Putative Phosphorylation Sites Research Articles

Genome-Wide Identification and Characterization of Salvia miltiorrhiza Laccases Reveal Potential Targets for Salvianolic Acid B Biosynthesis.

Laccases are widely distributed in plant kingdom catalyzing the polymerization of lignin monolignols. Rosmarinic acid (RA) has a lignin monolignol-like structure and is converted into salvianolic acid B (SAB), which is a representatively effective hydrophilic compound of a well-known medicinal plant Salvia miltiorrhiza and also the final compound of phenolic acids metabolism pathway in the plant. But the roles of laccases in the biosynthesis of SAB are poorly understood. This work systematically characterizes S. miltiorrhiza laccase (SmLAC) gene family and identifies the SAB-specific candidates. Totally, 29 laccase candidates (SmLAC1-SmLAC29) are found to contain three signature Cu-oxidase domains. They present relatively low sequence identity and diverse intron–exon patterns. The phylogenetic clustering of laccases from S. miltiorrhiza and other ten plants indicates that the 29 SmLACs can be divided into seven groups, revealing potential distinct functions. Existence of diverse cis regulatory elements in the SmLACs promoters suggests putative interactions with transcription factors. Seven SmLACs are found to be potential targets of miR397. Putative glycosylation sites and phosphorylation sites are identified in SmLAC amino acid sequences. Moreover, the expression profile of SmLACs in different organs and tissues deciphers that 5 SmLACs (SmLAC7/8/20/27/28) are expressed preferentially in roots, adding the evidence that they may be involved in the phenylpropanoid metabolic pathway. Besides, silencing of SmLAC7, SmLAC20 and SmLAC28, and overexpression of SmLAC7 and SmLAC20 in the hairy roots of S. miltiorrhiza result in diversification of SAB, signifying that SmLAC7 and SmLAC20 take roles in SAB biosynthesis. The results of this study lay a foundation for further elucidation of laccase functions in S. miltiorrhiza, and add to the knowledge for SAB biosynthesis in S. miltiorrhiza.

In silico analysis of putative polyphenol oxidases in olive using bioinformatics tools

Sequence, physicochemical, and three-dimensional structure properties of putative polyphenol oxidase proteins in olive (Olea europaea) using various bioinformatics tools were analyzed. The amino acid length varied from 469 to 582 amino acids. The molecular weights of the proteins (ALG62778.1, AFS28698.1 and AFS28697.1) were 65294.67 Da, 53324.79 Da and 53349.48 Da, and isoelectric points (pI) were 7.58, 7.24 and 6.86, respectively. Instability index values were 40.68, 37.52 and 36.89 while aliphatic index values were 72.08, 73.39 and 73.39 respectively. The GRAVY values were –0.540, –0.580 and –0.578, respectively. The most abundant amino acid was Asp (8.5%) while the least abundant one was Cys (1.4%). The putative phosphorylation sites of the polyphenol oxidase proteins were determined by NetPhos 2.0 and NetPhos 3.1. Based on the phylogenetic analysis, the tree constructed using polyphenol oxidase proteins is composed of two main clades. To predict the three dimensional (3D) structures of these proteins, Py MOL was used. The results of the present study provide insight into fundamental characteristics of putative polyphenol oxidase proteins in Olea europaea.

Role of the putative PKC phosphorylation sites of the type IIc sodium-dependent phosphate transporter in parathyroid hormone regulation.

Injection of parathyroid hormone (PTH) rapidly stimulates renal Pi excretion, in part by downregulating NaPi-IIa (Npt2a/SLC34A1) and NaPi-IIc (Npt2c/SLC34A3) transporters. The mechanisms underlying the effects of PTH on NaPi-IIc are not fully elucidated. We analyzed the effect of PTH on inorganic phosphate (Pi) reabsorption in Npt2a-/- mice to eliminate the influence of Npt2a on renal Pi reabsorption. In opossum kidney (OK) cells and Xenopus oocytes, we investigated the effect of NaPi-IIc transporter phosphorylation. Studies of mice with mutations of NaPi-IIc protein in which serine and threonine were replaced with either alanine (A), which prevents phosphorylation, or aspartic acid (D), which mimics the charged state of phosphorylated NaPi-IIc, were also performed to evaluate the involvement of phosphorylation in the regulation of transport function. The Npt2a-/- experiments showed that PTH administration rapidly inactivated NaPi-IIc function in the apical membrane of proximal tubular cells. Analysis of mutant proteins (S71, S138, T151, S174, T583) at putative protein kinase C sites, revealed that S138 markedly suppressed the function and cellular expression of mouse NaPi-IIc in Xenopus oocytes and OK cells. In addition, 138D had a short half-life compared with wild-type protein. The present study suggests that acute regulation of NaPi-IIc protein by PTH is involved in the inactivation of Na+-dependent Pi cotransporter activity and that phosphorylation of the transporter is involved in the rapid modification.

Modulation of GIRK Channels by Protein Kinase C

G protein-gated inwardly rectifying potassium channels (GIRK or Kir3) are crucial for the maintenance of the resting membrane potential and inhibitory post-synaptic potentials in the body. GIRK channel activity is increased by PIP2, sodium ions and Gβγ-subunits and is modulated by several factors. Protein Kinase C (PKC), which is widely reported to inhibit GIRK channels, has 14 different isoforms which are divided into conventional, novel and atypical sub-types. In this study, we evaluated PKC isoform-specific effects on GIRK active mutant subunits - GIRK1(F137S), GIRK4(S143T) and GIRK2(E152D) - and wild-type heteromers - GIRK1/2 and GIRK1/4 expressed in Xenopus oocytes and HEK293T cells. The conventional isoform, PKC-alpha, inhibited the activity of GIRK2(E152D) and GIRK4(S143T) homomeric channels. The novel isoform, PKC-epsilon, augmented the basal activity of GIRK1(F137S) and GIRK4(S143T) as well as channel activity in response to agonist stimulation of the Gi-coupled, muscarinic M2 receptor (M2R)/dopamine D2 receptor (D2R). Conversely, PKC-epsilon inhibited GIRK2(E152D) basal activity and diminished inward currents evoked through dopamine stimulation of D2R. Similar inhibitory effects by PKC-epsilon were obtained with the more distantly related IRK (Kir2) family members. The effect of PKC-epsilon on GIRK2, GIRK4 predominated over GIRK1 in heteromeric GIRK1/2 and GIRK1/4 channels. Due to the sequence homology between GIRK2 and GIRK4, and the differential effect of PKC-epsilon activity, we sought distinct, putative phosphorylation sites on each channel subunit. Phosphorylation sites for both PKC isoforms on GIRK2 and GIRK4 were predicted using the Group-based Prediction System software and represented on a model for the GIRK2 homomer and a homology model of GIRK4 based on the GIRK2 crystal structure. To converge on the phosphorylation site(s) on GIRK4, chimeras between GIRK4(S143T) and IRK1 are being employed concurrently with site-directed mutagenesis.

Tolerance to WIN55,212-2 is delayed in desensitization-resistant S426A/S430A mice

Tolerance to cannabinoid agonists can develop through desensitization of the cannabinoid receptor 1 (CB1) following prolonged administration. Desensitization results from phosphorylation of CB1 by a G protein-coupled receptor kinase (GRK), and subsequent association of the receptor with arrestin. Mice expressing a mutant form of CB1, in which the serine residues at two putative phosphorylation sites necessary for desensitization have been replaced by non-phosphorylatable alanines (S426A/S430A), display reduced tolerance to Δ9-tetrahydrocannabinol (Δ9-THC). Tolerance to the antinociceptive effects of WIN55,212-2 was delayed in S426A/S430A mutants using the tail-flick and formalin tests. However, tolerance to the antinociceptive effects of once daily CP55,940 injections was not significantly delayed in S426A/S430A mutant mice using either of these tests. Interestingly, the dose response curve shifts for the hypothermic and antinociceptive effects of CP55,940 that were induced by chronic treatment with this agonist in wild-type mice were blocked in S426A/S430A mutant mice. Assessment of mechanical allodynia in mice exhibiting chronic cisplatin-evoked neuropathic pain found that tolerance to the anti-allodynic effects WIN55,212-2 but not CP55,940 was delayed in S426A/S430A mice compared to wild-type littermates. Despite these deficits in tolerance, S426A/S430A mutant mice eventually developed tolerance to both WIN55,212-2 and CP55,940 for all pain assays that were examined, suggesting that other mechanisms likely contribute to tolerance for these cannabinoid agonists. These findings suggest that GRK- and βarrestin2-mediated desensitization of CB1 may strongly contribute to the rate of tolerance to the antinociceptive effects of WIN55,212-2, and raises the possibility of agonist-specific mechanisms of cannabinoid tolerance.

Phosphorylation of the phosphatase PTPROt at Tyr399 is a molecular switch that controls osteoclast activity and bone mass in vivo.

Bone resorption by osteoclasts is essential for bone homeostasis. The kinase Src promotes osteoclast activity and is activated in osteoclasts by the receptor-type tyrosine phosphatase PTPROt. In other contexts, however, PTPROt can inhibit Src activity. Through in vivo and in vitro experiments, we show that PTPROt is bifunctional and can dephosphorylate Src both at its inhibitory residue Tyr527 and its activating residue Tyr416 Whereas wild-type and PTPROt knockout mice exhibited similar bone masses, mice in which a putative C-terminal phosphorylation site, Tyr399, in endogenous PTPROt was replaced with phenylalanine had increased bone mass and reduced osteoclast activity. Osteoclasts from the knock-in mice also showed reduced Src activity. Experiments in cultured cells and in osteoclasts derived from both mouse strains demonstrated that the absence of phosphorylation at Tyr399 caused PTPROt to dephosphorylate Src at the activating site pTyr416 In contrast, phosphorylation of PTPROt at Tyr399 enabled PTPROt to recruit Src through Grb2 and to dephosphorylate Src at the inhibitory site Tyr527, thus stimulating Src activity. We conclude that reversible phosphorylation of PTPROt at Tyr399 is a molecular switch that selects between its opposing activities toward Src and maintains a coherent signaling output, and that blocking this phosphorylation event can induce physiological effects in vivo. Because most receptor-type tyrosine phosphatases contain potential phosphorylation sites at their C termini, we propose that preventing phosphorylation at these sites or its consequences may offer an alternative to inhibiting their catalytic activity to achieve therapeutic benefit.

Comparative and phylogenetic analysis of RuBisCO large subunit (rbcL) proteins in some Sideritis L. (Lamiaceae) species: A bioinformatic approach

The large subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) protein, which plays an important role in the photosynthesis reaction, are encoded by the chloroplast genome. Sideritis L., a medical and aromatic plant group, belongs to Lamiaceae family. In this study, we performed sequence, physicochemical, phylogenetic and three-dimensional (3D) bioinformatic analyses of RuBisCO large subunit (rbcL) proteins in the Sideritis ssp. using various bioinformatics tools. Physicochemical analyzes were performed by ExPASy - ProtParam. The putative phosphorylation sites of the rbcL proteins were determined by NetPhos 2.0 and NetPhos 3.1. Phylogenetic analyses were performed with the MEGA 6.0 software. To estimate 3D protein structures, PyMol program was used. At the end of the study, it was found that the amino acid number of stilbene synthase proteins ranged between 171 and 456, molecular weight ranged between 19002.67 and 50420.44 Da, instability index ranged between 27.30 to 40.70 and GRAVY values ranged between -0.394 to -0.226. While the highest average amino acid rate in the rbcL proteins was Gly (10.00%), the lowest amino acid ratio (1.4%) was determined as Trp. In phylogenetic analyses performed using protein sequences, maximum likelihood (ML) tree consisted of 2 large clades. Pairwise distance analysis based on Sideritis species? rbcL protein sequences was performed using MEGA 6.0. The lowest pairwise distance was 0.000, while the highest pairwise distance was 0.024. When the estimated 3D structures of the proteins were examined, the Gly residue, which plays an important role in the structure and function of the proteins, was detected as the least in S. libanotica subsp. kurdica species while it was the most abundant residue in S. cretica subsp. spicata. The results of our study provide insights into fundamental characteristics of rbcL proteins in Sideritis taxa.

Class III OFPs function in the ER signaling pathway to regulate plant growth and development in Arabidopsis

ABSTRACT Both OFPs and ERECTA (ER) regulate plant response to environmental stresses as well as plant growth and development. Blunt-endeded siliques, the characterized phenotype of the er mutants, was observed in transgenic plants overexpressing Class III OFP genes, including OFP13, OFP15, OFP16 and OFP18. We found here that overexpression of ER restored the blunt-endeded siliques phenotypes observed in 35S:OFP15 or 35S:OFP16 plants, whereas er ofp15 and er ofp16 double mutants showed an overall phenotype similar to er single mutants, but their siliques were not blunt-endeded. By generating and characterizing transgenic plants overexpressing putative MAPK phosphorylation sites mutated OFP15 and OPF18, we found that phosphorylation status in Class III OFPs may affect their function in regulating plant growth and development. RT-PCR analysis showed that the expression levels of OFP15, OFP16 and OFP18 were increased in er mutants. Taken together, these results suggest that functions of Class III OFPs may depend on ER signaling.

Cloning and molecular characterization of TaERF6, a gene encoding a bread wheat ethylene response factor.

Ethylene response factor proteins are important for regulating gene expression under different stresses. Different isoforms for ERF have previously isolated from bread wheat (Triticum aestivum L.) and related genera and called from TaERF1 to TaERF5. We isolated, cloned and molecular characterized a novel one based on TdERF1, an isoform in durum wheat (Triticum turgidum L.) and called TaERF6. Its cDNA was synthesized, sequenced and compared with genomic sequence to figure out intron and exon regions and determine coding sequence region. The length of TdERF1 gene was 1939 bp and cDNA was 1065 bp including two exons, the first one 259 bp and the second one 806 bp separated by a 874 bp intron with a 111 bp 5'-UTR (untranslated region) and 401 bp 3'-UTR. TaERF6 encodes a 353 amino acids protein with nearly 99% identity to TdERF1. Hydrophobic cluster analysis revealed an N-terminal hydrophobic domain contains a highly conserved motif with the consensus sequence of M [C/L/Y] [G/R] [G/R/P] [A/G/V/L/R] [I/L/R/S/P/Q] [L/I/R/H] and hydrophobic clusters in AP2/ERF domain of which tends to form -sheet. Three monopartite nuclear localization signals also identified in TaERF6 that play important role in getting back into the nucleus. The results showed several putative phosphorylation sites in TaERF6 that a motif from residues 246 to 266, the CMVII-4 motif, was predicted to phosphorylate by different kinase proteins and play important roles in TaERF6 function. Phylogenetic analysis showed 7 clusters (I to VII) and 10 subclusters according to their relatedness in Poaceae family.

LRRK2 impairs autophagy by mediating phosphorylation of leucyl-tRNA synthetase.

Leucine-rich repeat kinase 2 (LRRK2) is a causal gene of Parkinson disease. G2019S pathogenic mutation increases its kinase activity. LRRK2 regulates various phenotypes including autophagy, neurite outgrowth, and vesicle trafficking. Leucyl-tRNA synthetase (LRS) attaches leucine to tRNALeu and activates mTORC1. Down-regulation of LRS induces autophagy. We investigated the relationship between LRRK2 and LRS in regulating autophagy and observed interaction between endogenous LRRK2 and LRS proteins and LRS phosphorylation by LRRK2. Mutation studies implicated that T293 in the LRS editing domain was a putative phosphorylation site. Phospho-Thr in LRS was increased in cells overexpressing G2019S and dopaminergic neurons differentiated from induced pluripotent stem (iPS) cells of a G2019S carrier. It was decreased by treatment with an LRRK2 kinase inhibitor (GSK2578215A). Phosphomimetic T293D displayed lower leucine bindings than wild type (WT), suggesting its defective editing function. Cellular expression of T293D increased expression of GRP78/BiP, LC3B-II, and p62 proteins and number of LC3 puncta. Increase of GRP78 and phosphorylated LRS was diminished by treatment with GSK2578215A. Levels of LC3B, GRP78/BiP, p62, and α-synuclein proteins were also increased in G2019S transgenic (TG) mice. These data suggest that LRRK2-mediated LRS phosphorylation impairs autophagy by increasing protein misfolding and endoplasmic reticulum stress mediated by LRS editing defect. SIGNIFICANCE OF THE STUDY: Leucine-rich repeat kinase 2 (LRRK2) is the most common genetic cause of Parkinson disease (PD), and the most prevalent pathogenic mutation, G2019S, increases its kinase activity. In this study, we elucidated that leucyl-tRNA synthetase (LRS) was an LRRK2 kinase substrate and identified T293 as an LRRK2 phosphorylation site. LRRK2-meidated LRS phosphorylation or G2019S can lead to impairment of LRS editing, increased ER stress, and accumulation of autophagy markers. These results demonstrate that LRRK2 kinase activity can facilitate accumulation of misfolded protein, suggesting that LRRK2 kinase might be a potential PD therapeutic target along with previous studies.

Nucleocytoplasmic Shuttling of Geminivirus C4 Protein Mediated by Phosphorylation and Myristoylation Is Critical for Viral Pathogenicity

Many geminivirus C4 proteins induce severe developmental abnormalities in plants. We previously demonstrated that Tomato leaf curl Yunnan virus (TLCYnV) C4 induces plant developmental abnormalities at least partically by decreasing the accumulation of NbSKη, an ortholog of Arabidopsis BIN2 kinase involved in the brassinosteroid signaling pathway, in the nucleus through directing it to the plasma membrane. However, the molecular mechanism by which the membrane-associated C4 modifies the localization of NbSKη in the host cell remains unclear. Here, we show that TLCYnV C4 is a nucleocytoplasmic shuttle protein, and that C4 shuttling is accompanied by nuclear export of NbSKη. TLCYnV C4 is phosphorylated by NbSKη in the nucleus, which promotes myristoylation of the viral protein. Myristoylation of phosphorylated C4 favors its interaction with exportin-α (XPO I), which in turn facilitates nuclear export of the C4/NbSKη complex. Supporting this model, chemical inhibition of N-myristoyltransferases or exportin-α enhanced nuclear retention of C4, and mutations of the putative phosphorylation or myristoylation sites in C4 resulted in increased nuclear retention of C4 and thus decreased severity of C4-induced developmental abnormalities. The impact of C4 on development is also lessened when a nuclear localization signal or a nuclear export signal is added to its C-terminus, restricting it to a specific cellular niche and therefore impairing nucleocytoplasmic shuttling. Taken together, our results suggest that nucleocytoplasmic shuttling of TLCYnV C4, enabled by phosphorylation by NbSKη, myristoylation, and interaction with exportin-α, is critical for its function as a pathogenicity factor.

The organophosphate pesticide methamidophos opens the blood-testis barrier and covalently binds to ZO-2 in mice

Methamidophos (MET) is an organophosphate (OP) pesticide widely used in agriculture in developing countries. MET causes adverse effects in male reproductive function in humans and experimental animals, but the underlying mechanisms remain largely unknown. We explored the effect of MET on mice testes (5 mg/kg/day/4 days), finding that this pesticide opens the blood-testis barrier and perturbs spermatogenesis, generating the appearance of immature germ cells in the epididymis. In the seminiferous tubules, MET treatment changed the level of expression or modified the stage-specific localization of tight junction (TJ) proteins ZO-1, ZO-2, occludin, and claudin-3. In contrast, claudin-11 was barely altered. MET also modified the shape of claudin-11, and ZO-2 at the cell border, from a zigzag to a more linear pattern. In addition, MET diminished the expression of ZO-2 in spermatids present in seminiferous tubules, induced the phosphorylation of ZO-2 and occludin in testes and reduced the interaction between these proteins assessed by co-immunoprecipitation. MET formed covalent bonds with ZO-2 in serine, tyrosine and lysine residues. The covalent modifications formed on ZO-2 at putative phosphorylation sites might interfere with ZO-2 interaction with regulatory molecules and other TJ proteins. MET bonds formed at ZO-2 ubiquitination sites likely interfere with ZO-2 degradation and TJ sealing, based on results obtained in cultured epithelial cells transfected with ZO-2 mutated at a MET target lysine residue. Our results shed light on MET male reproductive toxicity and are important to improve regulations regarding the use of OP pesticides and to protect the health of agricultural workers.

Both Ser361 phosphorylation and the C-arrestin domain of thioredoxin interacting protein are important for cell cycle blockade at the G1/S checkpoint.

Thioredoxin interacting protein (TXNIP) is a novel tumor suppressor that is down‐regulated in several cancer tissues and tumor cell lines. Overexpression of TXNIP causes cell cycle arrest at the G1/S checkpoint in the hepatocellular carcinoma cell line HuH‐7. TXNIP contains putative phosphorylation sites, but the effects of its phosphorylation have not been fully characterized. TXNIP also contains two α‐arrestin domains (N‐arrestin and C‐arrestin) whose functions are not fully understood. Here, we reveal an association between TXNIP and cell cycle regulatory proteins (p27kip1, Jun activation domain‐binding protein 1 (JAB1), Cdk2, and cyclin E), suggesting its participation in cell cycle regulation. We observed phosphorylation of TXNIP and used both in vivo and in vitro kinase assays to demonstrate that TXNIP can be phosphorylated by p38 mitogen‐activated protein kinase. Furthermore, we also identified Ser361 in TXNIP as one of the major phosphorylation sites. Cell cycle analysis showed that Ser361 phosphorylation participates in TXNIP‐mediated cell cycle arrest. In addition, the C‐arrestin domain may also play an important role in cell cycle arrest. We also showed that phosphorylation at Ser361 may be important for the association of TXNIP with JAB1 and that the C‐arrestin domain is necessary for the nuclear localization of this molecule. Collectively, these studies reveal that TXNIP participates in cell cycle regulation through association with regulatory proteins, especially JAB1, and that C‐arrestin‐dependent nuclear localization is important for this function. This work may facilitate the development of a new cancer therapy strategy that targets TXNIP as a key molecule inhibiting cancer cell growth via cell cycle blockade at the G1/S checkpoint.

The tuberous sclerosis complex subunit TBC1D7 is stabilized by Akt phosphorylation–mediated 14-3-3 binding

The tuberous sclerosis complex (TSC) is a negative regulator of mTOR complex 1, a signaling node promoting cellular growth in response to various nutrients and growth factors. However, several regulators in TSC signaling still await discovery and characterization. Using pulldown and MS approaches, here we identified the TSC complex member, TBC1 domain family member 7 (TBC1D7), as a binding partner for PH domain and leucine-rich repeat protein phosphatase 1 (PHLPP1), a negative regulator of Akt kinase signaling. Most TBC domain-containing proteins function as Rab GTPase-activating proteins (RabGAPs), but the crystal structure of TBC1D7 revealed that it lacks residues critical for RabGAP activity. Sequence analysis identified a putative site for both Akt-mediated phosphorylation and 14-3-3 binding at Ser-124, and we found that Akt phosphorylates TBC1D7 at Ser-124. However, this phosphorylation had no effect on the binding of TBC1D7 to TSC1, but stabilized TBC1D7. Moreover, 14-3-3 protein both bound and stabilized TBC1D7 in a growth factor-dependent manner, and a phospho-deficient substitution, S124A, prevented this interaction. The crystal structure of 14-3-3ζ in complex with a phospho-Ser-124 TBC1D7 peptide confirmed the direct interaction between 14-3-3 and TBC1D7. The sequence immediately upstream of Ser-124 aligned with a canonical β-TrCP degron, and we found that the E3 ubiquitin ligase β-TrCP2 ubiquitinates TBC1D7 and decreases its stability. Our findings reveal that Akt activity determines the phosphorylation status of TBC1D7 at the phospho-switch Ser-124, which governs binding to either 14-3-3 or β-TrCP2, resulting in increased or decreased stability of TBC1D7, respectively.

Arabidopsis ETHYLENE RESPONSE FACTOR 8 (ERF8) has dual functions in ABA signaling and immunity

BackgroundETHYLENE RESPONSE FACTOR (ERF) 8 is a member of one of the largest transcription factor families in plants, the APETALA2/ETHYLENE RESPONSIVE FACTOR (AP2/ERF) superfamily. Members of this superfamily have been implicated in a wide variety of processes such as development and environmental stress responses.ResultsIn this study we demonstrated that ERF8 is involved in both ABA and immune signaling. ERF8 overexpression induced programmed cell death (PCD) in Arabidopsis and Nicotiana benthamiana. This PCD was salicylic acid (SA)-independent, suggesting that ERF8 acts downstream or independent of SA. ERF8-induced PCD was abolished by mutations within the ERF-associated amphiphilic repression (EAR) motif, indicating ERF8 induces cell death through its transcriptional repression activity. Two immunity-related mitogen-activated protein kinases, MITOGEN-ACTIVATED PROTEIN KINASE 4 (MPK4) and MPK11, were identified as ERF8-interacting proteins and directly phosphorylated ERF8 in vitro. Four putative MPK phosphorylation sites were identified in ERF8, one of which (Ser103) was determined to be the predominantly phosphorylated residue in vitro, while mutation of all four putative phosphorylation sites partially suppressed ERF8-induced cell death in N. benthamiana. Genome-wide transcriptomic analysis and pathogen growth assays confirmed a positive role of ERF8 in mediating immunity, as ERF8 knockdown or overexpression lines conferred compromised or enhanced resistance against the hemibiotrophic bacterial pathogen Pseudomonas syringae, respectively.ConclusionsTogether these data reveal that the ABA-inducible transcriptional repressor ERF8 has dual roles in ABA signaling and pathogen defense, and further highlight the complex influence of ABA on plant-microbe interactions.

Identification and characterization of a novel phosphoregulatory site on cyclin-dependent kinase 5

Cyclin-dependent kinase 5 (CDK5) is a serine/threonine kinase essential for embryonic development whose overactivation has been implicated in several pathologies including neurodegeneration, cancer cell metastasis and type II diabetes. Therefore, it is important to investigate molecular mechanism(s) that mediate regulation of CDK5 activity. Here we identify and characterize a novel phosphoregulatory site on CDK5. Our mass spectrometry analysis identified seven putative phosphorylation sites on CDK5. Using phosphomimetic and non-phosphorylatable mutants, we determined that phosphorylation of S47, one of the identified sites, renders the kinase catalytically inactive. The inactivation of the kinase due to the phosphomimetic change at S47 results from inhibition of its interaction with its cognate activator, p35. We connect the effect of this regulatory event to a cellular phenotype by showing that the S47D CDK5 mutant inhibits cell migration and promotes cell proliferation. Together, these results have uncovered a potential physiological mechanism to regulate CDK5 activity. The evolutionary placement of a phosphorylatable residue (S/T) at this position not only in CDK5 but also in the majority of other CDK family members suggests that this phosphosite may represent a shared regulatory mechanism across the CDK family.

Mechanisms of PKA-Dependent Potentiation of Kv7.5 Channel Activity in Human Airway Smooth Muscle Cells.

β-adrenergic receptor (βAR) activation promotes relaxation of both vascular and airway smooth muscle cells (VSMCs and ASMCs, respectively), though the signaling mechanisms have not been fully elucidated. We previously found that the activity of Kv7.5 voltage-activated potassium channels in VSMCs is robustly enhanced by activation of βARs via a mechanism involving protein kinase A (PKA)-dependent phosphorylation. We also found that enhancement of Kv7 channel activity in ASMCs promotes airway relaxation. Here we provide evidence that Kv7.5 channels are natively expressed in primary cultures of human ASMCs and that they conduct currents which are robustly enhanced in response to activation of the βAR/cyclic adenosine monophosphate (cAMP)/PKA pathway. MIT Scansite software analysis of putative PKA phosphorylation sites on Kv7.5 identified 8 candidate serine or threonine residues. Each residue was individually mutated to an alanine to prevent its phosphorylation and then tested for responses to βAR activation or to stimuli that elevate cAMP levels. Only the mutation of serine 53 (S53A), located on the amino terminus of Kv7.5, significantly reduced the increase in Kv7.5 current in response to these stimuli. A phospho-mimic mutation (S53D) exhibited characteristics of βAR-activated Kv7.5. Serine-to-alanine mutations of 6 putative PKA phosphorylation sites on the Kv7.5 C-terminus, individually or in combination, did not significantly reduce the enhancement of the currents in response to forskolin treatment (to elevate cAMP levels). We conclude that phosphorylation of S53 on the amino terminus of Kv7.5 is essential for PKA-dependent enhancement of channel activity in response to βAR activation in vascular and airway smooth muscle cells.

Context-dependent AMPK activation distinctly regulates TAp73 stability and transcriptional activity

TAp73, the homologue of the tumour suppressor p53, has dual roles in tumourigenesis: both as a tumour suppressor and as a promoter of tumour growth. We have recently shown that hypoxia, a condition prevalent in tumours, results in the stabilisation of TAp73 through a mechanism involving HIF-1α-mediated repression of the E3 ligase Siah1. Elevated TAp73 in turn regulates the angiogenic transcriptional programme, exemplified by vegf-A activation, thereby promoting angiogenesis and tumour growth. To further understand hypoxia-mediated TAp73 regulation, we have focused on the Adenosine monophosphate (AMP)-dependent protein kinase (AMPK) signalling pathway induced by hypoxia. We show that hypoxia-mediated AMPK activation is required for efficient TAp73 stabilisation, through multiple means by using AMPK-deficient cells or inhibiting its activity and expression. Conversely, direct AMPK activation using its activator AICAR is also sufficient to induce TAp73 stabilisation but this is independent of putative AMPK phosphorylation sites on TAp73, HIF-1α activation, and transcriptional repression of Siah1. Furthermore, while vegf-A up-regulation upon hypoxia requires AMPK, direct activation of AMPK by AICAR does not activate vegf-A. Consistently, supernatant from cells exposed to hypoxia, but not AICAR, was able to induce tube formation in HUVECs. These data therefore highlight that the processes of TAp73 stabilisation and transcriptional activation of angiogenic target genes by AMPK activation can be decoupled. Collectively, these results suggest that the context of AMPK activation determines the effect on TAp73, and proposes a model in which hypoxia-induced TAp73 stabilisation occurs by parallel pathways converging to mediate its transactivation potential.

A Novel Tomato Fusarium Wilt Tolerance Gene.

The reduced mycorrhizal colonization (rmc) tomato mutant is unable to form mycorrhiza and is more susceptible to Fusarium wilt compared with its wild-type isogenic line 76R. The rmc mutant has a chromosomal deletion affecting five genes, one of which is similar to CYCLOPS. Loss of this gene is responsible for non-mycorrhizality in rmc but not enhanced Fusarium wilt susceptibility. Here, we describe assessment of a second gene in the rmc deletion, designated Solyc08g075770 that is expressed in roots. Sequence analyses show that Solyc08g075770 encodes a small transmembrane protein with putative phosphorylation and glycosylation sites. It is predicted to be localized in the plasma membrane and may function in transmembrane ion transport and/or as a cell surface receptor. Complementation and knock-out strategies were used to test its function. Some putative CRISPR/Cas-9 knock-out transgenic events exhibited Fusarium wilt susceptibility like rmc and some putative complementation lines were 76R-like, suggesting that the tomato Solyc08g075770 functions in Fusarium wilt tolerance. This is the first study to demonstrate that Solyc08g075770 is the contributor to the Tfw locus, conferring tolerance to Fusarium wilt in 76R which was lost in rmc.

In silico assessment of human CD14 gene revealed high-risk single nucleotide polymorphisms and their impact on innate immune response against microbial pathogens

CD14 is a multifunctional pattern recognition receptor, which works in concert with several other toll-like receptors and activates downstream immune response. As CD14 polymorphisms have been associated with several diseases, a study of non-synonymous SNPs will be useful in understanding disease pathogenesis and developing molecular diagnostics. Out of 155 non-synonymous SNPs, 10 high-risk nsSNPs were identified using six different predictive algorithms, which were further examined structurally. The ConSurf server was used to derive the conservation scores of amino acids and identify structural and functional residues. Six of these high-risk nsSNPs – rs745518361, rs751442680, rs754968319, rs756150689, rs764993455 and rs767220208 were within the lysine-rich repeat domain of CD14, which facilitates protein-protein interactions. Putative phosphorylation, sumoylation and ubiquitination sites were also investigated using NetPhos, GPS, GPS-SUMO, SUMO Plot, BDM-PUB and UbPred servers. 6 high-risk SNPs were identified at conserved phosphorylation sites which could affect CD14 signaling. Protein-protein interaction analysis revealed association between CD14 and other mediators of innate immunity including lipopolysaccharide binding protein, Toll-like receptor 1 (TLR1), TLR2, TLR4, TLR6 and Integrins. In conclusion, our data suggests that disruption of CD14 structure by SNPs rs767220208, rs745518361, rs754968319, rs764993455 and rs749260147 could affect activation of the innate immunity.