IndraLab
Statements
NPM1 is modified
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rlimsp
"Other antibodies used include: Mouse monoclonal anti-p53 (DO-1) and either a rabbit or goat polyclonal anti-HA (HA-probe (Y-11); Santa Cruz Biotechnology, Santa Cruz, CA, USA); mouse monoclonal anti-actin (Ab-1; Calbiochem, La Jolla, CA, USA); mouse monoclonal anti-NPM (mouse anti-Nucleophosmin (FC-61991); Invitrogen, Carlsbad, CA, USA); rabbit monoclonal anti-pNPM(S4) (Phospho-NPM (Ser4) (D19C1) XP® Rabbit mAb), rabbit polyclonal anti-pNPM(T199) (Phospho-NPM (Thr199) Antibody), rabbit monoclonal anti-pCDC25C(S216) (Phospho-CDC25C (Ser216)(63F9) Rabbit mAb) and rabbit monoclonal anti-CDC25C (cdc25C (5H9) rabbit mAb; all from Cell Signaling Technology, Beverly, MA, USA); and rat monoclonal anti-HA (Anti-HA affinity clone3F10; Roche Applied Science, Indianapolis, IN, USA)."
rlimsp
"Phosphorylation regulates nucleophosmin targeting to the centrosome during mitosis as detected by cross-reactive phosphorylation-specific MKK1/MKK2 antibodies. Phosphorylation-specific antibodies provide a powerful tool for analysing the regulation and activity of proteins in the MAP (mitogen-activated protein) kinase and other signalling pathways. Using synchronized cells, it was observed that phosphorylation-specific antibodies developed against the active form of MKK1/MKK2 (MAP kinase kinase-1 and -2) reacted with a protein that was approx. 35 kDa during G2/M-phase of the cell cycle. Failure of the 35 kDa protein to react with phosphorylation-independent MKK1/MKK2 antibodies suggested that this protein was not related to MKK1 or MKK2. Thus the 35 kDa protein was isolated by immunoprecipitation with the phospho-MKK1/MKK2 antibody and identified by MS. Peptide sequence analysis revealed matches with NPM (nucleophosmin/B23), a phosphoprotein involved in nucleolar assembly, centrosome duplication and ribosome assembly and transport. Biochemical and immunocytochemistry analyses verified that the phospho-MKK1/MKK2 antibodies cross-reacted with NPM that was phosphorylated at Thr234 and Thr237 during G2/M-phase, which are the same sites that are targeted by Cdc2 (cell division cycle protein-2) during mitosis. Using phosphorylation site mutants, we show that phosphorylation of Thr234 and Thr237 is required for NPM immunoreactivity with the phospho-MKK1/MKK2 antibody. Moreover, phosphorylation of Thr234 and Thr237 was demonstrated to regulate NPM localization to the centrosome after nuclear envelope breakdown in mitotic cells."
rlimsp
"By overexpressing Flag-tagged NPM and its phosphorylation site mutant (Thr234/237A) into low p-NPM-Thr234/237 expressing cells (Hep3B and Huh7) using a lentiviral based approach, we demonstrated that p-NPM-Thr234/237 is critical in invasion and migration of HCC cells, and this effect was mediated by cyclin-dependent kinase 1 (CDK1)."
sparser
"Further, temporal analysis of phosphoproteome change revealed that phosphorylation of NPM S254 and COPA S173 was observed from the early (6 h) and late (24 h) time point after nocodazole treatment, respectively, suggesting that NPM S254 may be involved in the induction of M-phase arrest by nocodazole, whereas COPA S173 may be caused as a result of M-phase arrest."
rlimsp
"Total NPM1 expression did not appear to change upon N6L treatment neither with time of treatment nor with the concentration used whereas a substantial decrease in NPM1 phosphorylation was observed with 20 μmol/L N6L (63% for Thr199 and 86% for Thr234/237 of reduction at 48 hours) (Figure 2A and 2B)."
sparser
"Having established that the phosphorylation of NPM-Ser48 by AKT promotes ARF nucleoplasmic localization, MDM2 inhibition and the stabilization of p53 mut , we next wished to address if phosphorylation of NPM-Ser48 was a common phenomenon, and potentially contributing to the stabilization of p53 mut in human tumors."
sparser
"It has been reported that ATM-mediated phosphorylation of NPM1 can inhibit the binding of NPM1 with p14ARF, and thereby promoting the translocation of p14ARF to nucleoplasm. xref , xref Besides, AKT-mediated phosphorylation of NPM-Ser48 can prevent the oligomerization of NPM1, and subsequently results in nucleoplasmic localization of p14ARF, constitutive HDM2 inhibition and stabilization of p53. xref However, we observed no effect of SOX6 on the phosphorylation of NPM1."
sparser
"In protein spots that significantly contributed to our signature, we found that glyceraldehyde-3-phosphate dehydrogenase was N-terminally truncated, pyruvate kinase M2 and nucleoside diphosphate kinase A but not other isoforms of these proteins were of importance, and nucleophosmin phosphorylation at serine residues 106 and 125 were clearly identified."
rlimsp
"Furthermore, knockdown of PP1β, but not of PP1α or PP1γ, blocked the UV-induced dephosphorylation of NPM on Thr199 and Thr234/237 (Figure 2B and Supplemental Figure S3B). Similar outcomes of PP1β knockdown also were observed in 293T cells (Supplemental Figure S3C). Consistently, when PP1β was overexpressed in cells, phosphorylation at these threonine residues underwent considerable reduction (Supplemental Figure S3D)."
rlimsp
"(b) In vivo sequential phosphorylation of NPM. MCF-7 cells were transfected with either HA-NPM(WT) or HA-NPM(T199A) for 40 h then treated with 4 μg/ml nocodazole for 16 h. Lysates were then purified with mouse monoclonal anti-HA and analysed by Western blotting with polyclonal anti-HA and the same antibodies as Fig. 3. (c) Decrease of phosphorylated-CDC25C at Ser216 by PPM1D overexpression in H1299 clones."
sparser
"In this regard, PKA participates in the separation of the centrioles by phosphorylating its structural component centrin. xref It is probable that AKAP350 integrates different events involved in the initiation of centrosomal duplication, such as phosphorylation of centrin by PKA, and phosphorylation of NPM by Cdk2."
reach
"Furthermore, hyperactive Cdk2 and Cdk4 deregulate the licensing of the centrosome duplication cycle in p53-null cells by hyperphosphorylating nucleophosmin (NPM) at Thr199, as evidenced by observations that ablation of Cdk2, Cdk4, or both Cdk2 and Cdk4 abrogates that excessive phosphorylation."
"Both subtypes of B23 proteins were phosphorylated during mitosis by cyclin B/cdc2. The RNA binding activity of B23.1 was repressed through cyclin B/cdc2-mediated phosphorylation at specific sites in B23. Thus, the RNA binding activity of B23.1 is stringently modulated by its phosphorylation and subtype association."
"We have recently found that nucleophosmin (npm/b23), a phosphoprotein primarily found in nucleolus, associates with unduplicated centrosomes and is a direct substrate of cdk2-cyclin e in centrosome duplication. Upon phosphorylation by CDK2-cyclin E, NPM/B23 dissociates from centrosomes, which is a prerequisite step for centrosomes to initiate duplication."
rlimsp
"We also demonstrated for the first time that PPM1D up-regulated phosphorylation of Thr199 and Ser4 of NPM sequentially via the CDC25C-CDK1-PLK1 signalling cascade thereby modulating nucleolar formation (Fig. 6). While phosphorylation at Thr199 by CDK1 and Ser4 by PLK1 has been reported in separate studies27383940, an association between these sites has not previously been reported."
rlimsp
"We also demonstrated for the first time that PPM1D up-regulated phosphorylation of Thr199 and Ser4 of NPM sequentially via the CDC25C-CDK1-PLK1 signalling cascade thereby modulating nucleolar formation (Fig. 6). While phosphorylation at Thr199 by CDK1 and Ser4 by PLK1 has been reported in separate studies27383940, an association between these sites has not previously been reported. Our results suggested that phosphorylation of Thr199 by CDK1 was important for phosphorylation of Ser4 by PLK1. Phosphorylation at Thr199 by CDK1 was suggested to be involved in dissociation of NPM from the centrosome, leading to centrosome duplication40, and in regulation of RNA binding activity of NPM3839."
rlimsp
"NPM phosphorylation at Thr234/237 is the only NPM phosphorylation site that spotted on this phospho-peptide array which is specifically phosphorylated by CDK1 (Figure 1B). Western blot analysis confirmed elevated p-NPM-Thr234/237 and CDK1 in PLC/PRF/5-LM cells using a phospho-NPM (Thr234/237) specific antibody (Biolegend) (Figure 1C)."
"Both subtypes of B23 proteins were phosphorylated during mitosis by cyclin B/cdc2. The RNA binding activity of B23.1 was repressed through cyclin B/cdc2-mediated phosphorylation at specific sites in B23. Thus, the RNA binding activity of B23.1 is stringently modulated by its phosphorylation and subtype association."
"Both subtypes of B23 proteins were phosphorylated during mitosis by cyclin B/cdc2. The RNA binding activity of B23.1 was repressed through cyclin B/cdc2-mediated phosphorylation at specific sites in B23. Thus, the RNA binding activity of B23.1 is stringently modulated by its phosphorylation and subtype association."
rlimsp
"We also demonstrated for the first time that PPM1D up-regulated phosphorylation of Thr199 and Ser4 of NPM sequentially via the CDC25C-CDK1-PLK1 signalling cascade thereby modulating nucleolar formation (Fig. 6). While phosphorylation at Thr199 by CDK1 and Ser4 by PLK1 has been reported in separate studies27383940, an association between these sites has not previously been reported. Our results suggested that phosphorylation of Thr199 by CDK1 was important for phosphorylation of Ser4 by PLK1."
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"As phosphorylation of human NPM by cyclin E-cdk2 was reported to be essential for the initiation of centrosome duplication in late G 1, we considered that ARF might inhibit NPM phosphorylation in addition to retaining it in the nucleolus to arrest cell growth before S-phase entry."
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"The exact role of this viral protein in regulating KSHV life cycle is not fully understood but studies indicate that v, Cyclin, and CDK6 complex mediated phosphorylation of nucleophosmin (NPM) facilitates NPM-LANA interaction and recruitment of HDAC1 to promote KSHV latency [XREF_BIBR]."
sparser
"At late G 1 phase, NPM is phosphorylated by cyclin E-Cdk2, phosphorylation which induces NPM release from the centrosomes and the initiation of centrosomal duplication. xref We found that AKAP350CTD expression induced an increase in Cdk2 specific phosphorylation of NPM, indicating that AKAP350 enables Cdk2 centrosomal activity at the G 1 /S transition."
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"In transactivation activity dependent mechanism, p21 expression plays a key role in synchronizing DNA replication and centrosome duplication by inhibiting Cdk2 and Cyclin E activity which phosphorylates Nucleophosmin and NPM1 at centrosomes to promote its dissociation from the centrosomes to allow initiation of centrosome duplication."
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"Interestingly, a recent report demonstrated that ATM kinase negatively regulates ARF by ubiquitin dependent degradation : ATM activated protein phosphatase 1 (PP1) antagonized Nek2 dependent phosphorylation of nucleophosmin (NPM) to liberate ARF from NPM and render it susceptible to degradation by the ULF E3-ubiquitin ligase [XREF_BIBR]."
sparser
"Interestingly, a recent report demonstrated that ATM kinase negatively regulates ARF by ubiquitin-dependent degradation: ATM-activated protein phosphatase 1 (PP1) antagonized Nek2-dependent phosphorylation of nucleophosmin (NPM) to liberate ARF from NPM and render it susceptible to degradation by the ULF E3-ubiquitin ligase [ xref ]."
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"To determine whether IKKalpha phosphorylates NPM, we used different NPM deletion and mutation forms as kinase substrates and found that IKKalpha phosphorylated the amino acid (aa) 119-195 region, which contains five serine sites, but a kinase-dead IKKalpha mutant (IKKalpha-KA) did not phosphorylate NPM (XREF_FIG; XREF_SUPPLEMENTARY)."
sparser
"To determine whether IKKα phosphorylates NPM, we used different NPM deletion and mutation forms as kinase substrates and found that IKKα phosphorylated the amino acid (aa) 119–195 region, which contains five serine sites, but a kinase-dead IKKα mutant (IKKα-KA) did not phosphorylate NPM ( xref ; xref )."
sparser
"The NPM/ARF interaction can be disrupted by DNA damage as well as phosphorylation of NPM at Ser48 by Akt, both of which lead to ARF’s nucleoplasmic transition and interaction with MDM2. xref , xref Following DNA damage, cJun can interact with NPM and cause NPM and ARF redistribution, an event that requires JunB, JNK activation and its phosphorylation of cJun at Thr91 and Thr93. xref "
sparser
"Although more assays should be performed to identify the interaction between Aurora-B and NPM1, our results were consistent with Shandilya et al who found that NPM1 can be phosphorylated by Aurora-B and participate in mitotic progression. xref Further studies demonstrated that the inhibitory effects induced by Aurora-B silencing could be partially reversed by NPM1 overexpression."
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"Interestingly, CIGB-300 was shown to bind NPM1 and to inhibit CK2-mediated phosphorylation of NPM1 at Ser125 inducing damage to the nucleolar architecture and massive apoptosis [149], in line with the putative implication of NPM1 phospho-Ser125 in the maintenance of the nucleolar assembly, in ribosome biogenesis, and in cytokinesis [153,154,155]."
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"During interphase, NPM1 has also been reported to be phosphorylated by casein kinase 2 (CK2) and this is thought to have a role in regulating the nucleolar structure by modulating the dynamic localization of NPM1 between nucleolus and nucleoplasm (Szebeni et al. 2003; Negi and Olson 2006)."
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sparser
"CK2 phosphorylates NPM at Ser125, which is located in one of NPM’s acidic stretches, a region that, along with its N-terminal oligomerization domain, is essential for its chaperone activity. xref Thus, CK2 phosphorylation promotes the dissociation of substrates bound to NPM. xref Furthermore, this phosphorylation during interphase enhances NPM movement through the nucleolus. xref NPM is necessary not only for pre-rRNA processing, but for the transport and nuclear export of both 40S and 60S ribosomal subunits. xref , xref , xref CK2, along with CDK1, phosphorylation may help reduce NPM’s nucleolar localization during mitosis and interfere with its binding to rRNA or ribosomal components, thereby inhibiting ribosome biogenesis."
D1K2 affects NPM1
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8
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"The exact role of this viral protein in regulating KSHV life cycle is not fully understood but studies indicate that v-Cyclin mediates phosphorylation of nucleophosmin (NPM), through its association with CDK6 and facilitates NPM-LANA interaction and recruitment of HDAC1 to promote KSHV latency [XREF_BIBR]."
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"To confirm that NPM is phosphorylated by v-cyclin on Thr199 in our experiments, U2OS cells were transfected with expression vectors for Myc tagged v-cyclin or a vector control together with eGFP tagged wild-type NPM (eGFP-NPM), or its phosphorylation site mutant T4A, with threonine to alanine mutations of NPM codons of 199, 214, 234, 237, and mutant T3A in which all but threonine 199 is replaced by alanine XREF_BIBR."
Aurora-B affects NPM1
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"Both subtypes of B23 proteins were phosphorylated during mitosis by cyclin B/cdc2. The RNA binding activity of B23.1 was repressed through cyclin B/cdc2-mediated phosphorylation at specific sites in B23. Thus, the RNA binding activity of B23.1 is stringently modulated by its phosphorylation and subtype association."
"Upon phosphorylation by CDK2-cyclin E, NPM/B23 dissociates from centrosomes, which is a prerequisite step for centrosomes to initiate duplication. Here, we identified that threonine 199 (Thr(199)) of NPM/B23 is the major phosphorylation target site of CDK2-cyclin E in vitro, and the same site is phosphorylated in vivo. NPM/T199A, a nonphosphorylatable NPM/B23 substitution mutant (Thr(199) --> Ala) acts as dominant negative when expressed in cells, resulting in specific inhibition of centrosome duplication."
V-cyclin-CDK6 affects NPM1
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3
Salicylate affects NPM1
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3
Diafenthiuron affects NPM1
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Diafenthiuron leads to the phosphorylation of NPM1. 1 / 1
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Diafenthiuron phosphorylates NPM1 on S4. 1 / 1
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Diafenthiuron phosphorylates NPM1 on T199. 1 / 1
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Phosphatase affects NPM1
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"Both subtypes of B23 proteins were phosphorylated during mitosis by cyclin B/cdc2. The RNA binding activity of B23.1 was repressed through cyclin B/cdc2-mediated phosphorylation at specific sites in B23. Thus, the RNA binding activity of B23.1 is stringently modulated by its phosphorylation and subtype association."
"Both subtypes of B23 proteins were phosphorylated during mitosis by cyclin B/cdc2. The RNA binding activity of B23.1 was repressed through cyclin B/cdc2-mediated phosphorylation at specific sites in B23. Thus, the RNA binding activity of B23.1 is stringently modulated by its phosphorylation and subtype association."
PP1beta affects NPM1
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2
KSHV latent protein v-cyclin affects NPM1
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2
sparser
"Gsk3 DKO reduced phosphorylation of the splicing factors RBM8A, SRSF9, and PSF as well as the nucleolar proteins NPM1 and PHF6, and recombinant GSK-3β phosphorylated these proteins in vitro RNA-Seq of WT and Gsk3 DKO ESCs identified ∼190 genes that are alternatively spliced in a GSK-3-dependent manner, supporting a broad role for GSK-3 in regulating alternative splicing."
G2/M checkpoint kinase affects NPM1
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2
CDK1-PLK1 affects NPM1
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2
V-cyclin associated kinase affects NPM1
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1
Titanium dioxide affects NPM1
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PChk1 affects NPM1
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1
Nuclear kinase type II [40] affects NPM1
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1
Nuclear kinase II affects NPM1
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Kinase activity affects NPM1
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Kinase activity phosphorylates NPM1. 1 / 1
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1
HLtalpha affects NPM1
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1
Deoxynivalenol affects NPM1
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CyclinB1 affects NPM1
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1
Cyclin E affects NPM1
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1
Cupric oxide affects NPM1
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Bis(tri-n-butyltin)oxide affects NPM1
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RABL6A affects NPM1
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1
PKCiota affects NPM1
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1
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"In line with our conclusion that p110alpha H1047R expression leads to centrosome overduplication in S-phase, the activation of ROCK (as measured by the phosphorylation of MLC on Ser19) and the phosphorylation of Npm on T199 (a phosphorylation site of CDK2) 30 were enhanced in p110alpha mutant cells."
NPMc affects NPM1
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1
Lymphoma, Large-Cell, Anaplastic affects NPM1
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1
Lymphoma, Large-Cell, Anaplastic leads to the phosphorylation of NPM1. 1 / 1
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1
KSHV v-cyclin affects NPM1
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1
IKKalpha-KA affects NPM1
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1
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"To determine whether IKKalpha phosphorylates NPM, we used different NPM deletion and mutation forms as kinase substrates and found that IKKalpha phosphorylated the amino acid (aa) 119-195 region, which contains five serine sites, but a kinase-dead IKKalpha mutant (IKKalpha-KA) did not phosphorylate NPM (XREF_FIG; XREF_SUPPLEMENTARY)."
IKKalpha mutant affects NPM1
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1
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"To determine whether IKKalpha phosphorylates NPM, we used different NPM deletion and mutation forms as kinase substrates and found that IKKalpha phosphorylated the amino acid (aa) 119-195 region, which contains five serine sites, but a kinase-dead IKKalpha mutant (IKKalpha-KA) did not phosphorylate NPM (XREF_FIG; XREF_SUPPLEMENTARY)."
D1/K2 affects NPM1
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1
Cyclin E affects NPM1
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1
Cyclin D1 affects NPM1
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1
Cellular D1/K2 Complexes affects NPM1
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1
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"Therefore we conclude that, depletion of 14-3-3gamma and the premature hyper-activation of cdc25C, causes hyper-activation in cdk1 and hyper-phosphorylation of NPM1 at T199 residue and other centrosome associated proteins (XREF_FIG) during interphase leading to (i) reversal of centrosome clustering, (ii) generation of extensive spindle multipolarity, (iii) cell death in culture and (iv) inhibition of tumorigenesis in mice (XREF_FIG)."
Aurora kinases A affects NPM1
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1
AKAP350CTD affects NPM1
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1