PlantsP: a functional genomics database for plant phosphorylation

The PlantsP database is a curated database that combines information derived from sequences with experimental functional genomics information. PlantsP focuses on plant protein kinases and protein phosphatases. The database will specifically provide a resource for information on a collection of T-DNA insertion mutants (knockouts) in each protein kinase and phosphatase in Arabidopsis thaliana. PlantsP also provides a curated view of each protein that includes a comprehensive annotation of functionally related sequence motifs, sequence family definitions, alignments and phylogenetic trees, and descriptive information drawn directly from the literature. PlantsP is available at http://PlantsP.sdsc.edu.

BLNK mediates Syk-dependent Btk activation

Btk is a critical molecule in B cell antigen receptor (BCR)-coupled signaling, and its activity is regulated by Lyn and Syk. Although the molecular mechanism of Lyn-dependent Btk activation has been investigated, that of Syk-dependent Btk activation has remained unidentified. We have demonstrated that BLNK mediates Syk-dependent Btk activation. In a reconstitution cell system, coexpression of BLNK allows Syk to phosphorylate Btk on its tyrosine 551, leading to the enhancement of Btk activity. This phosphorylation depends on the interaction of Btk and BLNK by means of the Btk-Src homology 2 domain. The existence of such an activation mechanism is supported by the observation that the BCR-induced Btk phosphorylation and activation are significantly reduced in BLNK-deficient B cells as well as in Syk-deficient B cells. Although previous observations have identified the function of BLNK as the linker that integrates the action of Btk and Syk into downstream effectors such as phospholipase Cγ2, our present study indicates another function of BLNK that connects the activity of Syk to that of Btk.

Activity-dependent CREB phosphorylation: Convergence of a fast, sensitive calmodulin kinase pathway and a slow, less sensitive mitogen-activated protein kinase pathway

The cAMP-responsive element binding protein (CREB), a key regulator of gene expression, is activated by phosphorylation on Ser-133. Several different protein kinases possess the capability of driving this phosphorylation, making it a point of potential convergence for multiple intracellular signaling cascades. Previous work in neurons has indicated that physiologic synaptic stimulation recruits a fast calmodulin kinase IV (CaMKIV)-dependent pathway that dominates early signaling to CREB. Here we show in hippocampal neurons that the fast, CaMK-dependent pathway can be followed by a slower pathway that depends on Ras/mitogen-activated protein kinase (MAPK), along with CaMK. This pathway was blocked by dominant-negative Ras and was specifically recruited by depolarizations that produced strong intracellular Ca2+ transients. When both pathways were recruited, phosphorylated CREB (pCREB) formation was overwhelmingly dominated by the CaMK pathway between 0 and 10 min, and by the MAPK pathway at 60 min, whereas the two pathways acted in concert at 30 min. The Ca2+ signals that produced only rapid CaMK signaling to pCREB or both rapid CaMK and slow MAPK signaling deviated significantly for only ≈1 min, yet their differential impact on pCREB extended over a much longer period, between 20 and 60 min and beyond, which is of likely significance for gene expression. The CaMK-dependent MAPK pathway may inform the nucleus about stimulus amplitude. In contrast, the CaMKIV pathway may be well suited to conveying information on the precise timing of localized synaptic stimuli, befitting its greater speed and sensitivity, whereas the previously described calcineurin pathway may carry information about stimulus duration.

Mutant Caldesmon Lacking cdc2 Phosphorylation Sites Delays M-Phase Entry and Inhibits Cytokinesis

Caldesmon is phosphorylated by cdc2 kinase during mitosis, resulting in the dissociation of caldesmon from microfilaments. To understand the physiological significance of phosphorylation, we generated a caldesmon mutant replacing all seven cdc2 phosphorylation sites with Ala, and examined effects of expression of the caldesmon mutant on M-phase progression. We found that microinjection of mutant caldesmon effectively blocked early cell division of Xenopus embryos. Similar, though less effective, inhibition of cytokinesis was observed with Chinese hamster ovary (CHO) cells microinjected with 7th mutant. When mutant caldesmon was introduced into CHO cells either by protein microinjection or by inducible expression, delay of M-phase entry was observed. Finally, we found that 7th mutant inhibited the disassembly of microfilaments during mitosis. Wild-type caldesmon, on the other hand, was much less potent in producing these three effects. Because mutant caldesmon did not inhibit cyclin B/cdc2 kinase activity, our results suggest that alterations in microfilament assembly caused by caldesmon phosphorylation are important for M-phase progression.

Activation of Trk neurotrophin receptors in the absence of neurotrophins

Neurotrophins regulate neuronal cell survival and synaptic plasticity through activation of Trk receptor tyrosine kinases. Binding of neurotrophins to Trk receptors results in receptor autophosphorylation and downstream phosphorylation cascades. Here, we describe an approach to use small molecule agonists to transactivate Trk neurotrophin receptors. Activation of TrkA receptors in PC12 cells and TrkB in hippocampal neurons was observed after treatment with adenosine, a neuromodulator that acts through G protein-coupled receptors. These effects were reproduced by using the adenosine agonist CGS 21680 and were counteracted with the antagonist ZM 241385, indicating that this transactivation event by adenosine involves adenosine 2A receptors. The increase in Trk activity could be inhibited by the use of the Src family-specific inhibitor, PP1, or K252a, an inhibitor of Trk receptors. In contrast to other G protein-coupled receptor transactivation events, adenosine used Trk receptor signaling with a longer time course. Moreover, adenosine activated phosphatidylinositol 3-kinase/Akt through a Trk-dependent mechanism that resulted in increased cell survival after nerve growth factor or brain-derived neurotrophic factor withdrawal. Therefore, adenosine acting through the A2A receptors exerts a trophic effect through the engagement of Trk receptors. These results provide an explanation for neuroprotective actions of adenosine through a unique signaling mechanism and raise the possibility that small molecules may be used to elicit neurotrophic effects for the treatment of neurodegenerative diseases.

Insulin promotes rapid delivery of N-methyl-d- aspartate receptors to the cell surface by exocytosis

Insulin potentiates N-methyl-d-aspartate receptors (NMDARs) in neurons and Xenopus oocytes expressing recombinant NMDARs. The present study shows that insulin induced (i) an increase in channel number times open probability (nPo) in outside-out patches excised from Xenopus oocytes, with no change in mean open time, unitary conductance, or reversal potential, indicating an increase in n and/or Po; (ii) an increase in charge transfer during block of NMDA-elicited currents by the open channel blocker MK-801, indicating increased number of functional NMDARs in the cell membrane with no change in Po; and (iii) increased NR1 surface expression, as indicated by Western blot analysis of surface proteins. Botulinum neurotoxin A greatly reduced insulin potentiation, indicating that insertion of new receptors occurs via SNARE-dependent exocytosis. Thus, insulin potentiation occurs via delivery of new channels to the plasma membrane. NMDARs assembled from mutant subunits lacking all known sites of tyrosine and serine/threonine phosphorylation in their carboxyl-terminal tails exhibited robust insulin potentiation, suggesting that insulin potentiation does not require direct phosphorylation of NMDAR subunits. Because insulin and insulin receptors are localized to glutamatergic synapses in the hippocampus, insulin-regulated trafficking of NMDARs may play a role in synaptic transmission and plasticity, including long-term potentiation.

Stathmin/Op18 Phosphorylation Is Regulated by Microtubule Assembly

Stathmin/Op 18 is a microtubule (MT) dynamics-regulating protein that has been shown to have both catastrophe-promoting and tubulin-sequestering activities. The level of stathmin/Op18 phosphorylation was proved both in vitro and in vivo to be important in modulating its MT-destabilizing activity. To understand the in vivo regulation of stathmin/Op18 activity, we investigated whether MT assembly itself could control phosphorylation of stathmin/Op18 and thus its MT-destabilizing activity. We found that MT nucleation by centrosomes from Xenopus sperm or somatic cells and MT assembly promoted by dimethyl sulfoxide or paclitaxel induced stathmin/Op18 hyperphosphorylation in Xenopus egg extracts, leading to new stathmin/Op18 isoforms phosphorylated on Ser 16. The MT-dependent phosphorylation of stathmin/Op18 took place in interphase extracts as well, and was also observed in somatic cells. We show that the MT-dependent phosphorylation of stathmin/Op18 on Ser 16 is mediated by an activity associated to the MTs, and that it is responsible for the stathmin/Op18 hyperphosphorylation reported to be induced by the addition of “mitotic chromatin.” Our results suggest the existence of a positive feedback loop, which could represent a novel mechanism contributing to MT network control.

Role of the cytoplasmic tyrosines of β1A integrins in transformation by v-src

GD25 cells lacking β1 integrins or expressing β1A with mutations of conserved cytoplasmic tyrosines (Y783, Y795) to phenylalanine have poor directed migration to platelet-derived growth factor or lysophosphatidic acid when compared with GD25 cells expressing wild-type β1A. We studied the effects of v-src on these cells. Transformation with v-src caused tyrosine and serine phosphorylation of wild-type β1A but not of Y783/795F doubly mutated β1A. v-src-transformed cells had rounded and/or fusiform morphology and poor assembly of fibronectin matrix. Adhesion to fibronectin or laminin and coupling of focal contacts to actin-containing cytoskeleton were preserved in transformed Y783/795F cells but lost on transformation when β1A was wild type. Transformed Y783/795F cells also retained ability, albeit limited, to migrate across filters, whereas transformed cells with wild-type β1A were unable to transverse filters. Studies of single tyrosine mutants showed that the more important tyrosine for retaining ability to adhere, assemble focal contacts, and migrate is Y783. These results suggest that overactive phosphorylation of cytoplasmic residues of β1A, particularly Y783, accounts in part for the phenotype of v-src-transformed cells.

Chronic morphine induces the concomitant phosphorylation and altered association of multiple signaling proteins: A novel mechanism for modulating cell signaling

Traditional mechanisms thought to underlie opioid tolerance include receptor phosphorylation/down-regulation, G-protein uncoupling, and adenylyl cyclase superactivation. A parallel line of investigation also indicates that opioid tolerance development results from a switch from predominantly opioid receptor Giα inhibitory to Gβγ stimulatory signaling. As described previously, this results, in part, from the increased relative abundance of Gβγ-stimulated adenylyl cyclase isoforms as well as from a profound increase in their phosphorylation [Chakrabarti, S., Rivera, M., Yan, S.-Z., Tang, W.-J. & Gintzler, A. R. (1998) Mol. Pharmacol. 54, 655–662; Chakrabarti, S., Wang, L., Tang, W.-J. & Gintzler, A. R. (1998) Mol. Pharmacol. 54, 949–953]. The present study demonstrates that chronic morphine administration results in the concomitant phosphorylation of three key signaling proteins, G protein receptor kinase (GRK) 2/3, β-arrestin, and Gβ, in the guinea pig longitudinal muscle myenteric plexus tissue. Augmented phosphorylation of all three proteins is evident in immunoprecipitate obtained by using either anti-GRK2/3 or Gβ antibodies, but the phosphorylation increment is greater in immunoprecipitate obtained with Gβ antibodies. Analyses of coimmunoprecipitated proteins indicate that phosphorylation of GRK2/3, β-arrestin, and Gβ has varying consequences on their ability to associate. As a result, increased availability of and signaling via Gβγ could occur without compromising the membrane content (and presumably activity) of GRK2/3. Induction of the concomitant phosphorylation of multiple proteins in a multimolecular complex with attendant modulation of their association represents a novel mechanism for increasing Gβγ signaling and opioid tolerance formation.

Evidence for regulation of protein synthesis at the elongation step by CDK1/cyclin B phosphorylation

Eukaryotic elongation factor 1 (eEF-1) contains the guanine nucleotide exchange factor eEF-1B that loads the G protein eEF-1A with GTP after each cycle of elongation during protein synthesis. Two features of eEF-1B have not yet been elucidated: (i) the presence of the unique valyl-tRNA synthetase; (ii) the significance of target sites for the cell cycle protein kinase CDK1/cyclin B. The roles of these two features were addressed by elongation measurements in vitro using cell-free extracts. A poly(GUA) template RNA was generated to support both poly(valine) and poly(serine) synthesis and poly(phenylalanine) synthesis was driven by a poly(uridylic acid) template. Elongation rates were in the order phenylalanine > valine > serine. Addition of CDK1/cyclin B decreased the elongation rate for valine whereas the rate for serine and phenylalanine elongation was increased. This effect was correlated with phosphorylation of the eEF-1δ and eEF-1γ subunits of eEF-1B. Our results demonstrate specific regulation of elongation by CDK1/cyclin B phosphorylation.

Involvement of the Octadecanoid Pathway and Protein Phosphorylation in Fungal Elicitor-Induced Expression of Terpenoid Indole Alkaloid Biosynthetic Genes in Catharanthus roseus

Two key genes in terpenoid indole alkaloid biosynthesis, Tdc and Str, encoding tryptophan decarboxylase and strictosidine synthase, respectively, are coordinately induced by fungal elicitors in suspension-cultured Catharanthus roseus cells. We have studied the roles of the jasmonate biosynthetic pathway and of protein phosphorylation in signal transduction initiated by a partially purified elicitor from yeast extract. In addition to activating Tdc and Str gene expression, the elicitor also induced the biosynthesis of jasmonic acid. The jasmonate precursor α-linolenic acid or methyl jasmonate (MeJA) itself induced Tdc and Str gene expression when added exogenously . Diethyldithiocarbamic acid, an inhibitor of jasmonate biosynthesis, blocked both the elicitor-induced formation of jasmonic acid and the activation of terpenoid indole alkaloid biosynthetic genes. The protein kinase inhibitor K-252a abolished both elicitor-induced jasmonate biosynthesis and MeJA-induced Tdc and Str gene expression. Analysis of the expression of Str promoter/gusA fusions in transgenic C. roseus cells showed that the elicitor and MeJA act at the transcriptional level. These results demonstrate that the jasmonate biosynthetic pathway is an integral part of the elicitor-triggered signal transduction pathway that results in the coordinate expression of the Tdc and Str genes and that protein kinases act both upstream and downstream of jasmonates.

Characterization of Mutants with Alterations of the Phosphorylation Site in the D2 Photosystem II Polypeptide of Chlamydomonas reinhardtii1

We have changed the potential phosphorylation site, a threonine residue at position 2 of the D2 polypeptide of the photosystem II complex of Chlamydomonas reinhardtii, to alanine, valine, aspartate, proline, glycine, or glutamate. Mutants with neutral amino acid changes did not display any phenotype with regard to photoautotrophic growth, light sensitivity, fluorescence transients, or photoinhibition. Pulse labeling of these mutants with 32P indicated that a phosphorylated protein of the same size as D2 is absent in these mutants, suggesting that threonine-2 is indeed the unique phosphorylation site of D2. In contrast, mutants in which threonine-2 has been replaced with acidic residues are deficient in photosystem II. Use of chimeric genes containing the psbD 5′-untranslated region revealed that the initiation of translation was not affected in these mutants, but the mutations interfered with a later step of D2 synthesis and accumulation.

ADP-Dependent Phosphorylation Regulates Association of a DNA-Binding Complex with the Barley Chloroplast psbD Blue-Light-Responsive Promoter1

The chloroplast gene psbD encodes D2, a chlorophyll-binding protein located in the photosystem II reaction center. Transcription of psbD in higher plants involves at least three promoters, one of which is regulated by blue light. The psbD blue-light-regulated promoter (BLRP) consists of a −10 promoter element and an activating complex, AGF, that binds immediately upstream of −35. A second sequence-specific DNA-binding complex, PGTF, binds upstream of AGF between −71 and −100 in the barley (Hordeum vulgare) psbD BLRP. In this study we report that ADP-dependent phosphorylation selectively inhibits the binding of PGTF to the barley psbD BLRP. ATP at high concentrations (1–5 mm) inhibits PGTF binding, but in the presence of phosphocreatine and phosphocreatine kinase, this capacity is lost, presumably due to scavenging of ADP. ADP inhibits PGTF binding at relatively low concentrations (0.1 mm), whereas other nucleotides are unable to mediate this response. ADP-mediated inhibition of PGTF binding is reduced in the presence of the protein kinase inhibitor K252a. This and other results suggest that ADP-dependent phosphorylation of PGTF (or some associated protein) inhibits binding of PGTF to the psbD BLRP and reduces transcription. ADP-dependent phosphorylation is expected to increase in darkness in parallel with the rise in ADP levels in chloroplasts. ADP-dependent phosphorylation in chloroplasts may, therefore, in coordination, inactivate enzymes involved in carbon assimilation, protein synthesis, and transcription during diurnal light/dark cycles.

A Conserved Acidic Motif in the N-Terminal Domain of Nitrate Reductase Is Necessary for the Inactivation of the Enzyme in the Dark by Phosphorylation and 14-3-3 Binding1

It has previously been shown that the N-terminal domain of tobacco (Nicotiana tabacum) nitrate reductase (NR) is involved in the inactivation of the enzyme by phosphorylation, which occurs in the dark (L. Nussaume, M. Vincentz, C. Meyer, J.P. Boutin, and M. Caboche [1995] Plant Cell 7: 611–621). The activity of a mutant NR protein lacking this N-terminal domain was no longer regulated by light-dark transitions. In this study smaller deletions were performed in the N-terminal domain of tobacco NR that removed protein motifs conserved among higher plant NRs. The resulting truncated NR-coding sequences were then fused to the cauliflower mosaic virus 35S RNA promoter and introduced in NR-deficient mutants of the closely related species Nicotiana plumbaginifolia. We found that the deletion of a conserved stretch of acidic residues led to an active NR protein that was more thermosensitive than the wild-type enzyme, but it was relatively insensitive to the inactivation by phosphorylation in the dark. Therefore, the removal of this acidic stretch seems to have the same effects on NR activation state as the deletion of the N-terminal domain. A hypothetical explanation for these observations is that a specific factor that impedes inactivation remains bound to the truncated enzyme. A synthetic peptide derived from this acidic protein motif was also found to be a good substrate for casein kinase II.