signaling complex.

Neuronal activity augments maturation of mushroom-shaped spines to form excitatory synapses, thereby strengthening synaptic transmission. We have delineated a Ca(2+)-signaling pathway downstream of the NMDA receptor that stimulates calmodulin-dependent kinase kinase (CaMKK) and CaMKI to promote formation of spines and synapses in hippocampal neurons. CaMKK and CaMKI form a multiprotein signaling complex with the guanine nucleotide exchange factor (GEF) betaPIX and GIT1 that is localized in spines. CaMKI-mediated phosphorylation of Ser516 in betaPIX enhances its GEF activity, resulting in activation of Rac1, an established enhancer of spinogenesis. Suppression of CaMKK or CaMKI by pharmacological inhibitors, dominant-negative (dn) constructs and siRNAs, as well as expression of the betaPIX Ser516Ala mutant, decreases spine formation and mEPSC frequency. Constitutively-active Pak1, a downstream effector of Rac1, rescues spine inhibition by dnCaMKI or betaPIX S516A. This activity-dependent signaling pathway can promote synapse formation during neuronal development and in structural plasticity.

Regulatory mechanisms of AMPA receptors in synaptic plasticity.

Activity-dependent changes in the strength of excitatory synapses are a cellular mechanism for the plasticity of neuronal networks that is widely recognized to underlie cognitive functions such as learning and memory. AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid)-type glutamate receptors (AMPARs) are the main transducers of rapid excitatory transmission in the mammalian CNS, and recent discoveries indicate that the mechanisms which regulate AMPARs are more complex than previously thought. This review focuses on recent evidence that alterations to AMPAR functional properties are coupled to their trafficking, cytoskeletal dynamics and local protein synthesis. These relationships offer new insights into the regulation of AMPARs and synaptic strength by cellular signalling.

Activity-dependent dendritic arborization mediated by CaM-kinase I activation and enhanced CREB-dependent transcription of Wnt-2.

Members of the Wnt signaling family are important mediators of numerous developmental events, including activity-dependent dendrite development, but the pathways regulating expression and secretion of Wnt in response to neuronal activity are poorly defined. Here, we identify an NMDA receptor-mediated, Ca2+-dependent signaling pathway that couples neuronal activity to dendritic arborization through enhanced Wnt synthesis and secretion. Activity-dependent dendritic outgrowth and branching in cultured hippocampal neurons and slices is mediated through activation by CaM-dependent protein kinase kinase (CaMKK) of the membrane-associated gamma isoform of CaMKI. Downstream effectors of CaMKI include the MAP-kinase pathway of Ras/MEK/ERK and the transcription factor CREB. A serial analysis of chromatin occupancy screen identified Wnt-2 as an activity-dependent CREB-responsive gene. Neuronal activity enhances CREB-dependent transcription of Wnt-2, and expression of Wnt-2 stimulates dendritic arborization. This novel signaling pathway contributes to dynamic remodeling of the dendritic architecture in response to neuronal activity during development.

Extrasynaptic membrane trafficking regulated by GluR1 serine 845 phosphorylation primes AMPA receptors for long-term potentiation.

Enhancement of synaptic transmission, as occurs in long-term potentiation (LTP), can result from several mechanisms that are regulated by phosphorylation of the AMPA-type glutamate receptor (AMPAR). Using a quantitative assay of net serine 845 (Ser-845) phosphorylation in the GluR1 subunit of AMPARs, we investigated the relationship between phospho-Ser-845, GluR1 surface expression, and synaptic strength in hippocampal neurons. About 15% of surface AMPARs in cultured neurons were phosphorylated at Ser-845 basally, whereas chemical potentiation (forskolin/rolipram treatment) persistently increased this to 60% and chemical depression (N-methyl-D-aspartate treatment) decreased it to 10%. These changes in Ser-845 phosphorylation were paralleled by corresponding changes in the surface expression of AMPARs in both cultured neurons and hippocampal slices. For every 1% increase in net phospho-Ser-845, there was 0.75% increase in the surface fraction of GluR1. Phosphorylation of Ser-845 correlated with a selective delivery of AMPARs to extrasynaptic sites, and their synaptic localization required coincident synaptic activity. Furthermore, increasing the extrasynaptic pool of AMPA receptors resulted in stronger theta burst LTP. Our results support a two-step model for delivery of GluR1-containing AMPARs to synapses during activity-dependent LTP, where Ser-845 phosphorylation can traffic AMPARs to extrasynaptic sites for subsequent delivery to synapses during LTP.

Bidirectional regulation of cytoplasmic polyadenylation element-binding protein phosphorylation by Ca2+/calmodulin-dependent protein kinase II and protein phosphatase 1 during hippocampal long-term potentiation.

Induction of hippocampal long-term potentiation (LTP) requires activation of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), whereas maintenance of LTP additionally requires protein synthesis. We recently reported that CaMKII stimulates protein synthesis in depolarized hippocampal neurons through phosphorylation of the mRNA translation factor cytoplasmic polyadenylation element-binding protein (CPEB), and this phosphorylation is rapidly reversed by protein phosphatase 1 (PP1). Protein synthesis-dependent late-phase LTP (L-LTP) in the hippocampus requires calcium influx through the NMDA-type glutamate receptor (NMDA-R) to activate CaMKII as well as concomitant inhibition of PP1 mediated by protein kinase A. Therefore, we investigated the regulation of CPEB phosphorylation during L-LTP. Pharmacological stimulation of the NMDA-R in hippocampal slices to produce chemical long-term depression induced a brief dephosphorylation of CPEB. Modest LTP induction (once at 100 Hz), which induces a protein synthesis-independent early-phase LTP (E-LTP), resulted in a transient phosphorylation of CPEB. However, stronger stimulation (four times at 100 Hz), known to induce protein synthesis-dependent L-LTP, elicited a prolonged phosphorylation of CPEB. Furthermore, CPEB phosphorylation correlated with phosphorylation of PP1 inhibitor dopamine- and cAMP-regulated phosphoprotein, a known substrate for protein kinase A. These results evoke the hypothesis that bidirectional regulation of CPEB phosphorylation by CaMKII and protein phosphatases may serve as a mechanism to convert E-LTP into protein synthesis-dependent L-LTP by stimulating protein synthesis and thereby stabilizing synaptic enhancement.

Calmodulin-dependent kinase kinase/calmodulin kinase I activity gates extracellular-regulated kinase-dependent long-term potentiation.

Intracellular Ca2+ and protein phosphorylation play pivotal roles in long-term potentiation (LTP), a cellular model of learning and memory. Ca2+ regulates multiple intracellular pathways, including the calmodulin-dependent kinases (CaMKs) and the ERKs (extracellular signal-regulated kinases), both of which are required for LTP. However, the mechanism by which Ca2+ activates ERK during LTP remains unknown. Here, we describe a requirement for the CaMK-kinase (CaMKK) pathway upstream of ERK in LTP induction. Both the pharmacological inhibitor of CaMKK, STO-609, and dominant-negative CaMKI (dnCaMKI), a downstream target of CaMKK, blocked neuronal NMDA receptor-dependent ERK activation. In contrast, an inhibitor of CaMKII and nuclear-localized dnCaMKIV had no effect on ERK activation. NMDA receptor-dependent LTP induction robustly activated CaMKI, the Ca2+-stimulated Ras activator Ras-GRF1 (Ras-guanyl-nucleotide releasing factor), and ERK. STO-609 blocked the activation of all three enzymes during LTP without affecting basal synaptic transmission, activation of CaMKII, or cAMP-dependent activation of ERK. LTP induction itself was suppressed 50% by STO-609 in a manner identical to the ERK inhibitor U0126: either inhibitor occluded the effect of the other, suggesting they are part of the same signaling pathway in LTP induction. STO-609 also suppressed regulatory phosphorylation of two downstream ERK targets during LTP, the general translation factors eIF4E (eukaryotic initiation factor 4) and its binding protein 4E-BP1 (eukaryotic initiation factor 4E-binding protein 1). These data indicate an essential role for CaMKK and CaMKI to link NMDA receptor-mediated Ca2+ elevation with ERK-dependent LTP.

Inhibition of calcium/calmodulin-dependent protein kinase kinase by protein 14-3-3.

Intracellular calcium concentrations regulate diverse cellular events including cytoskeletal dynamics, gene transcription, and synaptic plasticity. The calcium signal is transduced in part by the calcium/calmodulin-dependent protein kinase (CaMK) cascade that is comprised of CaMK kinase (CaMKK) and its primary downstream substrates, CaMKI and CaMKIV. The CaMK cascade also participates in cross-talk with other signaling pathways: CaMKK/CaMKI can activate the mitogen-activated protein kinase pathway and cAMP-dependent protein kinase (PKA) can directly phosphorylate two inhibitory sites (Thr108 and Ser458) in CaMKK. Here we report an additional PKA-dependent regulation of CaMKK through its interaction with protein 14-3-3. CaMKK and 14-3-3 co-immunoprecipitated from co-transfected heterologous cells as well as from rat brain homogenate, and site-directed mutagenesis studies identified phospho-Ser74 in CaMKK as the primary 14-3-3 binding site. In cultured rat hippocampal neurons and acute hippocampal slices this interaction was robustly stimulated by activation of PKA through forskolin treatment and was blocked by inhibition of PKA. Interaction of 14-3-3 with CaMKK had two regulatory consequences in vitro. It directly inhibited CaMKK activity, and it also blocked dephosphorylation of Thr108, an inhibitory PKA phosphorylation site. In human embryonic kidney 293 cells transfected with CaMKK and stimulated with forskolin, co-transfection with 14-3-3 prevented dephosphorylation of Thr108 to the same extent as did inhibition of protein phosphatases with okadaic acid. We conclude that binding of 14-3-3 to CaMKK stabilizes its inhibition by PKA-mediated phosphorylation, which may have important consequences in the regulation of CaMKI, CaMKIV, protein kinase B, and ERK signaling pathways.