ADP-ribosylation Factor 6 (ARF6) Bidirectionally Regulates Dendritic Spine Formation Depending on Neuronal Maturation and Activity

Recent studies have reported conflicting results regarding the role of ARF6 in dendritic spine development, but no clear answer for the controversy has been suggested. We found that ADP-ribosylation factor 6 (ARF6) either positively or negatively regulates dendritic spine formation depending on neuronal maturation and activity. ARF6 activation increased the spine formation in developing neurons, whereas it decreased spine density in mature neurons. Genome-wide microarray analysis revealed that ARF6 activation in each stage leads to opposite patterns of expression of a subset of genes that are involved in neuronal morphology. ARF6-mediated Rac1 activation via the phospholipase D pathway is the coincident factor in both stages, but the antagonistic RhoA pathway becomes involved in the mature stage. Furthermore, blocking neuronal activity in developing neurons using tetrodotoxin or enhancing the activity in mature neurons using picrotoxin or chemical long term potentiation reversed the effect of ARF6 on each stage. Thus, activity-dependent dynamic changes in ARF6-mediated spine structures may play a role in structural plasticity of mature neurons.     

SNX26, a GTPase-activating Protein for Cdc42, Interacts with PSD-95 Protein and Is Involved in Activity-dependent Dendritic Spine Formation in Mature Neurons

SNX26, a brain-enriched RhoGAP, plays a key role in dendritic arborization during early neuronal development in the neocortex. In mature neurons, it is localized to dendritic spines, but little is known about its role in later stages of development. Our results show that SNX26 interacts with PSD-95 in dendritic spines of cultured hippocampal neurons, and as a GTPase-activating protein for Cdc42, it decreased the F-actin content in COS-7 cells and in dendritic spines of neurons. Overexpression of SNX26 resulted in a GTPase-activating protein activity-dependent decrease in total protrusions and spine density together with dramatic inhibition of filopodia-to-spine transformations. Such effects of SNX26 were largely rescued by a constitutively active mutant of Cdc42. Consistently, an shRNA-mediated knockdown of SNX26 significantly increased total protrusions and spine density, resulting in an increase in thin or stubby type spines at the expense of the mushroom spine type. Moreover, endogenous expression of SNX26 was shown to be bi-directionally modulated by neuronal activity. Therefore, we propose that in addition to its key role in neuronal development, SNX26 also has a role in the activity-dependent structural change of dendritic spines in mature neurons.     

Phosphorylation of Tubulin by Casein Kinase II Regulates Its Binding to a Neuronal Protein (NP185) Associated with Brain Coated Vesicles

We recently described a new protein associated exclusively with neuronal clathrin-coated vesicles (CCVs), and characterized two monoclonal antibodies that react with it (S-8G8 and S-6G7). In this report, the association of neuronal protein of 185 kilodaltons (NP185) with CCV kinases and its interaction with tubulin are described. The affinity of NP185 for tubulin is significantly enhanced when tubulin is phosphorylated by CCV-associated casein kinase II. In contrast, phosphorylation of tubulin by a kinase activity associated with purified brain tubulin decreases its affinity for NP185. Together, these data suggest that the interaction of NP185 with tubulin is modulated by protein phosphorylation. Recent evidence has suggested that tubulin is phosphorylated by casein kinase II during neurite development. The enhanced affinity of NP185 for tubulin phosphorylated by casein kinase II could be important for proper intracellular sorting of this protein in the developing neuron.     

Phosphorylation by Casein Kinase 2 Facilitates Psh1 Protein-assisted Degradation of Cse4 Protein

Cse4 is the centromeric histone H3 variant in budding yeast. Psh1 is an E3 ubiquitin ligase that controls Cse4 levels through proteolysis. Here we report that Psh1 is phosphorylated by the Cka2 subunit of casein kinase 2 (CK2) to promote its E3 activity for Cse4. Deletion of CKA2 significantly stabilized Cse4. Consistent with phosphorylation promoting the activity of Psh1, Cse4 was stabilized in a Psh1 phosphodepleted mutant strain in which the major phosphorylation sites were changed to alanines. Phosphorylation of Psh1 did not control Psh1-Cse4 or Psh1-Ubc3(E2) interactions. Although Cse4 was highly stabilized in a cka2Δ strain, mislocalization of Cse4 was mild, suggesting that Cse4 misincorporation was prevented by the intact Psh1-Cse4 association. Supporting this idea, Psh1 was also stabilized in a cka2Δ strain. Collectively our data suggest that phosphorylation is crucial in Psh1-assisted control of Cse4 levels and that the Psh1-Cse4 association itself functions to prevent Cse4 misincorporation.     

Increased Phosphorylation of a 17-kDa Protein Kinase C Substrate (P17) in Long-Term Potentiation

Hippocampal long-term potentiation (LTP) is a persistent increase in the efficacy of synaptic transmission, which is widely thought to be a cellular mechanism that could contribute to learning and memory. Studies on the biochemical mechanisms underlying LTP suggest the involvement of protein kinases in both LTP induction and maintenance. In this report we describe an LTP-associated increase in the phosphorylation in vitro of a 17-kDa protein kinase C (PKC) substrate protein, which we have termed P17, in homogenates from the CA1 region of rat hippocampal slices. This LTP-associated increase in phosphorylation was expressed independent of significant levels of free Ca2+, as phosphorylation reactions were performed in the presence of 500 microM EGTA. The increased phosphorylation of P17 was substantially inhibited by PKC(19-36), a selective inhibitor of PKC. These data support the model that persistent PKC activation contributes to the maintenance of LTP and implicate P17 as a potential target for PKC in the CA1 region of the hippocampus.     

A Protein Modulator Stimulates C Kinase-Dependent Phosphorylation of a 90K Substrate in Synaptic Membranes

We have identified and partially purified an acidic, heat-stable, noncalmodulin protein from bovine brain cytosol that stimulates Ca2+-dependent phosphorylation of an Mr 90K substrate in crude rat brain synaptic membranes. We show that this modulator of phosphorylation (MOP) enhances Ca2+- and phospholipid-dependent protein kinase (C kinase) phosphorylation of this 90K substrate. The 90K substrate is a higher Mr form of an 87K substrate that is a major C kinase substrate in rat brain. The Ca2+-dependent phosphorylation of both substrates is inhibited by the Ca2+-binding proteins S-100 and calmodulin. Both substrates yield phosphopeptide fragments of Mr 9K and 13K after limited proteolysis with V8 protease. Two-dimensional polyacrylamide gel electrophoresis reveals that they have similar acidic isoelectric points (pI 5.0). MOP enhances Ca2+-dependent phosphorylation of the 90K substrate whereas the phosphorylation of 87K is diminished. This reciprocal relationship suggests that the mobility of the 87K substrate in sodium dodecyl sulfate-polyacrylamide gels is decreased to 90K with increasing phosphorylation. MOP may be a novel protein modulator of C kinase-mediated phosphorylation in the nervous system.     

Rho-GTPase-activating Protein Interacting with Cdc-42-interacting Protein 4 Homolog 2 (Rich2): A NEW Ras-RELATED C3 BOTULINUM TOXIN SUBSTRATE 1 (Rac1) GTPase-ACTIVATING PROTEIN THAT CONTROLS DENDRITIC SPINE MORPHOGENESIS*

Development of dendritic spines is important for synaptic function, and alteration in spine morphogenesis is often associated with mental disorders. Rich2 was an uncharacterized Rho-GAP protein. Here we searched for a role of this protein in spine morphogenesis. We found that it is enriched in dendritic spines of cultured hippocampal pyramidal neurons during early stages of development. Rich2 specifically stimulated the Rac1 GTPase in these neurons. Inhibition of Rac1 by EHT 1864 increased the size and decreased the density of dendritic spines. Similarly, Rich2 overexpression increased the size and decreased the density of dendritic spines, whereas knock-down of the protein by specific si-RNA decreased both size and density of spines. The morphological changes were reflected by the increased amplitude and decreased frequency of miniature EPSCs induced by Rich2 overexpression, while si-RNA treatment decreased both amplitude and frequency of these events. Finally, treatment of neurons with EHT 1864 rescued the phenotype induced by Rich2 knock-down. These results suggested that Rich2 controls dendritic spine morphogenesis and function via inhibition of Rac1.     

Phosphorylation of Serine-880 in GluR2 by Protein Kinase C Prevents Its C Terminus from Binding with Glutamate Receptor-Interacting Protein

Phosphorylation of the glutamate receptor is an important mechanism of synaptic plasticity. Here, we show that the C terminus of GluR2 of the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor is phosphorylated by protein kinase C and that serine-880 is the major phosphorylation site. This phosphorylation also occurs in human embryonic kidney (HEK) cells by addition of 12-O-tetradecanoylphorbol 13-acetate. Our immunoprecipitation experiment revealed that the phosphorylation of serine-880 in GluR2 drastically reduced the affinity for glutamate receptor-interacting protein (GRIP), a synaptic PDZ domain-containing protein, in vitro and in HEK cells. This result suggests that modulation of serine-880 phosphorylation in GluR2 controls the clustering of AMPA receptors at excitatory synapses and consequently contributes to synaptic plasticity.     

Ca2+-independent Activation of Ca2+/Calmodulin-dependent Protein Kinase II Bound to the C-terminal Domain of CaV2.1 Calcium Channels

Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) forms a major component of the postsynaptic density where its functions in synaptic plasticity are well established, but its presynaptic actions are poorly defined. Here we show that CaMKII binds directly to the C-terminal domain of Ca_V2.1 channels. Binding is enhanced by autophosphorylation, and the kinase-channel signaling complex persists after dephosphorylation and removal of the Ca²⁺/CaM stimulus. Autophosphorylated CaMKII can bind the Ca_V2.1 channel and synapsin-1 simultaneously. CaMKII binding to Ca_V2.1 channels induces Ca²⁺-independent activity of the kinase, which phosphorylates the enzyme itself as well as the neuronal substrate synapsin-1. Facilitation and inactivation of Ca_V2.1 channels by binding of Ca²⁺/CaM mediates short term synaptic plasticity in transfected superior cervical ganglion neurons, and these regulatory effects are prevented by a competing peptide and the endogenous brain inhibitor CaMKIIN, which blocks binding of CaMKII to Ca_V2.1 channels. These results define the functional properties of a signaling complex of CaMKII and Ca_V2.1 channels in which both binding partners are persistently activated by their association, and they further suggest that this complex is important in presynaptic terminals in regulating protein phosphorylation and short term synaptic plasticity.     

Protein Kinase A-dependent Phosphorylation of Rap1 Regulates Its Membrane Localization and Cell Migration

The small G protein Rap1 can mediate “inside-out signaling” by recruiting effectors to the plasma membrane that signal to pathways involved in cell adhesion and cell migration. This action relies on the membrane association of Rap1, which is dictated by post-translational prenylation as well as by a stretch of basic residues within its carboxyl terminus. One feature of this stretch of acidic residues is that it lies adjacent to a functional phosphorylation site for the cAMP-dependent protein kinase PKA. This phosphorylation has two effects on Rap1 action. One, it decreases the level of Rap1 activity as measured by GTP loading and the coupling of Rap1 to RapL, a Rap1 effector that couples Rap1 GTP loading to integrin activation. Two, it destabilizes the membrane localization of Rap1, promoting its translocation into the cytoplasm. These two actions, decreased GTP loading and decreased membrane localization, are related, as the translocation of Rap1-GTP into the cytoplasm is associated with its increased GTP hydrolysis and inactivation. The consequences of this phosphorylation in Rap1-dependent cell adhesion and cell migration were also examined. Active Rap1 mutants that lack this phosphorylation site had a minimal effect on cell adhesion but strongly reduced cell migration, when compared with an active Rap1 mutant that retained the phosphorylation site. This suggests that optimal cell migration is associated with cycles of Rap1 activation, membrane egress, and inactivation, and requires the regulated phosphorylation of Rap1 by PKA.     

Protein Kinase CK2 Regulates the Dimerization of Histone Deacetylase 1 (HDAC1) and HDAC2 during Mitosis

Histone deacetylase 1 (HDAC1) and HDAC2 are components of corepressor complexes that are involved in chromatin remodeling and regulation of gene expression by regulating dynamic protein acetylation. HDAC1 and -2 form homo- and heterodimers, and their activity is dependent upon dimer formation. Phosphorylation of HDAC1 and/or HDAC2 in interphase cells is required for the formation of HDAC corepressor complexes. In this study, we show that during mitosis, HDAC2 and, to a lesser extent, HDAC1 phosphorylation levels dramatically increase. When HDAC1 and -2 are displaced from the chromosome during metaphase, they dissociate from each other, but each enzyme remains in association with components of the HDAC corepressor complexes Sin3, NuRD, and CoREST as homodimers. Enzyme inhibition studies and mutational analyses demonstrated that protein kinase CK2-catalyzed phosphorylation of HDAC1 and -2 is crucial for the dissociation of these two enzymes. These results suggest that corepressor complexes, including HDAC1 or HDAC2 homodimers, might target different cellular proteins during mitosis.     

Characterization of the Phosphorylation Site of PP59, a Substrate for Cyclic AMP-Dependent Protein Kinase That Is Enriched in the Synaptic Membrane Fraction of Rat Cerebellum

Abstract: We have identified previously a synaptic membrane-associated protein, PP59, that serves as a substrate for cyclic AMP-dependent protein kinase and is enriched in rat cerebellum. We show here that PP59 can be extracted from synaptic plasma membranes with a combination of 2% Triton X-100 plus 1 M KCl. A 290-fold purification of PP59 was achieved by selective solubilization, followed by continuous-elution preparative gel electrophoresis. To determine the amino acid sequence surrounding the cyclic AMP-dependent protein kinase phosphorylation site within PP59, the partially purified ³²P-phosphorylated protein was digested with chymotrypsin, and radiolabeled peptides were purified by sequential reversed-phase HPLC in two different solvent systems. Automated Edman degradation revealed a single phosphorylation site contained within the sequence Ala-Arg-Glu-Arg-Ser-Asp-Ser(P)-Thr-Gly-Ser-Ser-Ser-Val-Tyr. No strong sequence homology to this peptide fragment with other known peptides or proteins in the SwissProt, PIR, or GenPept databases could be found. A synthetic peptide containing this unique 14-amino acid sequence was used to develop polyclonal anti-peptide antibodies that were affinity-purified and shown to recognize intact PP59 as determined by western blotting. These antibodies specifically inhibited the phosphorylation of PP59 by cyclic AMP-dependent protein kinase in an in vitro phosphorylation assay containing synaptic plasma membranes.     

The Polarity Protein Partitioning-defective 1 (PAR-1) Regulates Dendritic Spine Morphogenesis through Phosphorylating Postsynaptic Density Protein 95 (PSD-95)

The polarity protein PAR-1 plays an essential role in many cellular contexts, including embryogenesis, asymmetric cell division, directional migration, and epithelial morphogenesis. Despite its known importance in different cellular processes, the role of PAR-1 in neuronal morphogenesis is less well understood. In particular, its role in the morphogenesis of dendritic spines, which are sites of excitatory synaptic inputs, has been unclear. Here, we show that PAR-1 is required for normal spine morphogenesis in hippocampal neurons. We further show that PAR-1 functions through phosphorylating the synaptic scaffolding protein PSD-95 in this process. Phosphorylation at a conserved serine residue in the KXGS motif in PSD-95 regulates spine morphogenesis, and a phosphomimetic mutant of this site can rescue the defects of kinase-dead PAR-1. Together, our findings uncover a role of PAR-1 in spine morphogenesis in hippocampal neurons through phosphorylating PSD-95.     

Phosphorylation of Synaptic GTPase-activating Protein (synGAP) by Ca2+/Calmodulin-dependent Protein Kinase II (CaMKII) and Cyclin-dependent Kinase 5 (CDK5) Alters the Ratio of Its GAP Activity toward Ras and Rap GTPases

synGAP is a neuron-specific Ras and Rap GTPase-activating protein (GAP) found in high concentrations in the postsynaptic density (PSD) fraction from the mammalian forebrain. We have previously shown that, in situ in the PSD fraction or in recombinant form in Sf9 cell membranes, synGAP is phosphorylated by Ca²⁺/calmodulin-dependent protein kinase II (CaMKII), another prominent component of the PSD. Here, we show that recombinant synGAP (r-synGAP), lacking 102 residues at the N terminus, can be purified in soluble form and is phosphorylated by cyclin-dependent kinase 5 (CDK5) as well as by CaMKII. Phosphorylation of r-synGAP by CaMKII increases its HRas GAP activity by 25% and its Rap1 GAP activity by 76%. Conversely, phosphorylation by CDK5 increases r-synGAP''s HRas GAP activity by 98% and its Rap1 GAP activity by 20%. Thus, phosphorylation by both kinases increases synGAP activity; CaMKII shifts the relative GAP activity toward inactivation of Rap1, and CDK5 shifts the relative activity toward inactivation of HRas. GAP activity toward Rap2 is not altered by phosphorylation by either kinase. CDK5 phosphorylates synGAP primarily at two sites, Ser-773 and Ser-802. Phosphorylation at Ser-773 inhibits r-synGAP activity, and phosphorylation at Ser-802 increases it. However, the net effect of concurrent phosphorylation of both sites, Ser-773 and Ser-802, is an increase in GAP activity. synGAP is phosphorylated at Ser-773 and Ser-802 in the PSD fraction, and its phosphorylation by CDK5 and CaMKII is differentially regulated by activation of NMDA-type glutamate receptors in cultured neurons.     

The Guanine Nucleotide Exchange Factor (GEF) Asef2 Promotes Dendritic Spine Formation via Rac Activation and Spinophilin-dependent Targeting

Dendritic spines are actin-rich protrusions that establish excitatory synaptic contacts with surrounding neurons. Reorganization of the actin cytoskeleton is critical for the development and plasticity of dendritic spines, which is the basis for learning and memory. Rho family GTPases are emerging as important modulators of spines and synapses, predominantly through their ability to regulate actin dynamics. Much less is known, however, about the function of guanine nucleotide exchange factors (GEFs), which activate these GTPases, in spine and synapse development. In this study we show that the Rho family GEF Asef2 is found at synaptic sites, where it promotes dendritic spine and synapse formation. Knockdown of endogenous Asef2 with shRNAs impairs spine and synapse formation, whereas exogenous expression of Asef2 causes an increase in spine and synapse density. This effect of Asef2 on spines and synapses is abrogated by expression of GEF activity-deficient Asef2 mutants or by knockdown of Rac, suggesting that Asef2-Rac signaling mediates spine development. Because Asef2 interacts with the F-actin-binding protein spinophilin, which localizes to spines, we investigated the role of spinophilin in Asef2-promoted spine formation. Spinophilin recruits Asef2 to spines, and knockdown of spinophilin hinders spine and synapse formation in Asef2-expressing neurons. Furthermore, inhibition of N-methyl-d-aspartate receptor (NMDA) activity blocks spinophilin-mediated localization of Asef2 to spines. These results collectively point to spinophilin-Asef2-Rac signaling as a novel mechanism for the development of dendritic spines and synapses.     

Phosphorylation of VACM-1/Cul5 by Protein Kinase A Regulates Its Neddylation and Antiproliferative Effect

Expression of the VACM-1/cul5 gene in endothelial and in cancer cell lines in vitro inhibits cellular proliferation and decreases phosphorylation of MAPK. Structure-function analysis of the VACM-1 protein sequence identified consensus sites specific for phosphorylation by protein kinases A and C (PKA and PKC) and a Nedd8 protein modification site. Mutations at the PKA-specific site in VACM-1/Cul5 (^S730AVACM-1) sequence resulted in increased cellular growth and the appearance of a Nedd8-modified VACM-1/Cul5. The aim of this study was to examine if PKA-dependent phosphorylation of VACM-1/Cul5 controls its neddylation status, phosphorylation by PKC, and ultimately growth. Our results indicate that in vitro transfection of rat adrenal medullary endothelial cells with anti-VACM-1-specific small interfering RNA oligonucleotides decreases endogenous VACM-1 protein concentration and increases cell growth. Western blot analysis of cell lysates immunoprecipitated with an antibody directed against a PKA-specific phosphorylation site and probed with anti-VACM-1-specific antibody showed that PKA-dependent phosphorylation of VACM-1 protein was decreased in cells transfected with ^S730AVACM-1 cDNA when compared with the cytomegalovirus-transfected cells. This change was associated with increased modification of VACM-1 protein by Nedd8. Induction of PKA activity with forskolin reduced modification of VACM-1 protein by Nedd8. Finally, rat adrenal medullary endothelial cells transfected with ^S730AVACM-1/cul5 cDNA and treated with phorbol 12-myristate 13-acetate (10 and 100 nm) to induce PKC activity grew significantly faster than the control cells. These results suggest that the antiproliferative effect of VACM-1/Cul5 is dependent on its posttranslational modifications and will help in the design of new anticancer therapeutics that target the Nedd8 pathway.     

Nucleotides and Phosphorylation Bi-directionally Modulate Ca2+/Calmodulin-dependent Protein Kinase II (CaMKII) Binding to the N-Methyl-d-aspartate (NMDA) Receptor Subunit GluN2B

The Ca²⁺/calmodulin (CaM)-dependent protein kinase II (CaMKII) and the NMDA-type glutamate receptor are key regulators of synaptic plasticity underlying learning and memory. Direct binding of CaMKII to the NMDA receptor subunit GluN2B (formerly known as NR2B) (i) is induced by Ca²⁺/CaM but outlasts this initial Ca²⁺-stimulus, (ii) mediates CaMKII translocation to synapses, and (iii) regulates synaptic strength. CaMKII binds to GluN2B around S1303, the major CaMKII phosphorylation site on GluN2B. We show here that a phospho-mimetic S1303D mutation inhibited CaM-induced CaMKII binding to GluN2B in vitro, presenting a conundrum how binding can occur within cells, where high ATP concentration should promote S1303 phosphorylation. Surprisingly, addition of ATP actually enhanced the binding. Mutational analysis revealed that this positive net effect was caused by four modulatory effects of ATP, two positive (direct nucleotide binding and CaMKII T286 autophosphorylation) and two negative (GluN2B S1303 phosphorylation and CaMKII T305/6 autophosphorylation). Imaging showed positive regulation by nucleotide binding also within transfected HEK cells and neurons. In fact, nucleotide binding was a requirement for efficient CaMKII interaction with GluN2B in cells, while T286 autophosphorylation was not. Kinetic considerations support a model in which positive regulation by nucleotide binding and T286 autophosphorylation occurs faster than negative modulation by GluN2B S1303 and CaMKII T305/6 phosphorylation, allowing efficient CaMKII binding to GluN2B despite the inhibitory effects of the two slower reactions.