Termination of TGF-β Superfamily Signaling Through SMAD Dephosphorylation——A Functional Genomic View

The transforming growth factor-β （TGF-β） and related growth factors activate a broad range of cellular responses in metazoan organisms via autocrine, paracrine, and endocrine modes. They play key roles in the pathogenesis of many diseases especially cancer, fibrotic diseases, autoimmune diseases and cardiovascular diseases. TGF-β receptor-mediated phosphorylation of R-SMADs represents the most critical step in the TGF-β signaling pathways that triggers a cascade of intracellular events from SMAD complex assembly in the cytoplasm to transcriptional control in the nucleus. Conversely, dephosphorylafion of R-SMADs is a key mechanism for terminating TGF-β signaling. Our labs have recently taken an integrated approach combining functional genomics, biochemistry and development biology to describe the isolation and functional characterization of protein phosphatase PPM1A in controlling TGF-β signaling. This article briefly reviews how dynamic phosphorylation and dephosphorylation of SMADs control or fine-tune the signaling strength and duration and ultimately the physiological consequences in TGF-β signaling.

Dephosphorylation of myristoylated alanine-rich C kinase substrate accelerates wound-induced migration of SH-SY5Y cells

Inflammation, which is induced after infection of bacteria and tissue injury, is one of the important early stages of wound healing. Bradykinin is increased during acute and chronic inflammation. We previously reported that bradykinin stimulation induces dephosphorylation of myristoylated alanine-rich C kinase substrate (MARCKS) after phosphorylation by ROCK leading neurite outgrowth in neuroblastoma SH-SY5Y cells. In this report we showed that knock-down of MARCKS by RNAi reduced cell migration. Wild-type MARCKS-overexpressed SH-SY5Y cells migrated faster than the control cells. Unphosphorylatable MARCKS-overexpressed cells notably migrated fast. Moreover, chronic MARCKS dephosphorylation by a ROCK inhibitor HA-1077 promoted the cell migration, on the other hand a PKC inhibitor Ro-31-8220 did not. After wounding, MARCKS was transiently phosphorylated and dephospho-rylated by 20 min. Immunocytochemistry showed that the dephosphorylated MARCKS was localized at neurite tips. These findings suggest that MARCKS dephosphorylation is important in wound-induced migration of SH-SY5Y cells. It indicates the possibility that MARCKS is associated with wound repair after inflammation.

Ca^(2+)-dependent TaCCD1 cooperates with TaSAUR215 to enhance plasma membrane H^(+)-ATPase activity and alkali stress tolerance by inhibiting PP2C-mediated dephosphorylation of TaHA2 in wheat

Alkali stress is a major constraint for crop production in many regions of saline-alkali land.However,little is known about the mechanisms through which wheat responds to alkali stress.In this study,we identified a calcium ion-binding protein from wheat,TaCCD1,which is critical for regulating the plasma membrane(PM)H^(+)-ATPase-mediated alkali stress response.PM H+-ATPase activity is closely related to alkali tolerance in the wheat variety Shanrong 4(SR4).We found that two D-clade type 2C protein phosphatases,TaPP2C.D1 and TaPP2C.D8(TaPP2C.D1/8),negatively modulate alkali stress tolerance by dephosphorylating the penultimate threonine residue(Thr926)of TaHA2 and thereby inhibiting PM H+-ATPase activity.Alkali stress induces the expression of TaCCD1 in SR4,and TaCCD1 interacts with TaSAUR215,an early auxin-responsive protein.These responses are both dependent on calcium signaling triggered by alkali stress.TaCCD1 enhances the inhibitory effect of TaSAUR215 on TaPP2C.D1/8 activity,thereby promoting the activity of the PM H^(+)-ATPase TaHA2 and alkali stress tolerance in wheat.Functional and genetic analyses verified the effects of these genes in response to alkali stress,indicating that TaPP2C.D1/8 function downstream of TaSAUR215 and TaCCD1.Collectively,this study uncovers a new signaling pathway that regulates wheat responses to alkali stress,in which Ca^(2+)-dependent TaCCD1 cooperates with TaSAUR215 to enhance PM H+-ATPase activity and alkali stress tolerance by inhibiting TaPP2C.D1/8-mediated dephosphorylation of PM H+-ATPase TaHA2 in wheat.

Damage Mechanism of CK2 and IKAROS in Philadelphia Like Acute Lymphoblastic Leukemia

Acute lymphoblastic leukemia (ALL) is characterized by immature and poorly differentiated B lymphocytes in large numbers in the blood. B cells are distinct from the cell types involved in their development (common lymphoid progenitor cells, pro-B cells, pre-B cells, and mature cells). The process of B cell maturation depends on precise communication within the cell: signals activate specific genes that are essential for proper development. Errors in this intricate signaling network can lead to issues with B cell function and contribute to disease. B-lineage acute lymphoid leukemias, malignancies of precursor-stage B lymphoid cells inhibit lymphoid differentiation, leading to abnormal cell proliferation and survival. The process of developing leukemia (leukemogenesis) can be triggered by an overproduction of both hematopoietic stem cells (the cells that form all blood cells) and the immature versions of white blood cells called lymphoblasts. Acute lymphoblastic leukemia (ALL) with the presence of the Philadelphia chromosome (ALL Ph) is classified as a high-risk manifestation of the disease, this chromosome is the product of the reciprocal translocation, whose product is a BCR-ABL fusion protein. It is a highly active tyrosine kinase that can transform hematopoietic cells into cytokine-independent. Hyperphosphorylation cascades inhibit the differentiating function of IKZF1 as a tumor suppressor gene which leads to an abnormal proliferation of B cells due to the presence of the Philadelphia chromosome;it inhibits the differentiating process, leukemogenesis involving immature B cells in the bloodstream can result from the uncontrolled growth and division of hematopoietic stem cells and immature lymphoblasts (the precursors to B cells).

Role of Wnt signaling pathway hypofunction mediated by dephosphorylation of β-catenin in impaired wound healing of type 1 diabetic rats

Objective To investigate the role of Wnt signaling pathway hypofunction mediated by dephosphorylation ofβ-catenin in the impaired wound healing of type 1 diabetic rats.Methods The back skin defect wounds were produced in rats with type 1 diabetes.These rats were divided into control,diabetes,lithium chloride treatment,and epidermal growth factor(EGF)

Anlotinib suppresses lymphangiogenesis and lymphatic metastasis in lung adenocarcinoma through a process potentially involving VEGFR-3 signaling

Objective:Lymphatic metastasis is one of the leading causes of malignancy dispersion in various types of cancer.However,few anti-lymphangiogenic drugs have been approved for clinical use to date.Therefore,new therapies to block lymphangiogenesis are urgently required.Methods:Immunohistochemistry,immunofluorescence,Western blot,migration assays,and lymphangiogenesis and lymphatic metastasis assays were used.Results:Anlotinib,a receptor tyrosine kinase inhibitor,suppressed the rate of new metastatic lesions(31.82%in the placebo arm and 18.18%in the anlotinib arm)in patients with advanced lung adenocarcinoma who were enrolled in our ALTER-0303 study.D2-40+-lymphatic vessel density was strongly correlated with disease stage,metastasis,and poor prognosis in 144 Chinese patients with lung adenocarcinoma.In mice bearing A549EGFP tumors,tumor lymphatic vessel density,tumor cell migration to lymph nodes,and the number of distant metastatic lesions were lower in the anlotinib group than in the controls.Anlotinib inhibited the growth and migration of human lymphatic endothelial cells(hLECs)and lymphangiogenesisin vitro andin vivo.Treatment of hLECs with anlotinib downregulated phosphorylated vascular endothelial growth factor receptor 3(VEGFR-3).Conclusions:Anlotinib inhibits lymphangiogenesis and lymphatic metastasis,probably through inactivating VEGFR-3 phosphorylation.The results indicate that anlotinib may be beneficial for treatment in avoiding lymphangiogenesis and distant lymphatic metastasis in lung adenocarcinoma.(Trial registration:ALTER0303;NCT02388919;March 17,2015.)

C60 Fullerenes Suppress Reactive Oxygen Species Toxicity Damage in Boar Sperm

We report the carboxylated C60 improved the survival and quality of boar sperm during liquid storage at 4°C and thus propose the use of carboxylated C60 as a novel antioxidant semen extender supplement.Our results demonstrated that the sperm treated with 2μg mL?1 carboxylated C60 had higher motility than the control group(58.6%and 35.4%,respectively;P?0.05).Moreover,after incubation with carboxylated C60 for 10 days,acrosome integrity and mitochondrial activity of sperm increased by 18.1%and 34%,respectively,compared with that in the control group.Similarly,the antioxidation abilities and adenosine triphosphate levels in boar sperm treated with carboxylated C60 significantly increased(P?0.05)compared with those in the control group.The presence of carboxylated C60 in semen extender increases sperm motility probably by suppressing reactive oxygen species(ROS)toxicity damage.Interestingly,carboxylated C60 could protect boar sperm from oxidative stress and energy deficiency by inhibiting the ROS-induced protein dephosphorylation via the cAMP-PKA signaling pathway.In addition,the safety of carboxylated C60 as an alternative antioxidant was also comprehensively evaluated by assessing the mean litter size and number of live offspring in the carboxylated C60 treatment group.Our findings confirm carboxylated C60 as a novel antioxidant agent and suggest its use as a semen extender supplement for assisted reproductive technology in domestic animals.

Src Is Dephosphorylated at Tyrosine 530 in Human Colon Carcinomas

Objective：Src is a protein tyrosine kinase that plays important roles in cancer development,and Src kinase activity has been found to be elevated in several types of cancers.However,the cause of the elevation of Src kinase activity in the majority of human colon carcinomas is still largely unknown.We aim at finding the cause of elevated Src kinase activity in human colon carcinomas.Methods：We employed normal colon epithelial FHC cells and examined Src activation in human colon carcinoma specimens from 8 patients.Protein expression levels were determined by Western blotting,and the activity of Src kinase by kinase assay.Results：Actin levels were different between tumor and normal tissues,demonstrating the complexities and inhomogeneities of the tissue samples.Src kinase activities were increased in the majority of the colon carcinomas as compared with normal colon epithelial cells （range 13-29）.Src protein levels were reduced in the colon carcinomas.Src Y530 phosphorylation levels were reduced to a higher extent than protein levels in the carcinomas.Conclusion：The results suggest that Src specific activities were highly increased in human colon carcinomas;phosphorylation at Src Y530 was reduced,contributing to the highly elevated Src specific activity and Src kinase activity.

Identification of the amino acid involved in the regulation of bacterial pyruvate, orthophosphate dikinase and phosphoenolpyruvate synthetase

Pyruvate, orthophosphate dikinase (PPDK) and phosphoenolpyruvate synthetase (PEPS) catalyze the conversion of pyruvate to phosphoenolpyruvate (PEP). Both are regulated by a phosphorylation-dephosphorylation mechanism involving a bifunctional serine/ threonine kinase and a pyrophosphorylase (PPDK regulatory protein, PDRP, and PEPS regulatory protein, PSRP, respectively). In plants the regulatory mechanism involves phosphorylation of a threonine residue that is separated by a single amino acid from the histidine residue that forms a phosphorylated intermediate during catalysis. Using antibodies, we demonstrated that the regulation of both Listeria monocytogenes PPDK and Escherichia coli PEP synthetase involves the phosphorylation of a threonine residue located close to the catalytic histidine residue. The amino acid located between the regulatory threonine and the catalytic histidine is highly conserved being serine in PPDK and cysteine in PEPS. Using site-directed mutagenesis we have shown that both PPDK and PEPS in which the serine and cysteine residues, respectively, were substituted with an alanine the enzymes could be regulated indicating that the serine and cysteine residues, respectively, are not essential for regulation. We also demonstrated that altering the intermediate amino acid did not alter the specificity of the regulatory proteins for their protein substrates.

Active zone stability:insights from fly neuromuscular junction

The presynaptic active zone is a dynamic structure that orchestrates regulated release of neurotrans- mitters. Developmental and aging processes, and changes in neuronal network activity can all modulate the number, size and composition of active zone and thereby synaptic efficacy. However, very little is known about the mechanism that controls the structural stability of active zone. By study- ing a model synapse, the Drosophila neuromuscular iunction, our recent work shed light on how two scaffolding proteins at the active zone regulate active zone stability by promoting a localized dephos- phorylation event at the nerve terminal. Here we discuss the major insights from our findings and their implications for future research.

Functional repertoire of protein kinases and phosphatases in synaptic plasticity and associated neurological disorders

Protein phosphorylation and dephosphorylation are two essential and vital cellular mechanisms that regulate many receptors and enzymes through kinases and phosphatases.Ca^2+- dependent kinases and phosphatases are responsible for controlling neuronal processing;balance is achieved through opposition.During molecular mechanisms of learning and memory,kinases generally modulate positively while phosphatases modulate negatively.This review outlines some of the critical physiological and structural aspects of kinases and phosphatases involved in maintaining postsynaptic structural plasticity.It also explores the link between neuronal disorders and the deregulation of phosphatases and kinases.

Dephosphorylated mutations affect the protein-protein interactions of ERF in Populus simonii x P.nigra

Phosphorylation is a common type of post-translational modification(PTM).It plays a vital role in many cellular processes.The reversible phosphorylation and dephosphorylation affect protein structures and protein-protein interactions.Previously,we obtained five proteins that interact with ethylene-responsive factor(ERF)from the cDNA library of Populus simonii x Populus nigra.To further investigate the effect of dephosphorylation of PsnERF on its protein binding ability,we generated different phosphorylation states of PsnERF and demonstrated their protein binding capacity by the yeast two-hybrid assay(Y2H).The secondary structures and 3D structures of PsnERF,ERFm,TrunERF,and psnerf^(197/198/202a) were predicted by homology modeling.The Y2H assay indicated that the deletion of serine-rich regions does not affect the interactions,while dephosphorylated mutations blocked the interactions.Homology modeling results suggested that the protein-binding activity was affected by dephosphorylation,and the S197/S198/S202 residues of PsnERF may be the key phosphorylation sites influencing its binding ability.

The relationship between cell apoptosis and dephosphorylated RB protein and proliferating cell nuclear antigen in human breast cancer

Objective:The aim of our study was to investigate the relationship between cell apoptosis and dephosphorylated RB protein and proliferating cell nuclear antigen(PCNA) in human breast cancer.Methods:MTT colorimetric assay was applied to examine the growth inhibition,and the apoptosis was determined by flow cytometry(FCM).The expressing quantity of dephosphorylated RB protein and PCNA pre-and post the action of ADR were detected with immunocytochemistry.Results:MTT assay revealed that ADR inhibited proliferation of MCF-7/S cells in a dose dependent manner,the 50% inhibition concentration(IC50) value was 0.128 mg/L.Tumor cell apoptotic rate(AR) in ADR group(χ= 0.259) was significantly higher than that in the control group(χ = 0.045)(P < 0.01).The expressive levels of dephosphorylated RB protein in ADR group(MOD × area = 986.8 ± 207.4) was significantly higher than that in control group(MOD × area =131.7 ± 31.9)(P < 0.01).PCNA positive expression rate in ADR group(χ = 0.3371) was significantly lower than that in the control group(χ = 0.5152)(P < 0.01).Conclusion:In ADR group,there was significant positive correlation between AR and the expressing quantity of dephosphorylated RB protein,but there was significant negative correlation between AR and PCNA.

Plasma Membrane H^＋-ATPase Regulation in the Center of Plant Physiology

The plasma membrane （PM） H^＋-ATPase is an important ion pump in the plant cell membrane. By extruding protons from the cell and generating a membrane potential, this pump energizes the PM, which is a prerequisite for growth. Modification of the autoinhibitory terminal domains activates PM H^＋-ATPase activity, and on this basis it has been hypothesized that these regulatory termini are targets for physiological factors that activate or inhibit proton pumping. In this review, we focus on the posttranslational regulation of the PM H＋-ATPase and place regulation of the pump in an evolutionary and physiological context. The emerging picture is that multiple signals regulating plant growth interfere with the posttranslational regulation of the PM H^＋-ATPase.

New Insight in Ethylene Signaling： Autokinase Activity of ETR1 Modulates the Interaction of Receptors and EIN2

Ethylene insensitive 2 （EIN2）, an integral membrane protein of the ER network, has been identified as the central regulator of the ethylene signaling pathway. Still, the mechanism by which the ethylene signal is transferred from the receptors to EIN2 has not been solved yet. Here, we show that protein phosphorylation is a key mechanism to control the interaction of EIN2 and the receptors. In vivo and in vitro fluorescence studies reveal that the kinase domain of the receptors is essential for the interaction. Cyanide, an ethylene agonist, which is known to reduce auto-phosphorylation of the ethylene receptor ethylene resistant 1 （ETR1） or a mutation in the kinase domain of ETR1 that prevents autophosphorylation （H353A）, increases the affinity of the receptors for EIN2. On the other hand, mimicking permanent auto-phosphorylation of ETR1 as in the mutant H353E releases the EiN2-ETR1 interaction from the control by the plant hormone. Based on our data, we propose a novel model on the integration of EIN2 in the ethylene signaling cascade.

Assembly and structure of protein phosphatase 2A

Protein phosphatase 2A (PP2A) represents a conserved family of important protein serine/threonine phosphatases in species ranging from yeast to human. The PP2A core enzyme comprises a scaffold subunit and a catalytic subunit. The heterotrimeric PP2A holoenzyme consists of the core enzyme and a variable regulatory subunit. The catalytic subunit of PP2A is subject to reversible methylation, medi-ated by two conserved enzymes. Both the PP2A core and holoenzymes are regulated through interac-tion with a large number of cellular cofactors. Recent biochemical and structural investigation reveals critical insights into the assembly and function of the PP2A core enzyme as well as two families of holoenzyme. This review focuses on the molecular mechanisms revealed by these latest advances.

Roles of Arabidopsis Patatin-Related Phospholipases A in Root Development Are Related to Auxin Responses and Phosphate Deficiency

Phospholipase A enzymes cleave phospho- and galactolipids to generate free fatty acids and lysolipids that function in animal and plant hormone signaling. Here, we describe three Arabidopsis patatin-related phospholipase A （pPLA） genes AtPLAIVA, AtPLAIVB, and AtPLAIVC and their corresponding proteins. Loss-of-function mutants reveal roles for these pPLAs in roots during normal development and under phosphate deprivation. AtPLAIVA is expressed strongly and exclusively in roots and AtplalVA-null mutants have reduced lateral root development, characteristic of an impaired auxin response. By contrast, AtPLAIVB is expressed weakly in roots, cotyledons, and leaves but is transcriptionally induced by auxin, although AtplalVB mutants develop normally. AtPLAIVC is expressed in the floral gynaecium and is induced by abscisic acid （ABA） or phosphate deficiency in roots. While an AtplalVC-1 loss-of-function mutant displays ABA respon- siveness, it exhibits an impaired response to phosphate deficiency during root development. Recombinant AtPLA proteins hydrolyze preferentially galactolipids and, less efficiently, phospholipids, although these enzymes are not localized in chloroplasts. We find that AtPLAIVA and AtPLAIVB are phosphorylated by calcium-dependent protein kinases in vitro and this enhances their activities on phosphatidylcholine but not on phosphatidylglycerol. Taken together, the data reveal novel functions of pPLAs in root development with individual roles at the interface between phosphate deficiency and auxin signaling.

Arabidopsis Protein Phosphatase 2C ABI1 Interacts with Type I ACC Synthases and Is Involved in the Regulation of Ozone-Induced Ethylene Biosynthesis

Ethylene plays a crucial role in various biological processes and therefore its biosynthesis is strictly regu- lated by multiple mechanisms. Posttranslational regulation, which is pivotal in controlling ethylene biosynthesis, impacts 1-aminocyclopropane 1-carboxylate synthase （ACS） protein stability via the complex interplay of specific factors. Here, we show that the Arabidopsis thaliana protein phosphatase type 2C, ABI1, a negative regulator of abscisic acid signaling, is involved in the regulation of ethylene biosynthesis under oxidative stress conditions. We found that ABI1 interacts with ACS6 and dephosphorylates its C-terminal fragment, a target of the stress-responsive mitogen-activated protein kinase, MPK6. In addition, ABI1 controls MPK6 activity directly and by this means also affects the ACS6 phosphorylation level. Consistently with this, ozone-induced ethylene production was significantly higher in an ABI1 knockout strain （abiltd） than in wild-type plants. Importantly, an increase in stress-induced ethylene production in the abiltd mutant was compen- sated by a higher ascorbate redox state and elevated antioxidant activities. Overall, the results of this study provide evi- dence that ABI1 restricts ethylene synthesis by affecting the activity of ACS6. The ABI1 contribution to stress phenotype underpins its role in the interplay between the abscisic acid （ABA） and ethylene signaling pathways.

The Chloroplast Kinase Network： New Insights from Large-Scale Phosphoproteome Profiling

Protein phosphorylation is one of the most important posttranslational modifications in eukaryotic cells and affects almost all basic cellular processes. The chloroplast as plant-specific cell organelle with important metabolic functions is integrated into the cellular signaling and phosphorylation network. Recent large-scale chloroplast phosphoproteome analyses in Arabidopsis have provided new information about phosphorylation targets and expanded the list of chloroplast metabolic and regulatory functions that are potentially controlled by protein phosphorylation. Phosphorylated peptides identified from chloroplast proteins provide new insights into phosphorylation motifs, protein kinase activities, and substrate utilization. Phosphorylation sites in protein kinases can reveal chloroplast phosphorylation cascades that may network different functions by integrating signaling chains. Our review provides a meta-analysis of currently available chloroplast phosphoproteome information and discusses biological insights from large-scale chloroplast phosphoprotein profiling as well as technological constraints of kinase network analysis.