Gary A. Wayman

Cross Talk between ERK and PKA Is Required for Ca2+ Stimulation of CREB-Dependent Transcription and ERK Nuclear Translocation

Although Ca2+-stimulated cAMP response element binding protein- (CREB-) dependent transcription has been implicated in growth, differentiation, and neuroplasticity, mechanisms for Ca2+-activated transcription have not been defined. Here, we report that extracellular signal-related protein kinase (ERK) signaling is obligatory for Ca2+-stimulated transcription in PC12 cells and hippocampal neurons. The sequential activation of ERK and Rsk2 by Ca2+ leads to the phosphorylation and transactivation of CREB. Interestingly, the Ca2+-induced nuclear translocation of ERK and Rsk2 to the nucleus requires protein kinase A (PKA) activation. This may explain why PKA activity is required for Ca2+-stimulated CREB-dependent transcription. Furthermore, the full expression of the late phase of long-term potentiation (L-LTP) and L-LTP-associated CRE-mediated transcription requires ERK activation, suggesting that the activation of CREB by ERK plays a critical role in the formation of long lasting neuronal plasticity.

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.

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.

Phosphorylation of CBP Mediates Transcriptional Activation by Neural Activity and CaM Kinase IV

Activity-regulated transcription has been implicated in adaptive plasticity in the CNS. In many instances, this plasticity depends upon the transcription factor CREB. Precisely how neuronal activity regulates CREB remains unclear. To address this issue, we examined the phosphorylation state of components of the CREB transcriptional pathway. We show that NMDA activates transcription of CREB-responsive genes in hippocampal neurons, with ERK responsible for persistent CREB phosphorylation and CaM kinase IV (CaMKIV) responsible for phosphorylating the CREB coactivator, CBP. Ser301 of CBP was identified as a major target of CaMKIV phosphorylation in vitro and in vivo. CaM kinase inhibitors attenuated phosphorylation at Ser301 and blocked CBP-dependent transcription. Additionally, mutation of Ser301 impaired NMDA- and CaMKIV-stimulated transcription. These findings demonstrate that activity-induced CaMKIV signaling contributes to CREB/CBP-dependent transcription by phosphorylating CBP at Ser301.

The WRP component of the WAVE-1 complex attenuates Rac-mediated signalling

WAVE-1, which is also known as Scar, is a scaffolding protein that directs actin reorganization by relaying signals from the GTPase Rac to the Arp2/3 complex. Although the molecular details of WAVE activation by Rac have been described, the mechanisms by which these signals are terminated remain unknown. Here we have used tandem mass spectrometry to identify previously unknown components of the WAVE signalling network including WRP, a Rac-selective GTPase-activating protein. WRP binds directly to WAVE-1 through its Src homology domain 3 and specifically inhibits Rac function in vivo. Thus, we propose that WRP is a binding partner of WAVE-1 that functions as a signal termination factor for Rac.

Activity-Dependent Synaptogenesis: Regulation by a CaM-Kinase Kinase/CaM-Kinase I/βPIX 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.

Regulation of axonal extension and growth cone motility by calmodulin-dependent protein kinase I.

Calcium and calmodulin (CaM) are important signaling molecules that regulate axonal or dendritic extension and branching. The Ca2+-dependent stimulation of neurite elongation has generally been assumed to be mediated by CaM-kinase II (CaMKII), although other members of the CaMK family are highly expressed in developing neurons. We have examined this assumption using a combination of dominant–negative CaMKs (dnCaMKs) and other specific CaMK inhibitors. Here we report that inhibition of cytosolic CaMKI, but not CaMKII or nuclear CaMKIV, dramatically decreases axonal outgrowth and branching in cultured neonatal hippocampal and postnatal cerebellar granule neurons. CaMKI is found throughout the cell cytosol, including the growth cone. Growth cones of neurons expressing dnCaMI or dnCaMKK, the upstream activator of CaMKI, exhibit collapsed morphology with a prominent reduction in lamellipodia. Live-cell imaging confirms that these morphological changes are associated with a dramatic decrease in growth cone motility. Treatment of neurons with 1,8-naphthoylene benzimidazole-3-carboxylic acid (STO-609), an inhibitor of CaMKK, causes a similar change in morphology and reduction in growth cone motility, and this inhibition can be rescued by transfection with an STO-609-insensitive mutant of CaMKK or by transfection with constitutively active CaMKI. These results identify CaMKI as a positive transducer of growth cone motility and axon outgrowth and provide a new physiological role for the CaMKK–CaMKI pathway.

Bone morphogenetic protein function is required for terminal differentiation of the heart but not for early expression of cardiac marker genes

To examine potential roles for bone morphogenetic proteins (BMPs) in cardiogenesis, we used intracellular BMP inhibitors to disrupt this signaling cascade in Xenopus embryos. BMP-deficient embryos showed endodermal defects, a reduction in cardiac muscle-specific gene expression, a decrease in the number of cardiomyocytes and cardia bifida. Early expression of markers of endodermal and precardiac fate, however, was not perturbed. Heart defects were observed even when BMP signal transduction was blocked only in cells that contribute primarily to endodermal, and not cardiac fates, suggesting a non-cell autonomous function. Our results suggest that BMPs are not required for expression of early transcriptional regulators of cardiac fate but are essential for migration and/or fusion of the heart primordia and cardiomyocyte differentiation.

Agonist-dependent desensitization of the kappa opioid receptor by G protein receptor kinase and beta-arrestin.

We used the Xenopus oocyte expression system to examine the regulation of rat κ opioid receptor (rKOR) function by G protein receptor kinases (GRKs). κ agonists increased the conductance of G protein-activated inwardly rectifying potassium channels in oocytes co-expressing KOR with Kir3.1 and Kir3.4. In the absence of added GRK and β-arrestin 2, desensitization of the κ agonist-induced potassium current was modest. Co-expression of either GRK3 or GRK5 along with β-arrestin 2 significantly increased the rate of desensitization, whereas addition of either β-arrestin 2, GRK3, or GRK5 alone had no effect on the KOR desensitization rate. The desensitization was homologous as co-expressed δ opioid receptor-evoked responses were not affected by KOR desensitization. The rate of GRK3/β-arrestin 2-dependent desensitization was reduced by truncation of the C-terminal 26 amino acids, KOR(Q355Δ). In contrast, substitution of Ala for Ser within the third intracellular loop [KOR(S255A,S260A,S262A)] did not reduce the desensitization rate. Within the C-terminal region, KOR(S369A) substitution significantly attenuated desensitization, whereas the KOR(T363A) and KOR(S356A,T357A) point mutations did not. These results suggest that co-expression of GRK3 or GRK5 and β-arrestin 2 produced homologous, agonist-induced desensitization of the κ opioid receptor by a mechanism requiring the phosphorylation of the serine 369 of rKOR.

Inhibitory cross-talk by cAMP kinase on the calmodulin-dependent protein kinase cascade

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. We have shown previously that activated CaM-KIV can activate the mitogen-activated protein kinases (Enslen, H., Tokumitsu, H., Stork, P. J. S., Davis, R. J., and Soderling, T. R. (1996) Proc. Natl. Acad. Sci. U.u2009S.u2009A. 93, 10803–10808), and the present paper describes a novel regulatory cross-talk between cAMP kinase (PKA) and CaM-KK. PKA gave rapid phosphorylation in vitro and in cells of recombinant CaM-KK, resulting in 50–75% inhibition of CaM-KK activity, part of which was 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 were deleted, was still partially suppressed by PKA-mediated phosphorylation. The second inhibitory site was identified as Thr108 by site-specific mutagenesis. Treatments of COS-7, PC12, hippocampal, or Jurkat cells with the PKA activators forskolin or isoproterenol gave 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.