Regulation of the p21-activated kinase (PAK) by a human Gβ-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 β-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 κ B signaling pathways. Our results demonstrate that hPIP1 is a negative regulator of PAK and PAK signaling pathways.

Nitric oxide and salicylic acid signaling in plant defense

Salicylic acid (SA) plays a critical signaling role in the activation of plant defense responses after pathogen attack. We have identified several potential components of the SA signaling pathway, including (i) the H2O2-scavenging enzymes catalase and ascorbate peroxidase, (ii) a high affinity SA-binding protein (SABP2), (iii) a SA-inducible protein kinase (SIPK), (iv) NPR1, an ankyrin repeat-containing protein that exhibits limited homology to IκBα and is required for SA signaling, and (v) members of the TGA/OBF family of bZIP transcription factors. These bZIP factors physically interact with NPR1 and bind the SA-responsive element in promoters of several defense genes, such as the pathogenesis-related 1 gene (PR-1). Recent studies have demonstrated that nitric oxide (NO) is another signal that activates defense responses after pathogen attack. NO has been shown to play a critical role in the activation of innate immune and inflammatory responses in animals. Increases in NO synthase (NOS)-like activity occurred in resistant but not susceptible tobacco after infection with tobacco mosaic virus. Here we demonstrate that this increase in activity participates in PR-1 gene induction. Two signaling molecules, cGMP and cyclic ADP ribose (cADPR), which function downstream of NO in animals, also appear to mediate plant defense gene activation (e.g., PR-1). Additionally, NO may activate PR-1 expression via an NO-dependent, cADPR-independent pathway. Several targets of NO in animals, including guanylate cyclase, aconitase, and mitogen-activated protein kinases (e.g., SIPK), are also modulated by NO in plants. Thus, at least portions of NO signaling pathways appear to be shared between plants and animals.

Suramin inhibits initiation of defense signaling by systemin, chitosan, and a β-glucan elicitor in suspension-cultured Lycopersicon peruvianum cells

Systemin-mediated defense signaling in tomato (Lycopersicon esculentum) plants is analogous to the cytokine-mediated inflammatory response in animals. Herein, we report that the initiation of defense signaling in suspension-cultured cells of Lycopersicon peruvianum by the peptide systemin, as well as by chitosan and β-glucan elicitor from Phytophtora megasperma, is inhibited by the polysulfonated naphtylurea compound suramin, a known inhibitor of cytokine and growth factor receptor interactions in animal cells. Using a radioreceptor assay, we show that suramin interfered with the binding of the systemin analog 125I-Tyr-2,Ala-15-systemin to the systemin receptor with an IC50 of 160 μM. Additionally, labeling of the systemin receptor with a photoaffinity analog of systemin was inhibited in the presence of suramin. Receptor-mediated tyrosine phosphorylation of a 48-kDa mitogen-activated protein kinase and alkalinization of the medium of suspension-cultured cells in response to systemin and carbohydrate elicitors were also inhibited by suramin. The inhibition of medium alkalinization by suramin was reversible in the presence of high concentrations of systemin and carbohydrate elicitors. Calyculin A and erythrosin B, intracellular inhibitors of phosphatases and plasma membrane proton ATPases, respectively, both induce medium alkalinization, but neither response was inhibited by suramin. The polysulfonated compound heparin did not inhibit systemin-induced medium alkalinization. NF 007, a suramin derivative, induced medium alkalinization, indicating that neither NF 007 nor heparin interact with elicitor receptors like suramin. The data indicate that cell-surface receptors in plants show some common structural features with animal cytokine and growth factor receptors that can interact with suramin to interfere with ligand binding.

Signaling in plants

Higher plants are sessile organisms that perceive environmental cues such as light and chemical signals and respond by changing their morphologies. Signaling pathways utilize a complex network of interactions to orchestrate biochemical and physiological responses such as flowering, fruit ripening, germination, photosynthetic regulation, and shoot or root development. In this session, the mechanisms of signaling systems that trigger plant responses to light and to the gaseous hormone, ethylene, were discussed. These signals are first sensed by a receptor and transmitted to the nucleus by a complex network. A signal may be transmitted to the nucleus by any of several systems including GTP binding proteins (G proteins), which change activity upon GTP binding; protein kinase cascades, which sequentially phosphorylate and activate a series of proteins; and membrane ion channels, which change ionic characteristics of the cells. The signal is manifested in the nucleus as a change in the activity of DNA-binding proteins, which are transcription factors that specifically interact and modulate the regulatory regions of genes. Thus, detection of an environmental signal is transmitted through a transduction pathway, and changes in transcription factor activity may coordinate changes in the expression of a portfolio of genes to direct new developmental programs.

Sonic hedgehog protein signals not as a hydrolytic enzyme but as an apparent ligand for Patched

The amino-terminal signaling domain of the Sonic hedgehog secreted protein (Shh-N), which derives from the Shh precursor through an autoprocessing reaction mediated by the carboxyl-terminal domain, executes multiple functions in embryonic tissue patterning, including induction of ventral and suppression of dorsal cell types in the developing neural tube. An apparent catalytic site within Shh-N is suggested by structural homology to a bacterial carboxypeptidase. We demonstrate here that alteration of residues presumed to be critical for a hydrolytic activity does not cause a loss of inductive activity, thus ruling out catalysis by Shh-N as a requirement for signaling. We favor the alternative, that Shh-N functions primarily as a ligand for the putative receptor Patched (Ptc). This possibility is supported by new evidence for direct binding of Shh-N to Ptc and by a strong correlation between the affinity of Ptc-binding and the signaling potency of Shh-N protein variants carrying alterations of conserved residues in a particular region of the protein surface. These results together suggest that direct Shh-N binding to Ptc is a critical event in transduction of the Shh-N signal.

CD95/Fas induces cleavage of the GrpL/Gads adaptor and desensitization of antigen receptor signaling

The balance between cell survival and cell death is critical for normal lymphoid development. This balance is maintained by signals through lymphocyte antigen receptors and death receptors such as CD95/Fas. In some cells, ligating the B cell antigen receptor can protect the cell from apoptosis induced by CD95. Here we report that ligation of CD95 inhibits antigen receptor-mediated signaling. Pretreating CD40-stimulated tonsillar B cells with anti-CD95 abolished B cell antigen receptor-mediated calcium mobilization. Furthermore, CD95 ligation led to the caspase-dependent inhibition of antigen receptor-induced calcium mobilization and to the activation of mitogen-activated protein kinase pathways in B and T cell lines. A target of CD95-mediated caspase 3-like activity early in the apoptotic process is the adaptor protein GrpL/Gads. GrpL constitutively interacts with SLP-76 via its C-terminal SH3 domain to regulate transcription factors such as NF-AT. Cleavage of GrpL removes the C-terminal SH3 domain so that it is no longer capable of recruiting SLP-76 to the membrane. Transfection of a truncated form of GrpL into Jurkat T cells blocked T cell antigen receptor-induced activation of NF-AT. These results suggest that CD95 signaling can desensitize antigen receptors, in part via cleavage of the GrpL adaptor.

PLS1, a gene encoding a tetraspanin-like protein, is required for penetration of rice leaf by the fungal pathogen Magnaporthe grisea

We describe in this study punchless, a nonpathogenic mutant from the rice blast fungus M. grisea, obtained by plasmid-mediated insertional mutagenesis. As do most fungal plant pathogens, M. grisea differentiates an infection structure specialized for host penetration called the appressorium. We show that punchless differentiates appressoria that fail to breach either the leaf epidermis or artificial membranes such as cellophane. Cytological analysis of punchless appressoria shows that they have a cellular structure, turgor, and glycogen content similar to those of wild type before penetration, but that they are unable to differentiate penetration pegs. The inactivated gene, PLS1, encodes a putative integral membrane protein of 225 aa (Pls1p). A functional Pls1p-green fluorescent protein fusion protein was detected only in appressoria and was localized in plasma membranes and vacuoles. Pls1p is structurally related to the tetraspanin family. In animals, these proteins are components of membrane signaling complexes controlling cell differentiation, motility, and adhesion. We conclude that PLS1 controls an appressorial function essential for the penetration of the fungus into host leaves.

SMART, a simple modular architecture research tool: Identification of signaling domains

Accurate multiple alignments of 86 domains that occur in signaling proteins have been constructed and used to provide a Web-based tool (SMART: simple modular architecture research tool) that allows rapid identification and annotation of signaling domain sequences. The majority of signaling proteins are multidomain in character with a considerable variety of domain combinations known. Comparison with established databases showed that 25% of our domain set could not be deduced from SwissProt and 41% could not be annotated by Pfam. SMART is able to determine the modular architectures of single sequences or genomes; application to the entire yeast genome revealed that at least 6.7% of its genes contain one or more signaling domains, approximately 350 greater than previously annotated. The process of constructing SMART predicted (i) novel domain homologues in unexpected locations such as band 4.1-homologous domains in focal adhesion kinases; (ii) previously unknown domain families, including a citron-homology domain; (iii) putative functions of domain families after identification of additional family members, for example, a ubiquitin-binding role for ubiquitin-associated domains (UBA); (iv) cellular roles for proteins, such predicted DEATH domains in netrin receptors further implicating these molecules in axonal guidance; (v) signaling domains in known disease genes such as SPRY domains in both marenostrin/pyrin and Midline 1; (vi) domains in unexpected phylogenetic contexts such as diacylglycerol kinase homologues in yeast and bacteria; and (vii) likely protein misclassifications exemplified by a predicted pleckstrin homology domain in a Candida albicans protein, previously described as an integrin.

Synaptic regulation of protein synthesis and the fragile X protein

Protein synthesis occurs in neuronal dendrites, often near synapses. Polyribosomal aggregates often appear in dendritic spines, particularly during development. Polyribosomal aggregates in spines increase during experience-dependent synaptogenesis, e.g., in rats in a complex environment. Some protein synthesis appears to be regulated directly by synaptic activity. We use “synaptoneurosomes,” a preparation highly enriched in pinched-off, resealed presynaptic processes attached to resealed postsynaptic processes that retain normal functions of neurotransmitter release, receptor activation, and various postsynaptic responses including signaling pathways and protein synthesis. We have found that, when synaptoneurosomes are stimulated with glutamate or group I metabotropic glutamate receptor agonists such as dihydroxyphenylglycine, mRNA is rapidly taken up into polyribosomal aggregates, and labeled methionine is incorporated into protein. One of the proteins synthesized is FMRP, the protein that is reduced or absent in fragile X mental retardation syndrome. FMRP has three RNA-binding domains and reportedly binds to a significant number of mRNAs. We have found that dihydroxyphenylglycine-activated protein synthesis in synaptoneurosomes is dramatically reduced in a knockout mouse model of fragile X syndrome, which cannot produce full-length FMRP, suggesting that FMRP is involved in or required for this process. Studies of autopsy samples from patients with fragile X syndrome have indicated that dendritic spines may fail to assume a normal mature size and shape and that there are more spines per unit dendrite length in the patient samples. Similar findings on spine size and shape have come from studies of the knockout mouse. Study of the development of the somatosensory cortical region containing the barrel-like cell arrangements that process whisker information suggests that normal dendritic regression is impaired in the knockout mouse. This finding suggests that FMRP may be required for the normal processes of maturation and elimination to occur in cerebral cortical development.

Carbon- and nitrogen-quality signaling to translation are mediated by distinct GATA-type transcription factors

The target of rapamycin (Tor) proteins sense nutrients and control transcription and translation relevant to cell growth. Treating cells with the immunosuppressant rapamycin leads to the intracellular formation of an Fpr1p-rapamycin-Tor ternary complex that in turn leads to translational down-regulation. A more rapid effect is a rich transcriptional response resembling that when cells are shifted from high- to low-quality carbon or nitrogen sources. This transcriptional response is partly mediated by the nutrient-sensitive transcription factors GLN3 and NIL1 (also named GAT1). Here, we show that these GATA-type transcription factors control transcriptional responses that mediate translation by several means. Four observations highlight upstream roles of GATA-type transcription factors in translation. In their absence, processes caused by rapamycin or poor nutrients are diminished: translation repression, eIF4G protein loss, transcriptional down-regulation of proteins involved in translation, and RNA polymerase I/III activity repression. The Tor proteins preferentially use Gln3p or Nil1p to down-regulate translation in response to low-quality nitrogen or carbon, respectively. Functional consideration of the genes regulated by Gln3p or Nil1p reveals the logic of this differential regulation. Besides integrating control of transcription and translation, these transcription factors constitute branches downstream of the multichannel Tor proteins that can be selectively modulated in response to distinct (carbon- and nitrogen-based) nutrient signals from the environment.

Phenserine regulates translation of β-amyloid precursor protein mRNA by a putative interleukin-1 responsive element, a target for drug development

The reduction in levels of the potentially toxic amyloid-β peptide (Aβ) has emerged as one of the most important therapeutic goals in Alzheimer's disease. Key targets for this goal are factors that affect the expression and processing of the Aβ precursor protein (βAPP). Earlier reports from our laboratory have shown that a novel cholinesterase inhibitor, phenserine, reduces βAPP levels in vivo. Herein, we studied the mechanism of phenserine's actions to define the regulatory elements in βAPP processing. Phenserine treatment resulted in decreased secretion of soluble βAPP and Aβ into the conditioned media of human neuroblastoma cells without cellular toxicity. The regulation of βAPP protein expression by phenserine was posttranscriptional as it suppressed βAPP protein expression without altering βAPP mRNA levels. However, phenserine's action was neither mediated through classical receptor signaling pathways, involving extracellular signal-regulated kinase or phosphatidylinositol 3-kinase activation, nor was it associated with the anticholinesterase activity of the drug. Furthermore, phenserine reduced expression of a chloramphenicol acetyltransferase reporter fused to the 5′-mRNA leader sequence of βAPP without altering expression of a control chloramphenicol acetyltransferase reporter. These studies suggest that phenserine reduces Aβ levels by regulating βAPP translation via the recently described iron regulatory element in the 5′-untranslated region of βAPP mRNA, which has been shown previously to be up-regulated in the presence of interleukin-1. This study identifies an approach for the regulation of βAPP expression that can result in a substantial reduction in the level of Aβ.

Control of Meristem Development by CLAVATA1 Receptor Kinase and Kinase-Associated Protein Phosphatase Interactions1

The CLAVATA1 (CLV1) gene encodes a putative receptor kinase required for the proper balance between cell proliferation and differentiation in Arabidopsis shoot and flower meristems. Impaired CLV1 signaling results in masses of undifferentiated cells at the shoot and floral meristems. Although many putative receptor kinases have been identified in plants, the mechanism of signal transduction mediated by plant receptor-like kinases is largely unknown. One potential effector of receptor kinase signaling is kinase-associated protein phosphatase (KAPP), a protein that binds to multiple plant receptor-like kinases in a phosphorylation-dependent manner. To examine a possible role for KAPP in CLV1-dependent plant development, the interaction of CLV1 and KAPP was investigated in vitro and in vivo. KAPP binds directly to autophosphorylated CLV1 in vitro and co-immunoprecipitates with CLV1 in plant extracts derived from meristematic tissue. Reduction of KAPP transcript accumulation in an intermediate clv1 mutant suppresses the mutant phenotype, and the degree of suppression is inversely correlated with KAPP mRNA levels. These data suggest that KAPP functions as a negative regulator of CLV1 signaling in plant development. This may represent a general model for the interaction of KAPP with receptor kinases.

Guard Cells Possess a Calcium-Dependent Protein Kinase That Phosphorylates the KAT1 Potassium Channel1

Increasing evidence suggests that changes in cytosolic Ca2+ levels and phosphorylation play important roles in the regulation of stomatal aperture and as ion transporters of guard cells. However, protein kinases responsible for Ca2+ signaling in guard cells remain to be identified. Using biochemical approaches, we have identified a Ca2+-dependent protein kinase with a calmodulin-like domain (CDPK) in guard cell protoplasts of Vicia faba. Both autophosphorylation and catalytic activity of CDPK are Ca2+ dependent. CDPK exhibits a Ca2+-induced electrophoretic mobility shift and its Ca2+-dependent catalytic activity can be inhibited by the calmodulin antagonists trifluoperazine and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide. Antibodies to soybean CDPKα cross-react with CDPK. Micromolar Ca2+ concentrations stimulate phosphorylation of several proteins from guard cells; cyclosporin A, a specific inhibitor of the Ca2+-dependent protein phosphatase calcineurin enhances the Ca2+-dependent phosphorylation of several soluble proteins. CDPK from guard cells phosphorylates the K+ channel KAT1 protein in a Ca2+-dependent manner. These results suggest that CDPK may be an important component of Ca2+ signaling in guard cells.

Mechanistic coupling of protease signaling and initiation of coagulation by tissue factor

The crucial role of cell signaling in hemostasis is clearly established by the action of the downstream coagulation protease thrombin that cleaves platelet-expressed G-protein-coupled protease activated receptors (PARs). Certain PARs are cleaved by the upstream coagulation proteases factor Xa (Xa) and the tissue factor (TF)–factor VIIa (VIIa) complex, but these enzymes are required at high nonphysiological concentrations and show limited recognition specificity for the scissile bond of target PARs. However, defining a physiological mechanism of PAR activation by upstream proteases is highly relevant because of the potent anti-inflammatory in vivo effects of inhibitors of the TF initiation complex. Activation of substrate factor X (X) by the TF–VIIa complex is here shown to produce enhanced cell signaling in comparison to the TF–VIIa complex alone, free Xa, or Xa that is generated in situ by the intrinsic activation complex. Macromolecular assembly of X into a ternary complex of TF–VIIa–X is required for proteolytic conversion to Xa, and product Xa remains transiently associated in a TF–VIIa–Xa complex. By trapping this complex with a unique inhibitor that preserves Xa activity, we directly show that Xa in this ternary complex efficiently activates PAR-1 and -2. These experiments support the concept that proinflammatory upstream coagulation protease signaling is mechanistically coupled and thus an integrated part of the TF–VIIa-initiated coagulation pathway, rather than a late event during excessive activation of coagulation and systemic generation of proteolytic activity.

The Caenorhabditis elegans hif-1 gene encodes a bHLH-PAS protein that is required for adaptation to hypoxia

Hypoxia-inducible factor, a heterodimeric transcription complex, regulates cellular and systemic responses to low oxygen levels (hypoxia) during normal mammalian development or tumor progression. Here, we present evidence that a similar complex mediates response to hypoxia in Caenorhabditis elegans. This complex consists of HIF-1 and AHA-1, which are encoded by C. elegans homologs of the hypoxia-inducible factor (HIF) α and β subunits, respectively. hif-1 mutants exhibit no severe defects under standard laboratory conditions, but they are unable to adapt to hypoxia. Although wild-type animals can survive and reproduce in 1% oxygen, the majority of hif-1-defective animals die in these conditions. We show that the expression of an HIF-1:green fluorescent protein fusion protein is induced by hypoxia and is subsequently reduced upon reoxygenation. Both hif-1 and aha-1 are expressed in most cell types, and the gene products can be coimmunoprecipitated. We conclude that the mechanisms of hypoxia signaling are likely conserved among metazoans. Additionally, we find that nuclear localization of AHA-1 is disrupted in an hif-1 mutant. This finding suggests that heterodimerization may be a prerequisite for efficient nuclear translocation of AHA-1.