Calcyon stimulates neuregulin 1 maturation and signaling.

Neuregulin1 (NRG1) is a single transmembrane protein that plays a critical role in neural development and synaptic plasticity. Both NRG1 and its receptor, ErbB4, are well-established risk genes of schizophrenia. The NRG1 ecto-domain (ED) binds and activates ErbB4 following proteolytic cleavage of pro-NRG1 precursor protein. Although several studies have addressed the function of NRG1 in brain, very little is known about the cleavage and shedding mechanism. Here we show that the neuronal vesicular protein calcyon is a potent activator and key determinant of NRG1 ED cleavage and shedding. Calcyon stimulates clathrin-mediated endocytosis and endosomal targeting; and its levels are elevated in postmortem brains of schizophrenics. Overexpression of calcyon stimulates NRG1 cleavage and signaling in vivo, and as a result, GABA transmission is enhanced in calcyon overexpressing mice. Conversely, NRG1 cleavage, ErbB4 activity and GABA transmission are decreased in calcyon null mice. Moreover, stimulation of NRG1 cleavage by calcyon was recapitulated in HEK 293 cells suggesting the mechanism involved is cell-autonomous. Finally, studies with site-specific mutants in calcyon and inhibitors for the major sheddases indicate that the stimulatory effects of calcyon on NRG1 cleavage and shedding depend on clathrin-mediated endocytosis, ß-secretase 1, and interaction with clathrin adaptor proteins. Together these results identify a novel mechanism for NRG1 cleavage and shedding.

Inhibition of PYK2-induced actin cytoskeleton reorganization, PYK2 autophosphorylation and focal adhesion targeting by FAK.

Focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (PYK2) are structurally related tyrosine kinases. They are implicated in regulating actin cytoskeleton organization, a process critical for cell migration, mitosis and tumor metastasis. In this paper, we demonstrate that, although both PYK2 and FAK were expressed and colocalized at focal adhesions in fibroblasts, microinjection of PYK2, but not FAK, in Swiss 3T3 fibroblastic cells led to reorganization of focal adhesions and cell rounding. PYK2-mediated actin cytoskeleton reorganization required the PYK2 N terminus, the focal adhesion targeting (FAT) domain, catalytic activity and autophosphorylation. Remarkably, FAK suppressed PYK2-mediated reorganization of focal adhesions and cell rounding. In addition, FAK inhibited PYK2 autophosphorylation and focal adhesion targeting, which might contribute to FAK-mediated suppression of PYK2's phenotypes. Further analyses demonstrated that the inhibition of PYK2 autophosphorylation required the FAK N terminus but not FAK tyrosine phosphorylation. The FAK FAT domain seemed to be critical for FAK-mediated suppression of PYK2 focal adhesion targeting. Taken together, these results demonstrate that FAK could inhibit PYK2 autophosphorylation, focal adhesion targeting and actin cytoskeleton reorganization, suggesting that the balance between FAK and PYK2 tyrosine kinases is important for regulating cellular morphology, cell migration and cell growth.

Signal transduction in neuronal migration: roles of GTPase activating proteins and the small GTPase Cdc42 in the Slit-Robo pathway.

The Slit protein guides neuronal and leukocyte migration through the transmembrane receptor Roundabout (Robo). We report here that the intracellular domain of Robo interacts with a novel family of Rho GTPase activating proteins (GAPs). Two of the Slit-Robo GAPs (srGAPs) are expressed in regions responsive to Slit. Slit increased srGAP1-Robo1 interaction and inactivated Cdc42. A dominant negative srGAP1 blocked Slit inactivation of Cdc42 and Slit repulsion of migratory cells from the anterior subventricular zone (SVZa) of the forebrain. A constitutively active Cdc42 blocked the repulsive effect of Slit. These results have demonstrated important roles for GAPs and Cdc42 in neuronal migration. We propose a signal transduction pathway from the extracellular guidance cue to intracellular actin polymerization.

Glycogen synthase kinase 3beta is tyrosine phosphorylated by PYK2.

Glycogen synthase kinase 3beta (GSK3beta) is a Ser/Thr kinase that is involved in numerous cellular activities. GSK3beta is activated by tyrosine phosphorylation. However, very little is known about the tyrosine kinases that are responsible for phosphorylating GSK3beta. In this report, we investigated the ability of the calcium-dependent tyrosine kinase, proline-rich tyrosine kinase 2 (PYK2) to tyrosine phosphorylate GSK3beta. In transfected CHO cells, it was demonstrated that PYK2 tyrosine phosphorylates GSK3beta in situ. The two kinases also coimmunoprecipitated. Furthermore, GSK3beta was tyrosine phosphorylated in vitro by an active, wild type PYK2, but not by the inactive, kinase dead form of PYK2. Therefore, this study is the first to demonstrate that GSK3beta is a substrate of PYK2 both in vitro and in situ.

Regulation of the p21-activated kinase (PAK) by a human Gbeta -like WD-repeat protein, hPIP1.

The family of p21-activated protein kinases (PAKs) is composed of serine-threonine kinases whose activity is regulated by the small guanosine triphosphatases (GTPases) Rac and Cdc42. In mammalian cells, PAKs have been implicated in the regulation of mitogen-activated protein cascades, cellular morphological and cytoskeletal changes, neurite outgrowth, and cell apoptosis. Although the ability of Cdc42 and Rac GTPases to activate PAK is well established, relatively little is known about the negative regulation of PAK or the identity of PAK cellular targets. Here, we describe the identification and characterization of a human PAK-interacting protein, hPIP1. hPIP1 contains G protein beta-like WD repeats and shares sequence homology with the essential fission yeast PAK regulator, Skb15, as well as the essential budding yeast protein, MAK11. Interaction of hPIP1 with PAK1 inhibits the Cdc42/Rac-stimulated kinase activity through the N-terminal regulatory domains of PAK1. Cotransfection of hPIP1 in mammalian cells inhibits PAK-mediated c-Jun N-terminal kinase and nuclear factor kappa B signaling pathways. Our results demonstrate that hPIP1 is a negative regulator of PAK and PAK signaling pathways.

Regulation of CDC42 GTPase by proline-rich tyrosine kinase 2 interacting with PSGAP, a novel pleckstrin homology and Src homology 3 domain containing rhoGAP protein.

Proline-rich tyrosine kinase 2 (PYK2), a tyrosine kinase structurally related to focal adhesion kinase (FAK), is implicated in regulating cytoskeletal organization. However, mechanisms by which PYK2 participates in and regulates cytoskeletal organization remain largely unknown. Here we report identification of PSGAP, a novel protein that interacts with PYK2 and FAK and contains multiple domains including a pleckstrin homology domain, a rhoGTPase-activating protein domain, and a Src homology 3 domain. PYK2 interacts with PSGAP Src homology 3 domain via the carboxyl-terminal proline-rich sequence. PSGAP is able to increase GTPase activity of CDC42 and RhoA in vitro and in vivo. Remarkably, PYK2, but not FAK, can activate CDC42 via inhibition of PSGAP-mediated GTP hydrolysis of CDC42. Moreover, PSGAP is localized at cell periphery in fibroblasts in a pleckstrin homology domain-dependent manner. Over expression of PSGAP in fibroblasts results in reorganization of cytoskeletal structures and changes of cellular morphology, which requires rhoGTPase-activating activity. Taken together, our results suggest that PSGAP is a signaling protein essential for PYK2 regulation of cytoskeletal organization via Rho family GTPases.

Erbin is a protein concentrated at postsynaptic membranes that interacts with PSD-95.

Neuregulin is a factor essential for synapse-specific transcription of acetylcholine receptor genes at the neuromuscular junction. Its receptors, ErbB receptor tyrosine kinases, are localized at the postjunctional membrane presumably to ensure localized signaling. However, the molecular mechanisms underlying synaptic localization of ErbBs are unknown. Our recent studies indicate that ErbB4 interacts with postsynaptic density (PSD)-95 (SAP90), a PDZ domain-containing protein that does not interact with ErbB2 or ErbB3. Using as bait the ErbB2 C terminus, we identified Erbin, another PDZ domain-containing protein that interacts specifically with ErbB2. Erbin is concentrated in postsynaptic membranes at the neuromuscular junction and in the central nervous system, where ErbB2 is concentrated. Expression of Erbin increases the amount of ErbB2 labeled by biotin in transfected cells, suggesting that Erbin is able to increase ErbB2 surface expression. Furthermore, we provide evidence that Erbin interacts with PSD-95 in both transfected cells and synaptosomes. Thus ErbB proteins can interact with a network of PDZ domain-containing proteins. This interaction may play an important role in regulation of neuregulin signaling and/or subcellular localization of ErbB proteins.

PI-3 kinase and IP3 are both necessary and sufficient to mediate NT3-induced synaptic potentiation.

Signaling mechanisms underlying neurotrophic regulation of synaptic transmission are not fully understood. Here we show that neurotrophin-3 (NT3)-induced potentiation of synaptic transmission at the neuromuscular synapses is blocked by inhibition of phosphoinositide-3 kinase, phospholipase C-gamma or the downstream IP3 receptors of phospholipase C-gamma, but not by inhibition of MAP kinase. However, neither stimulation of Ca2+ release from intracellular stores by photolysis of caged IP3, nor expression of a constitutively active phosphoinositide-3 kinase (PI3K*) in presynaptic motoneurons alone is sufficient to enhance transmission. Photo-uncaging of IP3 in neurons expressing PI3K* elicits a marked synaptic potentiation, mimicking the NT3 effect. These results reveal an involvement of PI3 kinase in transmitter release, and suggest that concomitant activation of PI3 kinase and IP3 receptors is both necessary and sufficient to mediate the NT3-induced synaptic potentiation.

Automated assay optimization with integrated statistics and smart robotics.

The transition from manual to robotic high throughput screening (HTS) in the last few years has made it feasible to screen hundreds of thousands of chemical entities against a biological target in less than a month. This rate of HTS has increased the visibility of bottlenecks, one of which is assay optimization. In many organizations, experimental methods are generated by therapeutic teams associated with specific targets and passed on to the HTS group. The resulting assays frequently need to be further optimized to withstand the rigors and time frames inherent in robotic handling. Issues such as protein aggregation, ligand instability, and cellular viability are common variables in the optimization process. The availability of robotics capable of performing rapid random access tasks has made it possible to design optimization experiments that would be either very difficult or impossible for a person to carry out. Our approach to reducing the assay optimization bottleneck has been to unify the highly specific fields of statistics, biochemistry, and robotics. The product of these endeavors is a process we have named automated assay optimization (AAO). This has enabled us to determine final optimized assay conditions, which are often a composite of variables that we would not have arrived at by examining each variable independently. We have applied this approach to both radioligand binding and enzymatic assays and have realized benefits in both time and performance that we would not have predicted a priori. The fully developed AAO process encompasses the ability to download information to a robot and have liquid handling methods automatically created. This evolution in smart robotics has proven to be an invaluable tool for maintaining high-quality data in the context of increasing HTS demands.

Regulation of neuregulin signaling by PSD-95 interacting with ErbB4 at CNS synapses.

Neuregulins (NRGs) and their receptors, the ErbB protein tyrosine kinases, are essential for neuronal development, but their functions in the adult CNS are unknown. We report that ErbB4 is enriched in the postsynaptic density (PSD) and associates with PSD-95. Heterologous expression of PSD-95 enhanced NRG activation of ErbB4 and MAP kinase. Conversely, inhibiting expression of PSD-95 in neurons attenuated NRG-mediated activation of MAP kinase. PSD-95 formed a ternary complex with two molecules of ErbB4, suggesting that PSD-95 facilitates ErbB4 dimerization. Finally, NRG suppressed induction of long-term potentiation in the hippocampal CA1 region without affecting basal synaptic transmission. Thus, NRG signaling may be synaptic and regulated by PSD-95. A role of NRG signaling in the adult CNS may be modulation of synaptic plasticity.

Identification of a novel cortactin SH3 domain-binding protein and its localization to growth cones of cultured neurons.

Cortactin is an actin-binding protein that contains several potential signaling motifs including a Src homology 3 (SH3) domain at the distal C terminus. Translocation of cortactin to specific cortical actin structures and hyperphosphorylation of cortactin on tyrosine have been associated with the cortical cytoskeleton reorganization induced by a variety of cellular stimuli. The function of cortactin in these processes is largely unknown in part due to the lack of information about cellular binding partners for cortactin. Here we report the identification of a novel cortactin-binding protein of approximately 180 kDa by yeast two-hybrid interaction screening. The interaction of cortactin with this 180-kDa protein was confirmed by both in vitro and in vivo methods, and the SH3 domain of cortactin was found to direct this interaction. Since this protein represents the first reported natural ligand for the cortactin SH3 domain, we designated it CortBP1 for cortactin-binding protein 1. CortBP1 contains two recognizable sequence motifs within its C-terminal region, including a consensus sequence for cortactin SH3 domain-binding peptides and a sterile alpha motif. Northern and Western blot analysis indicated that CortBP1 is expressed predominately in brain tissue. Immunofluorescence studies revealed colocalization of CortBP1 with cortactin and cortical actin filaments in lamellipodia and membrane ruffles in fibroblasts expressing CortBP1. Colocalization of endogenous CortBP1 and cortactin was also observed in growth cones of developing hippocampal neurons, implicating CortBP1 and cortactin in cytoskeleton reorganization during neurite outgrowth.

Expression and characterization of splice variants of PYK2, a focal adhesion kinase-related protein.

Focal adhesion kinase and the recently identified proline-rich tyrosine kinase 2 (PYK2), also known as cell adhesion kinase &bgr ;, related adhesion focal tyrosine kinase or calcium-dependent protein tyrosine kinase, define a new family of non-receptor protein tyrosine kinases. Activation of PYK2 has been implicated in multiple signaling events, including modulation of ion channels, T- and B-cell receptor signaling and cell death. Mechanisms underlying the functional diversity of PYK2 are unclear. Here, we provide evidence for two novel alternatively expressed isoforms of PYK2. One isoform, designated PYK2s (PYK2 splice form), appears to be a splice variant of PYK2 lacking 42 amino acids within the C-terminal domain. A second isoform, referred to as PRNK (PYK2-related non-kinase), appears to be specified by mRNAs that encode only part of the C-terminal domain of PYK2. Northern blot analysis indicates that the unspliced PYK2 is expressed at high levels in the brain and poorly expressed in the spleen, whereas PYK2s and PRNK are expressed in the spleen. In situ hybridization studies of rat brain demonstrate that the unspliced PYK2 is selectively expressed at high levels in hippocampus, cerebral cortex and olfactory bulb, whereas PYK2s and PRNK are expressed at low levels in all regions of rat brain examined. Immunofluorescence analysis of ectopically expressed PRNK protein shows that PRNK, in contrast to full-length PYK2, is localized to focal adhesions by sequences within the focal adhesion targeting domain. In addition, PYK2, but not PRNK, interacts with p130(cas )and Graf. These results imply that PRNK may selectively regulate PYK2 function in certain cells by binding to some but not all PYK2 binding partners, and the functional diversity mediated by PYK2 may be due in part to complex alternative splicing.

Crystallization and preliminary diffraction analysis of porcine heart mitochondrial NADP(+)-dependent isocitrate dehydrogenase.

Isocitrate dehydrogenases [isocitrate:NAD(P)(+) oxidoreductase (decarboxylating), E.C. 1.1.1.42] are ubiquitous metabolic enzymes which occur in all living organisms. The NADP(+)- dependent mitochondrial isocitrate dehydrogenase from pig heart has been crystallized from polyethylene glycol/sodium sulfate mixtures in the presence of Mg(2+) and isocitrate. The crystals belong to space group C2 with a = 137.0, b = 113.4, c = 65.0 A and beta = 98.5 degrees, and diffract to at least 2.4 A resolution. There are two protein monomers per asymmetric unit which are related by non-crystallographic twofold symmetry.

Defective glia induce neuronal apoptosis in the repo visual system of Drosophila.

The role of glia in suppressing neuronal cell death was investigated in the visual system of the Drosophila mutant, reversed polarity (repo). The repo locus encodes a glial-specific homeodomain protein expressed in the optic lobes. Here, we show that survival of the laminar neurons in the optic lobe depends on repo expression in the laminar glia, indicating that the laminar glia supply factors required for neuronal survival. The repo glia also underwent cell death, suggesting that the laminar neurons are required for survival of the glia or that repo expression is required to suppress an intrinsic cell suicide program. Subsequent to the laminar cell death, the retinal cells in the repo visual system also degenerated, indicating that the retinal cell death was due to retrograde degeneration.

repo encodes a glial-specific homeo domain protein required in the Drosophila nervous system.

We report the identification of a Drosophila locus, reversed polarity (repo). Weak repo alleles were viable but affected glia in the optic lobe, resulting in a reversal in polarity of the electrophysiological to light in the adult. Strong repo alleles caused defects in embryonic glia and resulted in embryonic lethality. Expression of repo appeared to be specific to glia throughout development. In the adult visual system, repo was expressed in laminal glia, medullar glia, and subretinal cells; in the embryo, repo was expressed in nearly all of the identified glia in the central and peripheral nervous systems except midline glia. The repo gene encoded a homeo domain protein suggesting that it might be a transcriptional regulator of genes required for glial development.

tramtrack is a transcriptional repressor required for cell fate determination in the Drosophila eye.

Cell fate determination in the Drosophila eye is mediated by inductive events between neighboring cells in the eye imaginal disc. These inductive signals lead to differential gene expression necessary for the elaboration of different cell types in the compound eye. Several putative transcription factors have been identified previously that may be required for expression of genes that specify cell fate in the compound eye. Repression of inappropriate gene expression may be as important as transcriptional activation in the determination of cell fate. We report the identification of a mutation in the Drosophila tramtrack (ttk) locus that is required for cell fate determination in the compound eye. ttk is expressed as two proteins, p69 and p88, shown previously to bind to the regulatory regions of several segmentation genes. In ttk1, an allele missing the mRNA encoding p88, many ommatidia contained supernumerary R7 cells and decreased numbers of R1-R6 cells. ttk1e11, which appears to disrupt both Ttk proteins, was characterized by early embryonic arrest as well as transformation of ommatidial cells into nonommatidial cell types in mosaic flies. Consistent with previous proposals that the Ttk proteins are transcriptional repressors of segmentation genes, we detected ectopic or increased expression of the segment polarity gene engrailed in several ttk1 larval tissues. We propose that p69 is required to repress expression of genes that are incompatible with development of photoreceptor cell fates, whereas p88 appears to be required to repress genes that promote the R7 cell fate.

Characterization of a [3H]-5-hydroxytryptamine binding site in rabbit caudate nucleus that differs from the 5-HT1A, 5-HT1B, 5-HT1C and 5-HT1D subtypes.

[3H]5-HT binding sites were analyzed in membranes prepared from the rabbit caudate nucleus (CN). [3H]5-HT labeled both 5-HT1A and 5-HT1C recognition sites, defined by nanomolar affinity for 8-OH-DPAT and mesulergine respectively; however, these represented only a fraction of total specific [3H]5-HT binding. Saturation experiments of [3H]5-HT binding in the presence of 100 nM 8-OH-DPAT and 100 nM mesulergine to block 5-HT1A and 5-HT1C sites revealed that non-5-HT1A/non-5-HT1C sites represented about 60% of the total 5-HT1 sites and that they exhibited saturable, high affinity, and homogeneous binding. The pharmacological profile of the non-5-HT1A/non-5-HT1C sites (designated 5-HT1R) also differed from that of 5-HT1B and 5-HT2 sites, but was similar to that of the 5-HT1D site. However, significant differences existed between the 5-HT1D and 5-HT1R sites for their Ki values for spiperone, spirilene (an analog of spiperone), metergoline, and methiothepin. The study of modulatory agents (calcium and GTP) also showed differences between the 5-HT1R and 5-HT1D sites. For example, the effects of GTP on agonist binding to the 5-HT1R sites were less than on the 5-HT1D sites in bovine caudate. In addition, calcium enhanced the effects of GTP on the 5-HT1R sites, whereas calcium inhibited the GTP effect on the 5-HT1D sites. The present findings demonstrate the presence of a high-affinity [3H]5-HT binding site in rabbit CN, designated 5-HT1R, that is different from previously defined 5-HT1A, 5-HT1B, 5-HT1C, 5-HT1D, and 5-HT2 sites.