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Intracellular calcium (Ca.sup.2+) signaling regulates many cellular functions, including cell proliferation and migration, in both normal cells and cancer cells. Store-operated Ca.sup.2+ entry (SOCE) is a major mechanism by which Ca.sup.2+ is imported from the extracellular space to the intracellular space, especially in nonexcitable cells. Store-operated Ca.sup.2+ entry (SOCE) is also a receptor-regulated Ca.sup.2+ entry pathway that maintains Ca.sup.2+ homeostasis by sensing reduced Ca.sup.2+ levels in the endoplasmic reticulum (ER). In general, the activation of G protein-coupled receptors (GPCRs) or immunoreceptors, such as T-cell, B-cell and Fc receptors, results in the production of inositol 1,4,5-trisphosphate (IP.sub.3). IP.sub.3 binds to IP.sub.3 receptors located in the ER membrane. The, IP.sub.3 receptors in the ER membrane trigger a rapid and transient release of Ca.sup.2+ from the ER store. The resulting depletion of ER Ca.sup.2+ concentrations is sensed by the EF-hand motif of stromal interaction molecule (STIM), i.e., calcium sensor, which then translocates to the plasma membrane (PM). STIM interacts with Orai Ca.sup.2+ channel subunits (also known as CRACM1) on the PM, leading to Ca.sup.2+ influx from the extracellular space to increase intracellular Ca.sup.2+ concentrations. The physiological functions of Orai and STIM have been studied mainly with respect to their roles in the immune system. Based on numerous previous studies, Orai channels (Orai1, Orai2 and Orai3 channels) control Ca.sup.2+ release-activated Ca.sup.2+ (CRAC) currents and contribute to SOCE currents in other types of cells, including various cancer cells. There are many reports that Orai1 is involved in cell proliferation, migration, metastasis, apoptosis and epithelial-mesenchymal transition (EMT) in various cancers. We previously found that Orai1 plays important roles in cell apoptosis and migration in melanoma. Recently, we reported novel evidence of Orai1 in human oral squamous cell carcinoma (OSCC) cells and human cardiac fibroblasts (HCFs). In this review, we present multiple physiological functions of Orai1 in various cancer cells and cardiac fibroblasts, including our findings. Keywords: Calcium, Orai1, EP4, Store-operated-calcium entry (SOCE), Cancer, Cardiac fibroblast, Author(s): Masanari Umemura[sup.1], Rina Nakakaji[sup.1] and Yoshihiro Ishikawa[sup.1] Introduction Calcium (Ca[sup.2+]) plays important roles as a second messenger in almost all cell types. Ca[sup.2+] release-activated Ca[sup.2+] (CRAC) channels mediate a [...]

Abstract. Barnacles permanently adhere to nearly any inert substrate using proteinaceous glue. The glue consists of at least ten major proteins, some of which have been isolated and sequenced. Questions [...]

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2010.12.001 Byline: Edward L. Huttlin (1), Mark P. Jedrychowski (1), Joshua E. Elias (1), Tapasree Goswami (1), Ramin Rad (2), Sean A. Beausoleil (1), Judit Villen (1), Wilhelm Haas (1), Mathew E. Sowa (1)(3), Steven P. Gygi (1)(2) Abstract: Although most tissues in an organism are genetically identical, the biochemistry of each is optimized to fulfill its unique physiological roles, with important consequences for human health and disease. Each tissue's unique physiology requires tightly regulated gene and protein expression coordinated by specialized, phosphorylation-dependent intracellular signaling. To better understand the role of phosphorylation in maintenance of physiological differences among tissues, we performed proteomic and phosphoproteomic characterizations of nine mouse tissues. We identified 12,039 proteins, including 6296 phosphoproteins harboring nearly 36,000 phosphorylation sites. Comparing protein abundances and phosphorylation levels revealed specialized, interconnected phosphorylation networks within each tissue while suggesting that many proteins are regulated by phosphorylation independently of their expression. Our data suggest that the 'typical' phosphoprotein is widely expressed yet displays variable, often tissue-specific phosphorylation that tunes protein activity to the specific needs of each tissue. We offer this dataset as an online resource for the biological research community. Author Affiliation: (1) Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA (2) Taplin Biological Mass Spectrometry Facility, Harvard Medical School, Boston, MA 02115, USA (3) Department of Pathology, Harvard Medical School, Boston, MA 02115, USA Article History: Received 16 June 2010; Revised 8 September 2010; Accepted 1 December 2010 Article Note: (miscellaneous) Published: December 23, 2010

Our group has previously shown that vasoconstrictors increase net actin polymerization in differentiated vascular smooth muscle cells (dVSMC) and that increased actin polymerization is linked to contractility of vascular tissue (Kim et al., Am J Physiol Cell Physiol 295: C768-778, 2008). However, the underlying mechanisms are largely unknown. Here, we evaluated the possible functions of the Ena/ vasodilator-stimulated phosphoprotein (VASP) family of actin filament elongation factors in dVSMC. Inhibition of actin filament elongation by cytochalasin D decreases contractility without changing myosin light-chain phosphorylation levels, suggesting that actin filament elongation is necessary for dVSM contraction. VASP is the only Ena/VASP protein highly expressed in aorta tissues, and VASP knockdown decreased smooth muscle contractility. VASP partially colocalizes with [alpha]-actinin and vinculin in dVSMC. Profilin, known to associate with G actin and VASP, also colocalizes with [alpha]-actinin and vinculin, potentially identifying the dense bodies and the adhesion plaques as hot spots of actin polymerization. The EVH1 domain of Ena/VASP is known to target these proteins to their sites of action. Introduction of an expressed EVH1 domain as a dominant negative inhibits stimulus-induced increases in actin polymerization. VASP phosphorylation, known to inhibit actin polymerization, is decreased during phenylephrine stimulation in dVSMC. We also directly visualized, for the first time, rhodamine-labeled actin incorporation in dVSMC and identified hot spots of actin polymerization in the cell cortex that colocalize with VASP. These results indicate a role for VASP in actin filament assembly, specifically at the cell cortex, that modulates contractility in dVSMC. Ena/VASP; EVH1; phosphorylation; profilin doi:10.1152/ajpcell.00431.2009

Vasodilator-stimulated phosphoprotein (VASP) is a major substrate for cyclic nucleotide-dependent kinases that has been implicated in cardiac pathology, yet many aspects of VASP's molecular regulation in cardiomyocytes are incompletely understood. In these studies, we explored the role of VASP, both in signaling pathways in isolated murine myocytes, as well as in a model of cardiac hypertrophy in [VASP.sup.null] mice. We found that the [beta]-adrenergic agonist isoproterenol promotes the rapid and reversible phosphorylation of VASP at Ser157 and Ser239. Forskolin and the cAMP analog 8-(4-chlorophenylthio)-cAMP promote a similar pattern of VASP phosphorylation at both sites. The effects of isoproterenol are blocked by atenolol and by compound H-89, an inhibitor of the cAMP-dependent protein kinase. By contrast, phosphorylation of VASP only at Ser239 is seen following activation of particulate guanylate cyclase by atrial natriuretic peptide, or following activation of soluble guanylate cyclase by sodium nitroprusside, or following treatment of myocytes with cGMP analog. We found that basal and isoproterenol-induced VASP phosphorylation is entirely unchanged in cardiomyocytes isolated from either endothelial or neuronal nitric oxide synthase knockout mice. In cardiomyocytes isolated from diabetic mice, only basal VASP phosphorylation is increased, whereas, in cells isolated from mice subjected to ascending aortic constriction (AAC), we found a significant increase in basal VASP expression, along with an increase in VASP phosphorylation, compared with cardiac myocytes isolated from sham-operated mice. Moreover, there is further increase in VASP phosphorylation in cells isolated from hypertrophic hearts following isoproterenol treatment. Finally, we found that [VASP.sup.null] mice subjected to transverse aortic constriction develop cardiac hypertrophy with a pattern similar to [VASP.sup.+/+] mice. Our findings establish differential receptor-modulated regulation of VASP phosphorylation in cardiomyocytes by cyclic nucleotides. Furthermore, these studies demonstrate for the first time that VASP expression is upregulated in hypertrophied heart. signal transduction; endothelial nitric oxide synthase; neuronal nitric oxide synthase; phosphoprotein doi: 10.1152/ajpheart.00595.2009.

Human leukocyte antigen (HLA) class I molecules present a variety of posttranslationally modified epitopes at the cell surface, although the consequences of such presentation remain largely unclear. Phosphorylation plays a critical cellular role, and deregulation in phosphate metabolism is associated with disease, including autoimmunity and tumor immunity. We have solved the high-resolution structures of 3 HLA A2-restricted phosphopeptides associated with tumor immunity and compared them with the structures of their nonphosphorylated counterparts. Phosphorylation of the epitope was observed to affect the structure and mobility of the bound epitope. In addition, the phosphoamino acid stabilized the HLA peptide complex in an epitope-specific manner and was observed to exhibit discrete flexibility within the antigen-binding cleft. Collectively, our data suggest that phosphorylation generates neoepitopes that represent demanding targets for T-cell receptor ligation. These findings provide insights into the mode of phosphopeptide presentation by HLA as well as providing a platform for the rational design of a generation of posttranslationally modified tumor vaccines. antigen presentation | HLA | phosphopeptide | T cells | X-ray crystallography

Increased pulmonary endothelial cGMP was shown to prevent endothelial barrier dysfunction through activation of protein kinase G ([PKG.sub.I]). Vasodilator-stimulated phosphoprotein (VASP) has been hypothesized to mediate [PKG.sub.I] barrier protection because VASP is a cytoskeletal phosphorylation target of [PKG.sub.I] expressed in cell-cell junctions. Unphosphorylated VASP was proposed to increase paracellular permeability through actin polymerization and stress fiber bundling, a process inhibited by [PKG.sub.I]-mediated phosphorylation of [Ser.sup.157] and [Ser.sup.239.] To test this hypothesis, we examined the role of VASP in the transient barrier dysfunction caused by [H.sub.2][O.sub.2] in human pulmonary artery endothelial cell (HPAEC) monolayers studied without and with [PKG.sub.I] expression introduced by adenoviral infection (Ad.PKG). In the absence of [PKG.sub.I] expression, [H.sub.2][O.sub.2] (100-250 [micro]M) caused a transient increased permeability and [pSer.sup.157]-VASP formation that were both attenuated by protein kinase C inhibition. Potentiation of VASP [Ser.sup.157] phosphorylation by either phosphatase 2B inhibition with cyclosporin or protein kinase A activation with forskolin prolonged, rather than inhibited, the increased permeability caused by [H.sub.2][O.sub.2]. With Ad.PKG infection, inhibition of VASP expression with small interfering RNA exacerbated [H.sub.2] [O.sub.2]-induced barrier dysfunction but had no effect on cGMP-mediated barrier protection. In addition, expression of a Ser-double phosphomimetic mutant VASP failed to reproduce the protective effects of activated [PKG.sub.I]. Finally, expression of a Ser-double phosphorylation-resistant VASP failed to interfere with the ability of cGMP/[PKG.sub.r] to attenuate [H.sub.2][O.sub.2]-induced disruption of VE-cadherin homotypic binding. Our results suggest that VASP phosphorylation does not explain the protective effect of cGMP/[PKG.sub.I] on [H.sub.2][O.sub.2]-induced endothelial barrier dysfunction in HPAEC. protein kinase G; cAMP; protein kinase A; small interfering RNA; [H.sub.2][O.sub.2]

We tested the hypotheses that the NO-cGMP-PKG pathway mediates inhibition of the store-operated cation channel (SOC) in human glomerular mesangial cells (HMC) and that TRPC4, a molecular component of SOC in HMC, is associated with PKG-phosphorylated vasodilator-stimulated phosphoprotein (VASP). Using fura 2 ratiometry, we measured intracellular [Ca.sup.2+] concentration [[[Ca.sup.2+]].sub.i] to determine whether sodium nitroprusside (SNP), an NO donor, and 8-Br-cGMP affected SOC-TRPC4 via PKG. We found that the SOC response in HMC was attenuated in the presence of 100 [micro]M SNP, an NO donor, or 100 [micro]M 8-Br-cGMP. Addition of DT-3 (2.5 [micro]M), a specific PKG-1[alpha] inhibitor, reversed the effects of 8-Br-cGMP on the SOC response. Application of 100 [micro]M cAMP did not significantly inhibit the SOC response. RT-PCR and Western blotting revealed PKG-1[alpha] transcript and protein in HMC. Immunocytochemical analysis localized PKG-1[alpha] to the cytoplasm and plasma membrane of HMC. Previous studies have shown that PKG-mediated phosphorylation of VASP attenuates cellular [Ca.sup.2+] entry, resulting in altered growth and proliferation. Therefore, we used Western blotting and immunocytochemistry to determine whether PKG-phosphorylated VASP associates with TRPC4. Western blot analysis revealed that 8-Br-cGMP enhanced the phosphorylation of VASP at serine 239 (Ser239), a known PKG phosphorylation site, in HMC within 5 min. Coimmunoprecipitation and coimmunostaining showed that P-Ser239-VASP associated with TRPC4. However, VASP that was unphosphorylated at Ser239 was not associated with TRPC4. These results indicate that VASP has a role in the NO/PKG1[alpha]-mediated inhibition of the TRPC4-SOC response in HMC. cGMP; SOC; nitric oxide; SNP

Many cellular processes are regulated by reversible protein phosphorylation, and the ability to broadly identify and quantify phosphoproteins from proteomes would provide a basis for gaining a better understanding of these dynamic cellular processes. However, such a sensitive, efficient, and global method capable of addressing the phosphoproteome has yet to be developed. Here we describe an improved stable-isotope labeling method using a phosphoprotein isotope-coded solid-phase tag (PhIST) for isolating and measuring the relative abundances of phosphorylated peptides from complex peptide mixtures resulting from the enzymatic digestion of extracted proteins. The PhIST approach is an extension of the previously reported phosphoprotein isotope-coded affinity tag (PhIAT) approach developed by our laboratory, (1,2) where phosphoseryl and phosphothreonyl residues were derivatized by hydroxide ion-mediated [beta]-elimination followed by the Michael addition of 1,2-ethanedithiol (EDT). Instead of using the biotin affinity tag, peptides containing the EDT moiety were captured and labeled in one step using isotope-coded solid-phase reagents containing either light ([sup.12][C.sub.6], [sup.14]N) or heavy ([sup.13][C.sub.6], [sub.15]N) stable isotopes. The captured peptides labeled with the isotope-coded tags were released from the solid-phase support by UV photocleavage and analyzed by capillary liquid chromatography--tandem mass spectrometry. The efficiency and sensitivity of the PhIST labeling approach for identification of phosphopeptides from mixtures were determined using casein proteins. Its utility for proteomic applications was demonstrated by the labeling of soluble phosphoproteins from a human breast cancer cell line.

The function of vasodilator-stimulated phosphoprotein (VASP) in motility is analyzed using a biomimetic motility assay in which ActA-coated microspheres propel themselves in a medium containing actin, the Arp2/3 complex, and three regulatory proteins in the absence or presence of VASP. Propulsion is linked to cycles of filament barbed end attachment-branching-detachment-growth in which the ActA-activated Arp2/3 complex incorporates at the junctions of branched filaments. VASP increases the velocity of beads. VASP increases branch spacing of filaments in the actin tail, as it does in lamellipodia in living cells. The effect of VASP on branch spacing of Arp2/3-induced branched actin arrays is opposed to the effect of capping proteins. However, VASP does not compete with capping proteins for binding barbed ends of actin filaments. VASP enhances branched actin polymerization only when ActA is immobilized on beads or on Listeria. VASP increases the rate of dissociation of the branch junction from immobilized ActA, which is the rate-limiting step in the catalytic cycle of site-directed filament branching.

Amyotrophic lateral sclerosis (ALS) is a human neurological disorder that results in progressive weakness and, ultimately, death. Although mutations in the SOD1 gene are found occasionally in ALS patients, there is no evidence of a heritable trait in the vast majority of ALS patients. Furthermore, variability is observed in the clinical presentation of ALS, raising the possibility that ALS is a heterogeneous disorder. In this article, we propose that several distinct initiating factors for ALS, including mutations in SOD1, trigger a common cascade of 'downstream' processes that result in neuronal death. Several studies demonstrate that abnormal expression or activities of protein kinases are common in ALS, and we hypothesize that some of these changes contribute to neuronal death. Phosphorylation of neuron-specific substrates can alter the binding and physiological properties of the substrate and might lead to neurotoxicity.

A filopodium protrudes by elongation of bundled actin filaments in its core. However, the mechanism of filopodia initiation remains unknown. Using live-cell imaging with GFP-tagged proteins and correlative electron microscopy, we performed a kinetic-structural analysis of filopodial initiation in B16F1 melanoma cells. Filopodial bundles arose not by a specific nucleation event, but by reorganization of the lamellipodial dendritic network analogous to fusion of established filopodia but occurring at the level of individual filaments. Subsets of independently nucleated lamellipodial filaments elongated and gradually associated with each other at their barbed ends, leading to formation of cone-shaped structures that we term [LAMBDA]-precursors. An early marker of initiation was the gradual coalescence of GFP-vasodilator-stimulated phosphoprotein (GFP-VASP) fluorescence at the leading edge into discrete foci. The GFP-VASP foci were associated with [LAMBDA]-precursors, whereas Arp2/3 was not. Subsequent recruitment of fascin to the clustered barbed ends of [LAMBDA]-precursors initiated filament bundling and completed formation of the nascent filopodium. We propose a convergent elongation model of filopodia initiation, stipulating that filaments within the lamellipodial dendritic network acquire privileged status by binding a set of molecules (including VASP) to their barbed ends, which protect them from capping and mediate association of barbed ends with each other.

It has been demonstrated previously that both the cystic fibrosis transmembrane conductance regulator (CFTR) and [[beta].sub.2] adrenergic receptor ([[beta].sub.2]AR) can bind ezrin/radixin/moesin-binding phosphoprotein 50 (EBP50, also referred to as NHERF) through their PDZ motifs. Here, we show that [[beta].sub.2] is the major adrenergic receptor isoform expressed in airway epithelia and that it colocalizes with CFTR at the apical membrane. [[beta].sub.2]AR stimulation increases CFTR activity, in airway epithelial cells, that is glybenclamide sensitive. Deletion of the PDZ motif from CFTR uncouples the channel from the receptor both physically and functionally. This uncoupling is specific to the [[beta].sub.2]AR receptor and does not affect CFTR coupling to other receptors (e.g., adenosine receptor pathway). Biochemical studies demonstrate the existence of a macromolecular complex involving CFTR-EBP50-[[beta].sub.2]AR through PDZ-based interactions. Assembly of the complex is regulated by PKA-dependent phosphorylation. Deleting the regulatory domain of CFTR abolishes PKA regulation of complex assembly. This report summarizes a macromolecular signaling complex involving CFTR, the implications of which may be relevant to CFTR-dysfunction diseases.

The membrane phosphoprotein GAP-43 is involved in axon growth and synaptic plasticity. In PC12 pheochromocytoma cells, induction of a neuronal phenotype by nerve growth factor (NGF) is accompanied by a marked increase in GAP-43 levels. NGF regulates GAP-43 expression by altering the half-life of its mRNA. We report here that the phosphoprotein ARPP-19 mediates this regulation. In an NGF-dependent manner, ARPP-19 bound to a region in the 3' end of GAP-43 mRNA previously found to be important for regulating the half-life of the mRNA. Overexpression of wild-type ARPP-19 in PC12 cells increased the NGF-dependent expression of a reporter construct linked to the critical 3' region of GAP-43 mRNA. Mutation of serine 104, the site of phosphorylation by protein kinase A in ARPP-19, to either alanine or aspartate abolished this regulation in PC12 cells. These findings demonstrate that ARPP-19 is an important link between NGF signaling and post-transcriptional control of neuronal gene expression.

Serotonin is implicated in the regulation of complex sensory, motor, affective, and cognitive functions. However, the biochemical mechanisms whereby this neurotransmitter exerts its actions remain largely unknown. DARPP-32 (dopamine- and cAMP-regulated phosphoprotein of molecular weight 32,000) is a phosphoprotein that has primarily been characterized in relation to dopaminergic neurotransmission. Here we report that serotonin regulates DARPP-32 phosphorylation both in vitro and in vivo. Stimulation of 5-hydroxytryptamine (5-H[T.sub.4] and 5-H[T.sub.6]) receptors causes an increased phosphorylation state at [Thr.sup.34]-DARPP-32, the protein kinase A site, and a decreased phosphorylation state at [Thr.sup.75]-DARPP-32, the cyclindependent kinase 5 site. Furthermore, stimulation of 5-H[T.sub.2] receptors increases the phosphorylation state of [Ser.sup.137]-DARPP-32, the casein kinase-1 site. Behavioral and gene transcriptional effects induced by compounds that selectively release serotonin were greatly reduced in DARPP-32 knockout mice. Our data indicate that DARPP-32 is essential not only for dopaminergic but also for serotonergic neurotransmission.

The use of a phosphoprotein isotope-coded affinity tag (PhIAT), which employs differential isotopic labeling and biotinylation, has been shown capable of enriching and identifying mixtures of low-abundance phosphopeptides. A denatured solution of [beta]-casein was labeled using the PhIAT method, and after proteolytic digestion, the labeled peptides were isolated using immobilized avidin. The recovered peptides were separated by capillary reversed-phase liquid chromatography and identified by tandem mass spectrometry. PhIAT-labeled peptides corresponding to known O-phosphorylated peptides from [beta]-casein were identified along with the phosphorylated peptides from [[alpha].sub.S1]-casein and [[alpha].sub.S2]-casein, known low-level (

Salt dependence of binding of tyrosyl-phosphorylated peptide binding to the tandem SH2 domain of the Syk kinase and the single SH2 domain of the Src kinase have been studied. The findings show that electrostatic interactions have important roles in mediating pY recognition and binding and also in the specificity of the interactions between tyrosyl phosphopeptides and SH2 domains.

There is strong evidence that growth-associated protein (GAP-43), a protein found only in the nervous system, regulates the response of neurons to axonal guidance signals. However, its role in complex spatial patterning in cerebral cortex has not been explored. We show that mice lacking GAP-43 expression (-/-) fail to establish the ordered whisker representation (barrel array) normally found in layer IV of rodent primary somatosensory cortex. Thalamocortical afferents to -/- cortex form irregular patches in layer IV within a poorly defined cortical field, which varies between hemispheres, rather than the stereotypic, whisker-specific, segregated map seen in normal animals. Furthermore, many thalamocortical afferents project abnormally to widely separated cortical targets. Taken together, our findings indicate a loss of identifiable whisker territories in the GAP-43 -/- mouse cortex. Here, we present a disrupted somatotopic map phenotype in cortex, in clear contrast to the blurring of boundaries within an ordered whisker map in other barrelless mutants. Our results indicate that GAP-43 expression is critical for the normal establishment of ordered topography in barrel cortex.

The role of nucleolar phosphoprotein Nopp140 as a molecular link between the nucleolus and coiled bodies has been studied through mutational analysis, transient transfections and the yeast two-hybrid system. Coiled bodies are small nuclear organelles that are abundant in small nuclear RNAs. Results show that exogenous expression of the conserved carboxy terminus of Nopp140 has a negative effect on endogenous Nopp140. It scatters the coiled bodies and drives antigens common to the coiled bodies out of the nucleolus. Nopp140 not only binds with nucleolar proteins but also with p80 coilin.

A study was conducted to characterize the isolation, microsequencing and cloning of a 40 kDa phosphoprotein from rabbit gastric parietal cells. Sequential pronae/collagenase digestion was carried out to obtain parietal and chief cells from the gastric mucosae of New Zealand White rabbits. Results suggested that the distribution of pp40/lasp-1 mRNA supports a critical regulatory function in other secretory cell types.

Long-term potentiation (LTP) is an increase in synaptic responsiveness thought to be involved in mammalian learning and memory. The localization (presynaptic and/or postsynaptic) of changes underlying LTP has been difficult to resolve with current electrophysiological techniques. Using a biochemical approach, we have addressed this issue and attempted to identify specific molecular mechanisms that may underlie LTP. We utilized a novel multiple-electrode stimulator to produce LTP in a substantial portion of the synapses in a hippocampal CA1 minislice and tested the effects of such stimulation on the presynaptic protein synapsin I. LTP-inducing stimulation produced a long-lasting 6-fold increase in the phosphorylation of synapsin I at its [Ca.sup.2+]/calmodulin-dependent protein kinase II (CaM kinase II) sites without affecting synapsin I levels. This effect was fully blocked by either the N-methyl-D-aspartate receptor antagonist D(-)-2-amino-5-phosphonopentanoic acid (APV) or the CaM kinase II inhibitor KN-62. Our results indicate that LTP expression is accompanied by persistent changes in presynaptic phosphorylation, and specifically that presynaptic CaM kinase II activity and synapsin I phosphorylation may be involved in LTP expression.

The role of ActA 639-amino acid transmembrane protein in the actin-based movement of Listeria monocytogenes was analyzed by utilizing bacterial cell lines with mutations in the ActA proline-rich repeats (PRR). The PRR motifs of ActA mediates the binding of the vasodilator-stimulated phosphoprotein (VASP) with ActA. Furthermore, actin-based motility of Listeria sp. is enhanced by the repeat motifs by increasing the recruitment of VASP to the bacterial surface.

The actin-driven migration of neuronal growth cones during axonal development is regulated by the murine protein, Mena, which exhibits a similar structure to the vasodilator-stimulated phosphoprotein. Mena promotes actin polymerization during axogenesis and induce the formation of F-actin-rich outgrowths in the fibroblasts. The actin-rich outgrowths the are produced by Mena reorganize actin polymerization for cell migration during axogenesis.

The nuclear phosphoprotein Fos gene expression in the ferret dorsal vagal complex and the area postrema has been found to be induced by intraduodenal hypertonic saline (IHS). Excitation of DVC after IHS treatment produced an emetic response that was accompanied by Fos expression. Vagal and splanchnic nerve section abolished the emetic response, suggesting that excitation of DVC is dependent on vagal afferents.

The cytosolic phosphoprotein stathmin is necessary for the differentiation of rat pheochromocytoma PC12 cells into sympathetic-like neurons induced by the nerve growth factor (NGF). The removal of stathmin by antisense oligonucleotides suppresses NGF-induced differentiation, but the expression of NGF-activated genes is unaffected. Stathmin is a substrate for the mitogen-activated protein kinase (MAPK) as blockage of MAPK suppresses the NGF-induced phosphorylation of stathmin.

Dynamin I is a nerve terminal phosphoprotein with intrinsic guanosine triphosphatase (GTPase) activity that is required for endocytosis. Upon depolarization and synaptic vesicle recycling, dynamin I undergoes a rapid dephosphorylation. [...]

A steady-state agarose gel system facilitates the analysis of the role of bone sialoprotein (BSP) and osteopontin in affecting the mineralization of bone through nucleation of carbonated hydroxyapatite (HA) crystals. BSP is identified as the phosphoprotein responsible for actuating the formation of HA. BSP and osteopontin, the essential phosphoproteins of mammalian bone, comprises tyrosine sulfates, acidic amino acids and an arginine glycine-asparagine cell adhering sequence. Peak nucleation of HA is observed at BSP concentrations of 2 microgram per multiliter.

The growth-associated protein GAP-43 is a neuron-specific phosphoprotein whose transcription is induced by nerve growth factor (NGF). The effects of NGF on the steady-state levels and rates of degradation of GAP-43 mRNA were investigated. The results showed that GAP-43 mRNAs are regulated and maintained in a translation-independent but protein kinase C-dependent manner. These results indicate that GAP-43 mRNA is stabilized through a novel mechanism.

The phosphorylation and dephosphorylation of the beta and gamma chains of the high-affinity IgE receptor upon engagement and disengagement with IgE was investigated. The study resulted in the identification of five distinct phosphoproteins comprising the IgE receptor. These phosphoproteins were proven to be phosphorylated and dephosphorylated in conjunction with IgE and IgE receptor engagement and disengagement.

Mutations in the gene coding for p53 phosphoprotein are those most commonly associated with human cancers. Previous studies have indicated that p53 is involved in the regulation of the cell cycle. An investigation was conducted to determine the precise phase within the cell cycle wherein p53 functions. The results showed that growth suppression mediated by p53 occurs before or near the R-point of the G1 phase of the cell cycle. Among the products of immediate-early genes, p53 was involved in the repression of B-MYC and RNA polymerase-alpha.

A live mouse neonatal calvarial bone organ cultures stimulated by parathyroid hormone (PTH) to undergo bone resorption was used to study the BSP mediated bone resorption inhibition. The study results provide direct evidence for the involvement of covalently bound phosphates on bone sialoprotein (BSP) in receptor mediated 'outside-in' signaling via transmembrane tyrosine phosphorylation with concurrent effects on downstream gene expressions.

Results reveal that Polo-box domain in Polo-like kinases recognize phosphoserine/threonine containing motiffs and the phosphopeptides bind to a positively charged cleft in the Polo-box interface. Data indicate that phosphopeptide binding to Polo-box domain stimulates kinase activity in Polo-like kinases, which undergo conformational switching for regulatory function.