Bidirectional changes in synapsin I phosphorylation at MAP kinase-dependent sites by acute neuronal excitation in vivo

Synapsin I is a synaptic vesicle-associated protein which is phosphorylated at multiple sites by various kinases. It has been proposed to play a role in the regulation of neurotransmitter release and the organization of cytoskeletal architecture in the presynaptic terminal. To better understand the physiological regulation of its phosphorylation in vivo, we induced acute, reversible neuronal excitation by electroconvulsive treatment (ECT) in rats, and studied its effects on synapsin I phosphorylation at sites 3, 4/5 and 6 by immunoblot analyses of homogenates from hippocampus and parietal cortex using phospho-site-specific antibodies. A decrease in phosphorylation at all sites was observed soon after the electrical stimulation, followed by a large increase in phosphorylation at site 4/5 peaking at 5 min and a moderate increase in phosphorylation at site 6 peaking at 20 min. Systemic injection of SL327, a mitogen-activated protein kinase (MAPK) kinase inhibitor, prior to ECT, suppressed the increase in phospho-site 4/5 level, as well as that in MAPK activity, but not that in phospho-site 6 level. Thus, phosphorylation at site 4/5 of synapsin I has been shown to be regulated by MAPK in vivo.     

Partial purification and characterization of a protein kinase that is activated by nuclear localization signal peptides

A nuclear localization signal (NLS) is required for the transport of karyophilic proteins from the cytoplasm to the nucleus. In this study, NLS was examined in terms of its effect on diverse cellular functions such as protein phosphorylation reactions. When synthetic peptides containing the NLS of SV40 T-antigen were injected into the cytoplasm of Xenopus oocytes, and the oocytes incubated with [³²P]phosphorus-containing medium, a 32 kDa protein was found to be preferentially phosphorylated in an NLS-dependent manner. The incubation of fractionated cytosolic extracts prepared from mouse Ehrlich ascites tumor cells with [γ-³²P]ATP in the presence of the NLS peptides, results in the stimulation of the phosphorylation of several proteins. Similar in vitro stimulation was observed by other functional NLS peptides such as those of polyoma virus T-antigen and nucleoplasmin. Little or no stimulation, however, was detected for peptides of mutant type and reverse type NLS of SV40 T-antigen, and the C-terminal portion of lamin B. Using an in vitro assay, the phosphorylation activity was fractionated chromatographically and a fraction was obtained which contained a high level of activity. The fraction was found to contain three major proteins having molecular masses of 95, 70, and 43 kDa. The in vivo and in vitro results are consistent with the existence of a protein kinase, called NLS kinase, that is specifically activated by NLS peptides.     

Pervanadate-induced reverse translocation and tyrosine phosphorylation of phorbol ester-stimulated protein kinase C betaII are mediated by Src-family tyrosine kinases in porcine neutrophils

Protein kinase C (PKC), upon activation, translocates from the cytosol to the plasma membrane. Phorbol 12-myristate 13-acetate (PMA), a potent PKC activator, is known to induce irreversible translocation of PKC to the plasma membrane, in contrast to the reversible translocation resulting from physiological stimuli and subsequent rapid return to the cytosol (reverse translocation). However, we have previously shown that tyrosine phosphatase (PTPase) inhibitors induce reverse translocation of PMA-stimulated PKCbetaII in porcine polymorphonuclear leukocytes (PMNs). In the present study, we showed that pervanadate, a potent PTPase inhibitor, also induces tyrosine phosphorylation of PMA-stimulated PKCbetaII in porcine PMNs. Furthermore, PP2, a specific inhibitor of Src-family tyrosine kinases (PTKs), was found to inhibit both pervanadate-induced reverse translocation and tyrosine phosphorylation of PMA-stimulated PKCbetaII, suggesting that these two pervanadate-induced responses are mediated by Src-family PTKs. Our findings provide novel insight into the relationship between the subcellular localization and tyrosine phosphorylation of PKC.     

Identification of phosphorylation sites on phosducin-like protein by QTOF mass spectrometry.

Post-translational modifications are used by cells to control the functions of proteins. Phosducin-like protein (PhLP) is a regulator of G-protein signaling that is post-translationally modified via phosphorylation. Phosphorylation of PhLP initiates its degradation by the 26S proteasome in serum-stimulated cells. In this report, we show that PhLP is phosphorylated in serum-stimulated Chinese hamster ovary (CHO) cells. Through the use of tandem mass spectrometry (MS/MS), the specific amino acids phosphorylated can be identified. A PhLP-myc-His construct was purified and phosphorylated by serum-stimulated CHO extract. The resulting protein was digested with trypsin and the peptides were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Automated collison-induced dissociation data acquisition was compared with LC-MS/MS of manually chosen parents. In general, LC-MS/MS is superior for parent ions chosen manually, with the notable exception that automated fragmentation employs dynamic collision energy, which can result in higher quality collison-induced dissociation. Using the LC-MS/MS methods, four phosphorylation sites on PhLP were positively identified.     

A novel nuclear trafficking module regulates the nucleocytoplasmic localization of the rabies virus interferon antagonist, p protein

Regulated nucleocytoplasmic transport of proteins is central to cellular function and dysfunction during processes such as viral infection. Active protein trafficking into and out of the nucleus is dependent on the presence within cargo proteins of intrinsic specific modular signals for nuclear import (nuclear localization signals, NLSs) and export (nuclear export signals, NESs). Rabies virus (RabV) phospho (P) protein, which is largely responsible for antagonising the host anti-viral response, is expressed as five isoforms (P1-P5). The subcellular trafficking of these isoforms is thought to depend on a balance between the activities of a dominant N-terminal NES (N-NES) and a distinct C-terminal NLS (C-NLS). Specifically, the N-NES-containing isoforms P1 and P2 are cytoplasmic, whereas the shorter P3-P5 isoforms, which lack the N-NES, are believed to be nuclear through the activity of the C-NLS. Here, we show for the first time that RabV P contains an additional strong NLS in the N-terminal region (N-NLS), which, intriguingly, overlaps with the N-NES. This arrangement represents a novel nuclear trafficking module where the N-NLS is inactive in P1 but becomes activated in P3, concomitant with truncation of the N-NES, to become the principal targeting signal conferring nuclear accumulation. Understanding this unique switch arrangement of overlapping, co-regulated NES/NLS sequences is vital to delineating the critical role of RabV P protein in viral infection.     

The nuclear localization of SET mediated by impalpha3/impbeta attenuates its cytosolic toxicity in neurons

SET is a multi-functional protein in proliferating cells. Some of the proposed functions of SET suggest an important nuclear role. However, the nuclear import pathway of SET is also unknown and the function of SET in neurons is unclear. Presently, using cortical neurons, we report that the nuclear import of SET is mediated by an impalpha/impbeta-dependent pathway. Nuclear localization signal, (168)KRSSQTQNKASRKR(181), in SET interacts with impalpha3, which recruits impbeta to form a ternary complex, resulting in efficient transportation of SET into nucleus. By in vitro nuclear import assay based on digitonin-permeabilized neurons, we further demonstrated that the nuclear import of SET relies on Ran GTPase. We provide evidence that this nuclear localization of SET is important in neuronal survival. Under basal conditions, SET is predominately nuclear. However, upon death induced by genotoxic stress, endogenous SET decreases in the nucleus and increases in the cytoplasm. Consistent with a toxic role of SET in the cytoplasm, targeted expression of SET to the cytoplasm exacerbates death compared to wild type SET expression which is protective following DNA damage. Taken together, our results indicate that SET is imported into the nucleus through its association with impalpha3/impbeta, and that localization of SET is important in regulation of neuronal death.     

LukS-PV,a component of Panton-Valentine leukocidin,exerts potent activity against acute myeloid leukemia in vitro and in vivo

LukS-PV, a component of Panton-Valentine leukocidin (PVL) secreted by Staphylococcus aureus, has been shown to inhibit proliferation and induce apoptosis in acute myeloid leukemia (AML) THP-1 cells. Here we investigated anti-leukemia activities of LukS-PV in HL-60 cells, using in vitro assays to assess the ability of LukS-PV to mediate cell viability, apoptosis and differentiation; and developing a Severe Combined Immunodeficiency (SCID) mouse model of disseminated AML with the HL-60 cells to examine in vivo anti-leukemia activity. LukS-PV inhibited viability and induced differentiation and apoptosis in the HL-60 AML cell line. In the SCID mice, LukS-PV potently inhibited tumor growth, decreased tumor cell infiltration into peripheral blood and tissues, and significantly increased mean survival time. No severe adverse effects, such as death, weight loss, or pathological changes in livers or spleens were observed in the toxicity test group. These results indicate that LukS-PV may be a novel and effective chemotherapeutic agent against AML.     

Prediction of kinase-specific phosphorylation sites with sequence features by a log-odds ratio approach

Protein phosphorylation plays important roles in a variety of cellular processes. Detecting possible phosphorylation sites and their corresponding protein kinases is crucial for studying the function of many proteins. This article presents a new prediction system, called PhoScan, to predict phosphorylation sites in a kinase-family-specific way. Common phosphorylation features and kinase-specific features are extracted from substrate sequences of different protein kinases based on the analysis of published experiments, and a scoring system is developed for evaluating the possibility that a peptide can be phosphorylated by the protein kinase at the specific site in its sequence context. PhoScan can achieve a specificity of above 90% with sensitivity around 90% at kinase-family level on the data experimented. The system is applied on a set of human proteins collected from Swiss-Prot and sets of putative phosphorylation sites are predicted for protein kinase A, cyclin-dependent kinase, and casein kinase 2 families. PhoScan is available at http://bioinfo.au.tsinghua.edu.cn/phoscan/.     

Nuclear protein phosphorylation in isolated nuclei from HeLa cells. Evidence that 32P incorporation from [γ-32P]GTP is catalyzed by nuclear kinase II

A nuclear system for studying nuclear protein phosphorylation is characterized, using as phosphate donor either low levels of [γ-³²P]GTP, low levels of [γ-³²P]ATP, or low levels of labeled ATP plus excess unlabeled GTP. Since nuclear casein kinase II is the only described nuclear protein kinase to use GTP with high affinity, low levels of GTP should specifically assay this enzyme. ATP should measure all kinases, and ATP plus unlabeled GTP should measure all kinases except nuclear casein kinase II (ATP-specific kinases). The results are consistent with these predictions. In contrast with the ATP-specific activity, endogenous phosphorylation with GTP was enhanced by 100 mM NaCl, inhibited by heparin and quercetin, stimulated by polyamines, and did not use exogenous histone as substrate. The GTP- and ATP-specific kinases phosphorylated different subsets of about 20 endogenous polypeptides each. Addition of purified casein kinase II enhanced the GTP-supported phosphorylation of the identical proteins that were phosphorylated by endogenous kinase. These results support the hypothesis that activity measured with GTP is catalyzed by nuclear casein kinase II, though other minor kinases which can use GTP are not ruled out. Preliminary observations with this system suggest that the major nuclear kinases exist in an inhibited state in nuclei, and that the effects of polyamines on nuclear casein kinase II activity are substrate specific. This nuclear system is used to determine if the C-proteins of hnRNP particles, previously shown to be substrates for nuclear casein kinase II in isolated particles, is phosphorylated by GTP in intact nuclei. The results demonstrate that the C-proteins are effectively phosphorylated by GTP, but in addition they are phosphorylated by ATP-specific kinase activity.     

SCCRO (DCUN1D1) promotes nuclear translocation and assembly of the neddylation E3 complex

SCCRO/DCUN1D1/DCN1 (squamous cell carcinoma-related oncogene/defective in cullin neddylation 1 domain containing 1/defective in cullin neddylation) serves as an accessory E3 in neddylation by binding to cullin and Ubc12 to allow efficient transfer of Nedd8. In this work we show that SCCRO has broader, pleiotropic effects that are essential for cullin neddylation in vivo. Reduced primary nuclear localization of Cul1 accompanying decreased neddylation and proliferation in SCCRO(-/-) mouse embryonic fibroblasts led us to investigate whether compartmentalization plays a regulatory role. Decreased nuclear localization, neddylation, and defective proliferation in SCCRO(-/-) mouse embryonic fibroblasts were rescued by transgenic expression of SCCRO. Expression of reciprocal SCCRO and Cul1-binding mutants confirmed the requirement for SCCRO in nuclear translocation and neddylation of cullins in vivo. Nuclear translocation of Cul1 by tagging with a nuclear localization sequence allowed neddylation independent of SCCRO, but at a lower level. We found that in the nucleus, SCCRO enhances recruitment of Ubc12 to Cul1 to promote neddylation. These findings suggest that SCCRO has an essential role in neddylation in vivo involving nuclear localization of neddylation components and recruitment and proper positioning of Ubc12.     

The molecular signature of oncofusion proteins in acute myeloid leukemia

Acute myeloid leukemia (AML) associated translocations often cause gene fusions that encode oncofusion proteins. Although many of the breakpoints involved in chromosomal translocations have been cloned, in most cases the role of the chimeric proteins in tumorigenesis is not elucidated. Here we will discuss the fusion proteins of the 4 most common translocations associated with AML as well as the common molecular mechanisms that these four and other fusion proteins utilize to transform progenitor cells. Intriguingly, although the individual partners within the fusion proteins represent a wide variety of cellular functions, at the molecular level many commodities can be found.     

Identification of survival-related alternative splicing signatures in acute myeloid leukemia

Aberrant RNA alternative splicing (AS) variants play critical roles in tumorigenesis and prognosis in human cancers. Here, we conducted a comprehensive profiling of aberrant AS events in acute myeloid leukemia (AML). RNA AS profile, including seven AS types, and the percent spliced in (PSI) value for each patient were generated by SpliceSeq using RNA-seq data from TCGA. Univariate followed by multivariate Cox regression analysis were used to identify survival-related AS events and develop the AS signatures. A nomogram was developed, and its predictive efficacy was assessed. About 27,892 AS events and 3,178 events were associated with overall survival (OS) after strict filtering. Parent genes of survival-associated AS events were mainly enriched in leukemia-associated processes including chromatin modification, autophagy, and T-cell receptor signaling pathway. The 10 AS signature based on seven types of AS events showed better efficacy in predicting OS of patients than those built on a single AS event type. The area under curve (AUC) value of the 10 AS signature for 3-year OS was 0.91. Gene set enrichment analysis (GSEA) confirmed that these survival-related AS events contribute to AML progression. Moreover, the nomogram showed good predictive performance for patient''s prognosis. Finally, the correlation network of AS variants with splicing factor genes found potential important regulatory genes in AML. The present study presented a systematic analysis of survival-related AS events and developed AS signatures for predicting the patient’s survival. Further studies are needed to validate the signatures in independent AML cohorts and might provide a promising perspective for developing therapeutic targets.     

Identification of the major sites of phosphorylation in IGF binding protein-3

Insulin-like growth factor binding protein-3 (IGFBP-3) is the major carrier of insulin-like growth dactor I and II in the circulation. IGFBP-3 is secreted by various tissues and cell lines as a glycosylated phosphoprotein. We have identified two major serine phosphorylation sites located at amino acids 111 and 113 of the human protein. These serine residues ngighboring amino acids potentially involved in defining a protrein kinase recognition sequence were mutated to alanine using PCR. Single and double point mutants were stably transfected into CHO-cells and analyzed for their level of phosphorylation. Mutation of both serines reduced phosphorylation by 80% in the full-length protein and completely abolished phosphorylation in a 17 kDa IGFBP-3 fragment, derived from digestion with EndoProteinase Lys-C. The 17kDa fragment containd serines 111 and 113. S111A/S113A, a double serine-to-alanine mutant at positions 111 and 113, showed a strongly reduced glycosylation pattern that appears to be the result of anino acid substitutions rather tha n lack of phosphrylation. Mutant S111A/S113A, despite being non-phosphorylated and non-glycosylated, is functionally similar to the wild-type IGFBP-3 in terms of IGF-1 binding. These results enhance our non-glycosylated, is functionally similar to the wild-type IGFBP-3 in terms of IGF-I binding. These results enhance our understanding on the functional role of glycosylation and phosphorylation of IGFBP-3.     

PLMT家族成员SET7/9的非组蛋白甲基化作用

SET7/9是蛋白赖氨酸甲基化转移酶（protein lysine methyltransferases,PLMTs或PKMTs）家族成员,具有SET结构域。现已发现SET7/9是一种赖氨酸单甲基化转移酶,除了能使组蛋白H3第四位赖氨酸（lysine4 of histone 3,H3K4）单甲基化外,更重要的能使一些转录因子、肿瘤抑制因子、膜相关受体等非组蛋白单甲基化,其甲基化作用主要与蛋白稳定和转录活化有关。该效应受赖氨酸特异性去甲基酶1（lysine specifcdemethylase,LSD1）的抑制。SET7/9与LSD1两者效应的平衡对维持体内活性蛋白质含量、调节基因表达具有重要意义。     

3D environment controls H3K4 methylation and the mechanical response of the nucleus in acute lymphoblastic leukemia cells

Acute lymphoblastic leukemia (ALL) is the most common pediatric cancer, and the infiltration of leukemic cells is critical for disease progression and relapse. Nuclear deformability plays a critical role in cancer cell invasion through confined spaces; however, the direct impact of epigenetic changes on the nuclear deformability of leukemic cells remains unclear. Here, we characterized how 3D collagen matrix conditions induced H3K4 methylation in ALL cell lines and clinical samples. We used specific shRNA and chemical inhibitors to target WDR5 (a core subunit involved in H3K4 methylation) and determined that targeting WDR5 reduced the H3K4 methylation induced by the 3D environment and the invasiveness of ALL cells in vitro and in vivo. Intriguingly, targeting WDR5 did not reduce the adhesion or the chemotactic response of leukemia cells, suggesting a different mechanism by which H3K4 methylation might govern ALL cell invasiveness. Finally, we conducted biochemical, and biophysical experiments to determine that 3D environments promoted the alteration of the chromatin, the morphology, and the mechanical behavior of the nucleus in ALL cells. Collectively, our data suggest that 3D environments control an upregulation of H3K4 methylation in ALL cells, and targeting WDR5 might serve as a promising therapeutic target against ALL invasiveness in vivo.     

Adenylate effects on protein phosphorylation in the interenvelope lumen of pea chloroplasts

A 64-kilodalton (kDa) protein, situated in the lumen between the inner and outer envelopes of pea (Pisum sativum L.) chloroplasts (Soll and Bennett 1988, Eur. J. Biochem., 175, 301–307) is shown to undergo reversible phosphorylation in isolated mixed envelope vesicles. It is the most conspicuously labelled protein after incubation of envelopes with 33 nmol·1^-1 [-³²P]ATP whereas incubation with 50 mol·1^-1 [-³²P]ATP labels most prominently two outer envelope proteins (86 and 23 kDa). Half-maximum velocity for phosphorylation of the 64-kDa protein occurs with 200 nmol·1^-1 ATP, and around 40 mol·1^-1 ATP for phosphorylation of the 86- and 23-kDa proteins, indicating the operation of two distinct kinases. GGuanosine-, uridine-, cytidine 5-triphosphate and AMP are poor inhibitors of the labelling of the 64-kDa protein with [-³²P]ATP. On the other hand, ADP has a potent influence on the extent of labelling (half-maximal inhibition at 1–5 mol·1^-1). The ADP-dependent appearance of ³²P in ATP indicates that ADP acts by reversal of kinase activity and not as a competitive inhibitor. However, the most rapid loss of ³²P from pre-labelled 64-kDa protein occurs when envelope vesicles are incubated with ATP t1/2=15 s at 20 molsd1^-1 ATP). This induced turnover of phosphate appears to be responsible for the rapid phosphoryl turnover seen in situ.Abbreviations LHCP ligh-harvesting chlorophyll-a/b-binding protein - S_0.5 concentration giving half-maximal phosphorylation - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - Tricine N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine     

Identification of a phosphoprotein in the nuclear matrix by monoclonal antibodies.

Previous work in this laboratory has established that a rat liver nuclear phosphoprotein (B2:Mr 68,000, pI 6.5-8.2) is associated with actively transcribed nucleosomes, as demonstrated by its preferential release after mild treatment with micrococcal nuclease. In the present report we provide further immunological evidence ('Western Blot' analysis, solid-phase radioimmunoassay and indirect immunofluorescence) that in addition establishes the presence of this phosphoprotein in the nuclear-matrix protein fraction. This paradoxical localization suggests that this phosphoprotein may function in two separate and distinct roles within the realm of nuclear organization.     

Initial identification of a phosphoprotein that appears to be involved in the induction of somatic embryogenesis in carrot

We examined changes in the absence versus presence patterns of phosphoproteins with respect to the acquisition of embryogenic competence during somatic embryogenensis in carrot (Daucus carota L.). To characterize a possible correlation between the induction of embryogenic competence and protein phosphorylation, we examined the patterns of protein phosphorylation in embryogenic cells (EC) and non-embryogenic cells (NC) that had lost the ability to form somatic embryos. Two-dimensional polyacrylamide gel electrophoresis and subsequent autoradiography revealed the presence of 31 phosphoproteins in EC but not in NC. Furthermore, when we examined the induction of somatic embryogenesis by certain stress compounds in the absence of phytohormones, we identified one specific phosphoprotein (ECPP-44). ECPP-44 was found to be induced in all treatments that resulted in embryogenic competence. The partial amino acid and nucleotide sequence of ECPP-44 shows partial homology to two dehydrins (ERD10 and ERD14) from Arabidopsis. Received: 1 October 1999 · Accepted: 3 November 1999