The mechanism of PAK activation. Autophosphorylation events in both regulatory and kinase domains control activity

The p21-activated kinases (PAKs), in common with many kinases, undergo multiple autophosphorylation events upon interaction with appropriate activators. The Cdc42-induced phosphorylation of PAK serves in part to dissociate the kinase from its partners PIX and Nck. Here we investigate in detail how autophosphorylation events affect the catalytic activity of PAK by altering the autophosphorylation sites in both alpha- and betaPAK. Both in vivo and in vitro analyses demonstrate that, although most phosphorylation events in the PAK N-terminal regulatory domain play no direct role in activation, a phosphorylation of alphaPAK serine 144 or betaPAK serine 139, which lie in the kinase inhibitory domain, significantly contribute to activation. By contrast, sphingosine-mediated activation is independent of this residue, indicating a different mode of activation. Thus two autophosphorylation sites direct activation while three others control association with focal complexes via PIX and Nck.     

Involvement of CaM kinase II in gonadotropin-releasing hormone-induced activation of MAP kinase in cultured hypothalamic neurons

Gonadotropin-releasing hormone (GnRH) is secreted from hypothalamic GnRH neurons. There is accumulating evidence that GnRH neurons have GnRH receptors and that the autocrine action of GnRH activates MAP kinase. In this study, we found that KN93, an inhibitor of Ca(2+)/calmodulin-dependent protein kinases (CaM kinases), inhibited the GnRH-induced activation of MAP kinase in immortalized GnRH neurons (GT1-7 cells). Immunoblot analysis indicated that the CaM kinase IIdelta2 isoform (CaM kinase IIdelta2) and synapsin I were expressed in GT1-7 cells. GnRH treatment rapidly increased phosphorylation of synapsin I at serine 603, a specific phosphorylation site for CaM kinase II, suggesting that GnRH treatment rapidly activated CaM kinase IIdelta2. In addition, when we stably overexpressed CaM kinase IIdelta2 in GT1-7 cells, the activation of MAP kinase was strongly enhanced. These results suggest that CaM kinase IIdelta2 was involved in the GnRH-induced activation of MAP kinase in GT1-7 cells.     

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

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

The T-type voltage-gated calcium channel as a molecular target of the novel cognitive enhancer ST101: enhancement of long-term potentiation and CaMKII autophosphorylation in rat cortical slices

In this study, we report that spiro[imidazo[1,2-a]pyridine-3,2-indan]-2(3H)-one (ST101; previously coded as ZSET1446) targets T-type voltage-gated calcium channels in mediating improved cognition in the CNS. We prepared rat somatosensory cortical and hippocampal slices, treated them with 0.01 to 100 nM ST101, and performed immunoblotting and electrophysiological analyses using various voltage-gated calcium channel (VGCC) inhibitors. Treatment of rat cortical slices with a range of ST101 concentrations significantly increased calcium/calmodulin-dependent protein kinase II (CaMKII) autophosphorylation following a bell-shaped dose-response curve, with 0.1 nM ST101 representing the maximally effective concentration. protein kinase Cα autophosphorylation was also significantly increased by 0.1 nM ST101 treatment. ST101 treatment had a moderate effect on CaMKII autophosphorylation but no effect on hippocampal protein kinase Cα autophosphorylation in slice preparations. Consistent with increased cortical CaMKII autophosphorylation, AMPA-type glutamate receptor subunit 1 (Ser-831) phosphorylation as a CaMKII post-synaptic substrate was significantly increased by treatment with 0.1-1 nM ST101, whereas phosphorylation of the pre-synaptic substrate synapsin I (Ser-603) remained unchanged. Notably, enhanced CaMKII autophosphorylation seen following 0.1 nM ST101 treatment was significantly inhibited by pre-treatment with 1 μM mibefradil, a T-type VGCC inhibitor, but not with N-type (ω-conotoxin), P/Q-type (ω-agatoxin) or L-type (nifedipine) VGCC inhibitors. Similarly, 0.1 nM ST101 significantly potentiated long-term potentiation in cortical but not hippocampal slices. Enhanced long-term potentiation in cortical slices was totally inhibited by 1 μM mibefadil treatment. Finally, whole-cell patch-clamp analysis of Neuro2A cells over-expressing recombinant human Ca(V) 3.1 (α1G) T-channels and treated with 0.1 nM ST101 showed significant increases in T-type VGCC currents. These results indicate that T-type VGCCs are direct molecular targets for the novel cognitive enhancer ST101, a potential Alzheimer disease therapeutic.     

Depolarization-induced differentiation of PC12 cells is mediated by phospholipase D2 through the transcription factor CREB pathway

The present study examined the role of phospholipase D2 (PLD2) in the regulation of depolarization-induced neurite outgrowth and the expression of growth-associated protein-43 (GAP-43) and synapsin I in rat pheochromocytoma (PC12) cells. Depolarization of PC12 cells with 50 mmol/L KCl increased neurite outgrowth and elevated mRNA and protein expression of GAP-43 and synapsin I. These increases were suppressed by inhibition of Ca²⁺-calmodulin-dependent protein kinase II (CaMKII), PLD, or mitogen-activated protein kinase kinase (MEK). Knockdown of PLD2 by small interfering RNA (siRNA) suppressed the depolarization-induced neurite outgrowth, and the increase in GAP-43 and synapsin I expression. Depolarization evoked a Ca²⁺ rise that activated various signaling enzymes and the cAMP response element-binding protein (CREB). Silencing CaMKIIδ by siRNA blocked KCl-induced phosphorylation of proline-rich protein tyrosine kinase 2 (Pyk2), Src kinase, and extracellular signal-regulated kinase (ERK). Inhibition of Src or MEK abolished phosphorylation of ERK and CREB. Furthermore, phosphorylation of Pyk2, ERK, and CREB was suppressed by the PLD inhibitor, 1-butanol and transfection of PLD2 siRNA, whereas it was enhanced by over-expression of wild-type PLD2. Depolarization-induced PLD2 activation was suppressed by CaMKII and Src inhibitors, but not by MEK or protein kinase A inhibitors. These results suggest that the signaling pathway of depolarization-induced PLD2 activation was downstream of CaMKIIδ and Src, and upstream of Pyk2(Y881) and ERK/CREB, but independent of the protein kinase A. This is the first demonstration that PLD2 activation is involved in GAP-43 and synapsin I expression during depolarization-induced neuronal differentiation in PC12 cells.     

Interaction between PAK and nck: a template for Nck targets and role of PAK autophosphorylation

The kinase PAK binds tightly to the SH3 domain of its partner PIX via a central proline-rich sequence. A different N-terminal sequence allows alphaPAK to bind an SH3 domain of the adaptor Nck. The Nck SH3[2] domain interacts equally with an 18-mer PAK-derived peptide and full-length alphaPAK. Detailed analysis of this binding by saturation substitution allows related Nck targets to be accurately identified from sequence characteristics alone. All Nck SH3[2] binding proteins, including PAK, NIK, synaptojanin, PRK2, and WIP, possess the motif PXXPXRXXS; in the case of PAK, serine phosphorylation at this site negatively regulates binding. We show that kinase autophosphorylation blocks binding by both Nck and PIX to alphaPAK, thus providing a mechanism to regulate PAK interactions with its SH3-containing partners. One cellular consequence of the regulatable binding of PAK is facilitation of its cycling between cytosolic and focal complex sites.     

Decreased calcium/calmodulin-dependent protein kinase II and protein kinase C activities mediate impairment of hippocampal long-term potentiation in the olfactory bulbectomized mice

Olfactory bulbectomized (OBX) mice showed significant impairment of learning and memory-related behaviors 14 days after olfactory bulbectomy, as measured by passive avoidance and Y-maze tasks. We here observed a large impairment of hippocampal long-term potentiation (LTP) in the OBX mice. Concomitant with decreased acetylcholinesterase expression, protein kinase C (PKC)alpha autophosphorylation and NR1(Ser-896) phosphorylation significantly decreased in the hippocampal CA1 region of OBX mice. Both PKCalpha and NR1(Ser-896) phosphorylation significantly increased following LTP in the control mice, whereas increases were not observed in OBX mice. Like PKC activities, calcium/calmodulin-dependent protein kinase II (CaMKII) autophosphorylation significantly decreased in the hippocampal CA1 region of OBX mice as compared with that of control mice. In addition, increased CaMKII autophosphorylation following LTP was not observed in OBX mice. Finally, the impairment of CaMKII autophosphorylation was closely associated with reduced pGluR1(Ser-831) phosphorylation, without change in synapsin I (site 3) phosphorylation in the hippocampal CA1 region of OBX mice. Taken together, in OBX mice NMDA receptor hypofunction, possibly through decreased PKCalpha activity, underlies decreased CaMKII activity in the post-synaptic regions, thereby impairing LTP induction in the hippocampal CA1 region. Both decreased PKC and CaMKII activities with concomitant LTP impairment account for the learning disability observed in OBX mice.     

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

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

Characterization of a central Ca2+/calmodulin-dependent protein kinase IIalpha/beta binding domain in densin that selectively modulates glutamate receptor subunit phosphorylation

The densin C-terminal domain can target Ca(2+)/calmodulin-dependent protein kinase IIα (CaMKIIα) in cells. Although the C-terminal domain selectively binds CaMKIIα in vitro, full-length densin associates with CaMKIIα or CaMKIIβ in brain extracts and in transfected HEK293 cells. This interaction requires a second central CaMKII binding site, the densin-IN domain, and an "open" activated CaMKII conformation caused by Ca(2+)/calmodulin binding, autophosphorylation at Thr-286/287, or mutation of Thr-286/287 to Asp. Mutations in the densin-IN domain (L815E) or in the CaMKIIα/β catalytic domain (I205/206K) disrupt the interaction. The amino acid sequence of the densin-IN domain is similar to the CaMKII inhibitor protein, CaMKIIN, and a CaMKIIN peptide competitively blocks CaMKII binding to densin. CaMKII is inhibited by both CaMKIIN and the densin-IN domain, but the inhibition by densin is substrate-selective. Phosphorylation of a model peptide substrate, syntide-2, or of Ser-831 in AMPA receptor GluA1 subunits is fully inhibited by densin. However, CaMKII phosphorylation of Ser-1303 in NMDA receptor GluN2B subunits is not effectively inhibited by densin in vitro or in intact cells. Thus, densin can target multiple CaMKII isoforms to differentially modulate phosphorylation of physiologically relevant downstream targets.     

Regulation of the Ca2+/CaM-responsive pool of CaMKII by scaffold-dependent autophosphorylation

CaMKII is critical for structural and functional plasticity. Here we show that Camguk (Cmg), the Drosophila homolog of CASK/Lin-2, associates in an ATP-regulated manner with CaMKII to catalyze formation of a pool of calcium-insensitive CaMKII. In the presence of Ca(2+)/CaM, CaMKII complexed to Cmg can autophosphorylate at T287 and become constitutively active. In the absence of Ca(2+)/CaM, ATP hydrolysis results in phosphorylation of T306 and inactivation of CaMKII. Cmg coexpression suppresses CaMKII activity in transfected cells, and the level of Cmg expression in Drosophila modulates postsynaptic T306 phosphorylation. These results suggest that Cmg, in the presence of Ca(2+)/CaM, can provide a localized source of active kinase. When Ca(2+)/CaM or synaptic activity is low, Cmg promotes inactivating autophosphorylation, producing CaMKII that requires phosphatase to reactivate. This interaction provides a mechanism by which the active postsynaptic pool of CaMKII can be controlled locally to differentiate active and inactive synapses.     

G protein-coupled receptor activation rapidly stimulates focal adhesion kinase phosphorylation at Ser-843. Mediation by Ca2+, calmodulin, and Ca2+/calmodulin-dependent kinase II

A rapid increase in the tyrosine phosphorylation of focal adhesion kinase (FAK) has been extensively documented in cells stimulated by multiple signaling molecules, but little is known about the regulation of FAK phosphorylation at serine residues. Stimulation of Swiss 3T3 cells with the G protein-coupled receptor agonists bombesin, vasopressin, or bradykinin induced an extremely rapid (within 5 s) increase in FAK phosphorylation at Ser-843. The phosphorylation of this residue preceded FAK phosphorylation at Tyr-397, the major autophosphorylation site, and FAK phosphorylation at Ser-910. Treatment of intact cells with ionomycin stimulated a rapid increase in FAK phosphorylation at Ser-843, indicating that an increase in intracellular Ca2+ concentration ([Ca2+]i) is a potential pathway leading to FAK-Ser-843 phosphorylation. Indeed, treatment with agents that prevent an agonist-induced increase in [Ca2+]i (e.g. thapsigargin or BAPTA (1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid)), interfere with calmodulin function (e.g. trifluoperazine, W13, and W7), or block Ca2+/calmodulin-dependent protein kinase II (CaMKII) activation (KN93) or expression (small interfering RNA) abrogated the rapid FAK phosphorylation at Ser-843 induced by bombesin, bradykinin, or vasopressin. Furthermore, activated CaMKII directly phosphorylated the recombinant COOH-terminal region of FAK at a residue equivalent to Ser-843. Thus, our results demonstrate that G protein-coupled receptor activation induces rapid FAK phosphorylation at Ser-843 through Ca2+, calmodulin, and CaMKII.     

Nitric oxide-mediated modulation of calcium/calmodulin-dependent protein kinase II

The mechanisms of NO inhibition of CaMK [Ca(2+)/CaM (calmodulin)-dependent protein kinase] II activity were studied. In rat pituitary tumour GH3 cells, TRH [thyrotrophin (TSH)-releasing hormone]-stimulated phosphorylation of nNOS [neuronal NOS (NO synthase)] at Ser(847) was sensitive to an inhibitor of CaMKs, KN-93, and was enhanced by inhibition of nNOS with 7NI (7-nitroindazole). Enzyme activity of CaMKII following in situ treatment with 7NI was also increased. The in vitro activity of CaMKII was inhibited by co-incubation either with nNOS and L-arginine or with NO donors SNAP (S-nitroso-N-acetyl-DL-penicillamine) and DEA-NONOate [diethylamine-NONOate (diazeniumdiolate)]. Once inhibited by these treatments, CaMKII was observed to undergo full reactivation on the addition of a reducing reagent, DTT (dithiothreitol). In transfected cells expressing CaMKII and nNOS, treatment with the calcium ionophore A23187 further revealed nNOS phosphorylation at Ser(847), which was enhanced by 7NI and CaMKII S-nitrosylation. Mutated CaMKII (C6A), in which Cys(6) was substituted with an alanine residue, was refractory to 7NI-induced enhancement of nNOS phosphorylation or to CaMKII S-nitrosylation. Furthermore, we could identify Cys(6) as a direct target for S-nitrosylation of CaMKII using MS. In addition, treatment with glutamate caused an increase in CaMKII S-nitrosylation in rat hippocampal slices. This glutamate-induced S-nitrosylation was blocked by 7NI. These results suggest that inactivation of CaMKII mediated by S-nitrosylation at Cys(6) may contribute to NO-induced neurotoxicity in the brain.     

AlphaCaMKII binding to the C-terminal tail of NMDA receptor subunit NR2A and its modulation by autophosphorylation.

Ca2+/calmodulin-dependent protein kinase II (CaMKII), a multifunctional, widely distributed enzyme, is enriched in post-synaptic densities (PSDs). Here, we demonstrate that CaMKII binds to a discrete C-terminal region of the NR2A subunit of NMDA receptors and promotes the phosphorylation of a Ser residue of this NMDA receptor subunit. Glutathione S-transferase (GST)-NR2A(1349-1464) binds native CaMKII from solubilised hippocampal PSDs in 'pull-out' and overlay experiments and this binding is competed by recombinant alphaCaMKII(1-315). The longer GST-NR2A(1244-1464), although containing the CaMKII phosphosite Ser-1289, binds the kinase with a lower efficacy. CaMKII association to NR2A(1349-1464) is positively modulated by kinase autophosphorylation in the presence of Ca2+/calmodulin. These data provide direct evidence for a mechanism modulating the synaptic strength.     

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

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

Nav1.5-dependent persistent Na+ influx activates CaMKII in rat ventricular myocytes and N1325S mice

Late Na(+) current (I(NaL)) and Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) are both increased in the diseased heart. Recently, CaMKII was found to phosphorylate the Na(+) channel 1.5 (Na(v)1.5), resulting in enhanced I(NaL). Conversely, an increase of I(NaL) would be expected to cause elevation of intracellular Ca(2+) and activation of CaMKII. However, a relationship between enhancement of I(NaL) and activation of CaMKII has yet to be demonstrated. We investigated whether Na(+) influx via Na(v)1.5 leads to CaMKII activation and explored the functional significance of this pathway. In neonatal rat ventricular myocytes (NRVM), treatment with the I(NaL) activators anemone toxin II (ATX-II) or veratridine increased CaMKII autophosphorylation and increased phosphorylation of CaMKII substrates phospholamban and ryanodine receptor 2. Knockdown of Na(v)1.5 (but not Na(v)1.1 or Na(v)1.2) prevented ATX-II-induced CaMKII phosphorylation, providing evidence for a specific role of Na(v)1.5 in CaMKII activation. In support of this view, CaMKII activity was also increased in hearts of transgenic mice overexpressing a gain-of-function Na(v)1.5 mutant (N(1325)S). The effects of both ATX-II and the N(1325)S mutation were reversed by either I(NaL) inhibition (with ranolazine or tetrodotoxin) or CaMKII inhibition (with KN93 or autocamtide 2-related inhibitory peptide). Furthermore, ATX-II treatment also induced CaMKII-Na(v)1.5 coimmunoprecipitation. The same association between CaMKII and Na(v)1.5 was also found in N(1325)S mice, suggesting a direct protein-protein interaction. Pharmacological inhibitions of either CaMKII or I(NaL) also prevented ATX-II-induced cell death in NRVM and reduced the incidence of polymorphic ventricular tachycardia induced by ATX-II in rat perfused hearts. Taken together, these results suggest that a Na(v)1.5-dependent increase in Na(+) influx leads to activation of CaMKII, which in turn phosphorylates Na(v)1.5, further promoting Na(+) influx. Pharmacological inhibition of either CaMKII or Na(v)1.5 can ameliorate cardiac dysfunction caused by excessive Na(+) influx.     

GIT1 activates p21-activated kinase through a mechanism independent of p21 binding

p21-activated kinases (PAKs) associate with a guanine nucleotide exchange factor, Pak-interacting exchange factor (PIX), which in turn binds the paxillin-associated adaptor GIT1 that targets the complex to focal adhesions. Here, a detailed structure-function analysis of GIT1 reveals how this multidomain adaptor also participates in activation of PAK. Kinase activation does not occur via Cdc42 or Rac1 GTPase binding to PAK. The ability of GIT1 to stimulate alphaPAK autophosphorylation requires the participation of the GIT N-terminal Arf-GAP domain but not Arf-GAP activity and involves phosphorylation of PAK at residues common to Cdc42-mediated activation. Thus, the activation of PAK at adhesion complexes involves a complex interplay between the kinase, Rho GTPases and protein partners that provide localization cues.     

Mechanism of fluoride-induced MAP kinase activation in pulmonary artery endothelial cells

In this study, we demonstrate that challenge of endothelial cells (EC) with NaF, a recognized G protein activator and protein phosphatase inhibitor, leads to a significant Erk activation, with increased phosphorylation of the well-known Erk substrate caldesmon. Inhibition of the Erk MAPK, MEK, by U0126 produces a marked decrease in NaF-induced caldesmon phosphorylation. NaF transiently increases the activity of the MEK kinase known as Raf-1 (approximately 3- to 4-fold increase over basal level), followed by a sustained Raf-1 inhibition (approximately 3- to 4-fold decrease). Selective Raf-1 inhibitors (ZM-336372 and Raf-1 inhibitor 1) significantly attenuate NaF-induced Erk and caldesmon phosphorylation. Because we have previously shown that Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) participates in Erk activation in thrombin-challenged cells, we next explored if CaMKII is involved in NaF-induced EC responses. We found that in NaF-treated EC, CaMKII activity increases in a time-dependent manner with maximal activity at 10 min (approximately 4-fold increase over a basal level). Pretreatment with KN93, a specific CaMKII inhibitor, attenuates NaF-induced barrier dysfunction and Erk phosphorylation. The Rho inhibitor C3 exotoxin completely abolishes NaF-induced CaMKII activation. Collectively, these data suggest that sequential activation of Raf-1, MEK, and Erk is modulated by Rho-dependent CaMKII activation and represents important NaF-induced signaling response. Caldesmon phosphorylation occurring by an Erk-dependent mechanism in NaF-treated pulmonary EC may represent a link between NaF stimulation and contractile responses of endothelium.     

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

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

Phosphorylation of RhoGDI1 by p21-activated kinase 2 mediates basic fibroblast growth factor-stimulated neurite outgrowth in PC12 cells

We previously showed that p21-activated kinase 2 (PAK2), a major PAK isoform expressed in PC12 cells, mediates neurite outgrowth via Rac1 GTPase. RhoGDI1 forms a complex with Rac1, resulting in its inhibition. Rac1 activation requires dissociation from RhoGDI1. Here, we show that PAK2 mediates basic fibroblast growth factor (bFGF)-stimulated neurite outgrowth via phosphorylation of RhoGDI1. RhoGDI1 was shown to be associated with PAK2, with phosphorylation of Ser34 and Ser101 by active PAK2 evident in vitro and in vivo. A RhoGDI1 phosphomimetic mutant (S34E/S101E) was dissociated from Rac1/Cdc42, whereas the wild-type or a nonphosphorylatable mutant (S34A/S101A) formed a tight complex. Consistent with this, PC12 cells expressing the phosphomimetic mutant displayed Rac1/Cdc42 activation in response to bFGF stimulation. Neurite outgrowth was also enhanced in PC12 cells expressing the phosphomimetic mutant. These results suggest that PAK2-mediated RhoGDI1 phosphorylation stimulates dissociation of RhoGDI1-Rac1/Cdc42 complex accompanied by relief of inhibitory effect on Rac1/Cdc42, which promotes neuronal differentiation.