Identification of neuronal substrates implicates Pak5 in synaptic vesicle trafficking

Synaptic transmission is mediated by a complex set of molecular events that must be coordinated in time and space. While many proteins that function at the synapse have been identified, the signaling pathways regulating these molecules are poorly understood. Pak5 (p21-activated kinase 5) is a brain-specific isoform of the group II Pak kinases whose substrates and roles within the central nervous system are largely unknown. To gain insight into the physiological roles of Pak5, we engineered a Pak5 mutant to selectively radiolabel its substrates in murine brain extract. Using this approach, we identified two novel Pak5 substrates, Pacsin1 and Synaptojanin1, proteins that directly interact with one another to regulate synaptic vesicle endocytosis and recycling. Pacsin1 and Synaptojanin1 were phosphorylated by Pak5 and the other group II Paks in vitro, and Pak5 phosphorylation promoted Pacsin1-Synaptojanin1 binding both in vitro and in vivo. These results implicate Pak5 in Pacsin1- and Synaptojanin1-mediated synaptic vesicle trafficking and may partially account for the cognitive and behavioral deficits observed in group II Pak-deficient mice.     

The effects of muscarine and atropine reveal that inhibitory autoreceptors are present on frog motor nerve terminals but are not activated during transmission

Summary The role of presynaptic muscarinic receptors in modulating neuromuscular transmission was studied in the isolated sartorius muscle of the frog using electrophysiological techniques. In low calcium solutions muscarine reduced mEPP frequency and the quantal of EPPs. In solutions containing the normal calcium concentration the effect of muscarine on quantal content, but not the effect on mEPP frequency, was somewhat attenuated. Muscarine-induced reductions in the parameters of ACh release were prevented by atropine. Irrespective of the calcium concentration, atropine had no effect on mEPP frequency except where fibres were pretreated with glycerol. In experiments where evoked acetylcholine release was maintained at physiologically relevant levels, atropine had no effect on the quantal content of EPPs evoked at low frequency or on the extent of rundown in trains of EPPs evoked at high frequency. Send offprint requests to the author at Department of Pharmacology, Faculty of Basic Medical Sciences, Queen Mary and Westfield College, University of London, Mile End Road, London E1 4NS, UK     

5-HT1B receptor regulation of serotonin (5-HT) release by endogenous 5-HT in the substantia nigra

Axonal release of serotonin (5-hydroxytryptamine, 5-HT) in the CNS is typically regulated by presynaptic 5-HT autoreceptors. Release of 5-HT in substantia nigra pars reticulata (SNr), a principal output from the basal ganglia, has seemed an interesting exception to this rule. The SNr receives one of the highest densities of 5-HT innervation in mammalian brain and yet negative feedback regulation of axonal 5-HT release by endogenous 5-HT has not been identified here. We explored whether we could identify autoregulation of 5-HT release by 5-HT_1B receptors in rat SNr slices using fast-scan cyclic voltammetry at carbon-fiber microelectrodes to detect 5-HT release evoked by discrete stimuli (50 Hz, 20 pulses) paired over short intervals (1–10 s) within which any autoreceptor control should occur. Evoked 5-HT release exhibited short-term depression after an initial stimulus that recovered by 10 s. Antagonists for 5-HT_1B receptors, isamoltane (1 μM) or SB 224-289 (1 μM), did not modify release during a stimulus train, but rather, they modestly relieved depression of subsequent release evoked after a short delay (≤2 s). Release was not modified by antagonists for GABA (picrotoxin, 100 μM, saclofen, 50 μM) or histamine-H₃ (thioperamide, 10 μM) receptors. These data indicate that 5-HT release can activate a 5-HT_1B-receptor autoinhibition of subsequent release, which is mediated directly via 5-HT axons and not via GABAergic or histaminergic inputs. These data reveal that 5-HT release in SNr is not devoid of autoreceptor regulation by endogenous 5-HT, but rather is under modest control which only weakly limits 5-HT signaling.     

Structural basis for Mob1-dependent activation of the core Mst–Lats kinase cascade in Hippo signaling

The Mst–Lats kinase cascade is central to the Hippo tumor-suppressive pathway that controls organ size and tissue homeostasis. The adaptor protein Mob1 promotes Lats activation by Mst, but the mechanism remains unknown. Here, we show that human Mob1 binds to autophosphorylated docking motifs in active Mst2. This binding enables Mob1 phosphorylation by Mst2. Phosphorylated Mob1 undergoes conformational activation and binds to Lats1. We determine the crystal structures of phospho-Mst2–Mob1 and phospho-Mob1–Lats1 complexes, revealing the structural basis of both phosphorylation-dependent binding events. Further biochemical and functional analyses demonstrate that Mob1 mediates Lats1 activation through dynamic scaffolding and allosteric mechanisms. Thus, Mob1 acts as a phosphorylation-regulated coupler of kinase activation by virtue of its ability to engage multiple ligands. We propose that stepwise, phosphorylation-triggered docking interactions of nonkinase elements enhance the specificity and robustness of kinase signaling cascades.     

Double inhibition and activation mechanisms of Ephexin family RhoGEFs

Ephexin family guanine nucleotide exchange factors (GEFs) transfer signals from Eph tyrosine kinase receptors to Rho GTPases, which play critical roles in diverse cellular processes, as well as cancers and brain disorders. Here, we elucidate the molecular basis underlying inhibition and activation of Ephexin family RhoGEFs. The crystal structures of partially and fully autoinhibited Ephexin4 reveal that the complete autoinhibition requires both N- and C-terminal inhibitory modes, which can operate independently to impede Ras homolog family member G (RhoG) access. This double inhibition mechanism is commonly employed by other Ephexins and SGEF, another RhoGEF for RhoG. Structural, enzymatic, and cell biological analyses show that phosphorylation of a conserved tyrosine residue in its N-terminal inhibitory domain and association of PDZ proteins with its C-terminal PDZ-binding motif may respectively relieve the two autoinhibitory modes in Ephexin4. Our study provides a mechanistic framework for understanding the fine-tuning regulation of Ephexin4 GEF activity and offers possible clues for its pathological dysfunction.

Rho GTPases are master regulators of cytoskeletal dynamics and play pivotal roles in diverse cellular processes, including cell polarity, cell motility, cell division, and synaptic signaling (1–3). Typically, Rho GTPases function as molecular switches that cycle between an active guanosine triphosphate (GTP)-bound form and an inactive guanosine diphosphate (GDP)-bound form. Upon activation, they interact with a wide range of downstream effectors, such as actin cytoskeletal regulators, kinases, and scaffold proteins, to drive essential changes in cytoskeletal architecture necessary for corresponding physiological functions (4, 5). Rho GTPases are activated by guanine nucleotide exchange factors (GEFs) and inactivated by GTPase-activating proteins (GAPs) (6, 7). These regulatory proteins are precisely controlled so that Rho GTPase activities are spatiotemporally initiated or suppressed in response to various upstream signals from cell-surface receptors, such as integrins, growth factors, and tyrosine kinase receptors, among others (4).Eph-interacting exchange protein (Ephexin) family RhoGEFs activate Rho GTPases, including RhoA, Rac, Cdc42, and RhoG (8). This family consists of five known members in most vertebrate species (Ephexin1 to -5). A common feature of the Ephexin family proteins is that they all associate with and act downstream of Eph receptors, the largest subfamily of tyrosine kinase receptors that are activated by Ephrins and participate in various cellular processes (8–11). Specifically, Ephexin1 (also known as NGEF or ARHGEF27) regulates axon growth cone dynamics and spine morphogenesis via binding to EphA4 and activation of RhoA (9, 12–14). Ephexin4 (also named as ARHGEF16) activates RhoG by interacting with EphA2, which promotes RhoG/ELMO/DOCK/Rac signaling and regulates cell migration (15, 16). Ephexin5 (also known as Vsm-RhoGEF or ARHGEF15) functions together with EphB2 to regulate excitatory synapse development (17). Notably, the biological functions of Ephexin2 and Ephexin3 remain elusive although they are known to activate RhoA (8). Therefore, the Ephexin family RhoGEFs serve as the regulatory hubs that link Ephrin-Eph signaling with cytoskeletal dynamics through spatiotemporal regulation of Rho GTPases. Dysfunctions of Eph-Ephexin–mediated Rho signaling have been associated with a variety of diseases, ranging from cancers to brain disorders (18–22).Each member of the Ephexin family proteins contains a Dbl homology (DH) domain, responsible for catalyzing guanine nucleotide exchange, and an adjacent regulatory pleckstrin homology (PH) domain. In addition, all members possess an Src homology 3 (SH3) domain C-terminal to the DH–PH domain tandem, except Ephexin5 (Fig. 1A). Ephexin4 contains an additional type-I PDZ-binding motif (PBM) at its very C terminus (Fig. 1A). Previous work has demonstrated that elimination of the C-terminal SH3 domain in Ephexin4 can significantly increase its GEF activity toward RhoG, suggesting that Ephexin4 may adopt an autoinhibited conformation via SH3-mediated intra- or intermolecular interactions (23, 24). Intriguingly, such an SH3-mediated autoinhibition mechanism may be also applicable to Ephexin1 and Ephexin3 (25). In addition to the C-terminal inhibition, several lines of evidence have suggested that an evolutionarily conserved helix preceding the catalytic DH domain (referred to as inhibitory helix [IH]) (Fig. 1A) also contributes to the inhibitory functions in Ephexin1 to -3, by binding directly to DH (9, 25, 26). Phosphorylation of a conserved tyrosine residue within the IH could potentially relieve the autoinhibition and activate Ephexin GEF activity (9, 25). However, the molecular basis underlying these autoinhibition and activation events is not well understood. The topic of whether a common regulatory mechanism is shared by all Ephexins remains an intriguing and potentially informative aspect of Rho GTPase biology.Open in a separate windowFig. 1.Crystal structure of Ephexin4^DPSH. (A) Schematic diagrams showing the conserved domain organizations of Ephexins and SGEF. The PBM sequences of Ephexin4 and SGEF are shown. The domain color coding is consistent throughout this paper. The domain keys are also shown here. (B) Ribbon diagram representation of the Ephexin4^DPSH structure. (C) Surface representation showing the overall architecture of the Ephexin4^DPSH. (D) Superposition of ARHGEF11 DH-PH/RhoA (PDB ID code: 1XCG) and the Ephexin4^DPSH (this study) structures. (E) Schematic representation of autoinhibited Ephexin4^DPSH. The circle indicates the DH active site.In the present study, we report crystal structures of Ephexin4 in its partially and fully autoinhibited states. Structural analyses show that the complete inhibition of Ephexin4 involves both N- and C-terminal inhibitory modes. We further demonstrate that this double inhibitory mechanism is conserved among the Ephexin family proteins. Interestingly, association of PDZ proteins with Ephexin4 relieves its C-terminal inhibition while N-terminal inhibition may be regulated by phosphorylation of a conserved tyrosine residue preceding its DH–PH catalytic domain tandem. Moreover, we predict and verify that another RhoGEF, SGEF (also known as ARHGEF26), also adopts a similar autoinhibited architecture and can be activated by binding to PDZ protein. In addition, our study provides a mechanistic clue for how a cancer-associated variant may lead to aberrant Ephexin4 GEF activity.     

A channelopathy mechanism revealed by direct calmodulin activation of TrpV4

Ca²⁺-calmodulin (CaM) regulates varieties of ion channels, including Transient Receptor Potential vanilloid subtype 4 (TrpV4). It has previously been proposed that internal Ca²⁺ increases TrpV4 activity through Ca²⁺-CaM binding to a C-terminal Ca²⁺-CaM binding domain (CBD). We confirmed this model by directly presenting Ca²⁺-CaM protein to membrane patches excised from TrpV4-expressing oocytes. Over 50 TRPV4 mutations are now known to cause heritable skeletal dysplasia (SD) and other diseases in human. We have previously examined 14 SD alleles and found them to all have gain-of-function effects, with the gain of constitutive open probability paralleling disease severity. Among the 14 SD alleles examined, E797K and P799L are located immediate upstream of the CBD. They not only have increase basal activity, but, unlike the wild-type or other SD-mutant channels examined, they were greatly reduced in their response to Ca²⁺-CaM. Deleting a 10-residue upstream peptide (Δ795–804) that covers the two SD mutant sites resulted in strong constitutive activity and the complete lack of Ca²⁺-CaM response. We propose that the region immediately upstream of CBD is an autoinhibitory domain that maintains the closed state through electrostatic interactions, and adjacent detachable Ca²⁺-CaM binding to CBD sterically interferes with this autoinhibition. This work further supports the notion that TrpV4 mutations cause SD by constitutive leakage. However, the closed conformation is likely destabilized by various mutations by different mechanisms, including the permanent removal of an autoinhibition documented here.Transient Receptor Potential (TRP) channels are a diverse family of cation-nonselective Ca²⁺-permeable channels with only limited sequence homology. They are tetramers of subunits that have the 6-TM membrane topology common to cation channels. TRPs tend to be polymodal, activated by a wide variety of chemical and physical stimuli. TrpV4 (vanilloid subfamily, type 4), cloned on the bases of its response to hypotonicity, can also be activated by direct membrane stretch, mild heat, and by endogenous metabolites as well as synthetic chemicals (1–3). The most potent agonist, GSK1016790A (abbreviated as GSK herein) (4) can drive the TrpV4 open probability (Po) to almost 100% (5).Ca²⁺ regulations of TRP channels are varied and complex. They often show Ca²⁺-dependent desensitizations to various stimuli by multiple mechanisms including the action of Ca²⁺-calmodulin (6, 7). In the case of TrpV4, however, Ca²⁺ increases (potentiates) channel response to hypotonicity or to phorbol esters (4αPDD or 4αPMA), although the activated current is followed by a Ca²⁺-dependent inactivation (8, 9). Strotmann et al. (9) found that the potentiation by Ca²⁺ requires an intact calmodulin (CaM) binding site (CBD) within Q806–E831 in the C-terminal tail. They later proposed that, without Ca²⁺-CaM, the CBD docks on an N-terminal site causing closure (10).At least 57 mutations in TRPV4 have been found to underlie dominantly heritable forms of skeletal dysplasia (SD) with several clinically distinguishable presentations, adult neuro-muscular degenerations, as well as being associated with other genetic predispositions. See ref. 3 for a review. We have shown that of 14 representative SD-causing TRPV4 alleles all encode channels with increased constitutive Po when expressed in Xenopus oocytes and that the level of basal leakage correlated with the clinical severity of the SD they caused (5). We therefore believe that SD mutations are likely all gain-of-function (GOF) alleles. None of the disease alleles falls within Q806–E831, the putative CBD. However, two GOFs we examined, E797K and P799L, are immediately upstream from this CBD (5). Fig. 5A diagrams this region of interest. E797K has been repeatedly discovered in different families (3, 11). P799 appears to be a hot spot with several known alleles: P799A, P799S, P799R, and the first-reported P799L (11, 12), causing different types of SD. Here, we scrutinized E797K, P799L, and several engineered mutations to further our understanding of TrpV4’s Ca²⁺-CaM regulation and its relationship to channelopathy.Open in a separate windowFig. 5.Autoinhibitory and CaM-binding domains in carboxyl tail. (A) Topological cartoon illustrating key regions of the carboxyl domain of TrpV4 considered in this work including the CaM-binding domain (CBD, green) and the proposed AID (blue). Also shown are the locations of the human SD mutations E799K and P799L as well as the engineered CaM-binding mutation W822A. (B) Alignment of the entire carboxyl termini of TrpV1 (Upper) and V4 (Lower). Identical residues (red) indicated conservation through but not beyond the AID. The extent of the known cryo structure of TrpV1 is indicated by the vertical dotted line (in A as well). Extended β-sheet (arrows) secondary structures predicted using PSSpred (Y. Zhang, zhanglab.ccmb.med.umich.edu/PSSpred) are indicated above and below the primary sequences for TrpV1 and TrpV4, respectively, with the red arrow highlighting the likely undefined β sheet from the cryo structure. Point mutants examined in this study are indicated by yellow boxes, the blue bar indicates the extent of the AID deletant and the green to the CBD deletant.To avoid the complexity of the cytoplasm in whole cells, we here directly present CaM protein to membrane patches excised from TRPV4-expressing oocytes. Channel activation by such presentation was first demonstrated with patches excised from Paramecium (13). Rosenbaum et al. (6) showed that direct application of Ca²⁺-CaM reduces capsaicin-activated current of TrpV1 in expressing oocyte patches. Thus, CaM can be regarded as a detachable channel subunit in these cases. We found Ca²⁺-CaM alone robustly activates wild-type TrpV4 current in the absence of added chemical or physical stimuli such as GSK or stretch force, and therefore refer to the effect as activation instead of potentiation. We found that E797K and P799L channels are uniquely defective in activation by Ca²⁺-CaM. Further investigation led us to postulate that the peptide at the I795-I804 region, which covers these mutations, likely binds to other gating elements to stabilize the closed state(s) and that Ca²⁺-CaM release the binding to open TrpV4. This is akin to the classical disinhibition mechanism of enzyme activation, in which an autoinhibitory peptide is removed by Ca²⁺-CaM to activate.     

Autoinhibitory regulation of SCF-mediated ubiquitination by human cullin 1's C-terminal tail

SCF (Skp1·CUL1·F-box protein·ROC1) E3 ubiquitin ligase and Cdc34 E2-conjugating enzyme catalyze polyubiquitination in a precisely regulated fashion. Here, we describe biochemical evidence suggesting an autoinhibitory role played by the human CUL1 ECTD (extreme C-terminal domain; spanning the C-terminal 50 amino acids), a region that is predicted to contact the ROC1 RING finger protein by structural studies. We showed that ECTD did not contribute to CUL1's stable association with ROC1. Remarkably, deletion of ECTD, or missense mutations designed to disrupt the predicted ECTD·ROC1 interaction, markedly increased the ability of SCF^βTrCP2 to promote IκBα polyubiquitination and polyubiquitin chain assembly by Cdc34 in vitro. Thus, disruption of ECTD yields in vitro effects that parallel SCF activation by Nedd8 conjugation to CUL1. We propose that SCF may be subject to autoinhibitory regulation, in which Nedd8 conjugation acts as a molecular switch to drive the E3 into an active state by diminishing the inhibitory ECTD·ROC1 interaction.     

Dealing with Time-Dependent Pharmacokinetics during the Early Clinical Development of a New Leukotriene B4 Synthesis Inhibitor

Purpose The aim of this study was to explore the possibility of achieving a practical dosing regimen for 2,4,6-triiodophenol (AM-24), a new leukotriene B₄ (LTB₄) synthesis inhibitor. First, a model capable of dealing with the nonlinearity in its pharmacokinetic profile was built, and then it was combined with a pharmacodynamic model previously established with data from earlier phase I trials. Methods One week after the first 240-, 350-, or 500-mg oral dose of AM-24, six additional doses were given to 24 healthy volunteers once daily. A total of 33 blood samples were obtained from each individual. Different models, including enzyme turnover models, were fitted to the data by using the software NONMEM. Results Drug absorption was modeled with a first-order process. Drug disposition was described with a one-compartment model, and elimination with an (auto)inhibited and a noninhibited clearance. AM-24 inhibited the enzyme production rate to a maximum of 98%. Relative bioavailability was independent of the decrease in the amount of enzyme. The estimate of the enzyme turnover half-life was 8.5 h. Conclusions Simulations have shown that steady-state conditions eliciting 90% of maximal LTB₄ synthesis inhibition can be reached after 3 weeks during an oral treatment with AM-24 administered at the dosage of 500 mg once daily.