IndraLab
Statements
rlimsp
"NPM1 may play a more direct role in centrosome duplication in some cells given evidence that NPM1 associates with the unduplicated centrosome in interphase and is released by cdk2/cyclin E mediated phosphorylation on NPM1 residue Thr199 leading to initiation of centrosome duplication [117]."
rlimsp
"Specific phosphorylation of nucleophosmin on Thr(199) by cyclin-dependent kinase 2-cyclin E and its role in centrosome duplication. The kinase activity of cyclin-dependent kinase 2 (CDK2)-cyclin E is required for centrosomes to initiate duplication. 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. 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. As expected, NPM/T199A remains associated with the centrosomes. These observations provide direct evidence that the CDK2-cyclin E-mediated phosphorylation on Thr(199) determines association and dissociation of NPM/B23 to the centrosomes, which is a critical control for the centrosome to initiate duplication."
sparser
"To investigate the cell’s presumed requirement for NPM-Thr 198 phosphorylation in promoting the processes of growth and proliferation, we examined the effects of a non-phosphorylatable NPM mutant, T198A, in a clean cell system in which endogenous NPM had been removed by RNA interference."
sparser
"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."
sparser
"Considering the essential role of LANA in KSHV latency and suppression of lytic viral transcription, as well as the observation that v-cyclin promotes LANA-NPM interaction, we sought to address whether there is a correlation between NPM Thr199 phosphorylation, v-cyclin expression, and the extent of spontaneous lytic replication in four different patient-derived KSHV-infected PEL lines (BC-3, BCBL-1, BC-1, JSC-1), IHH (a KSHV-positive lymphoblastoid cell line), and IHE (a KSHV-negative cell line)."
rlimsp
"However, independent groups have demonstrated the roles of NPM, and its phosphorylation at Thr199 in the process of centrosome duplication does not account for the observed increase in cell growth and proliferation [23]. Salvianolic acid B stimulates the proliferation and tube formation of a murine simian virus resistance endothelial cell line through the sequential upregulation of the genes encoding MMP-2, VEGF, VEGF receptor 2, and Tie-1 [24]. To date, phosphorylation of NPM-Thr199 continues to be the subject of discussion and debate in the field."
sparser
"To obtain further evidence about the correlation between, v-cyclin expression levels, extent of NPM phosphorylation on Thr199, and spontaneous viral reactivation we over-expressed v-cyclin in BCBL-1 and JSC-1 cells using retroviruses expressing v-cyclin and GFP (KpBMN) or GFP (pBMN) as a control."
rlimsp
"Kanellis et al. have found that in malignant cells and normal fibroblasts a fraction of TPL2 resides in the nucleolus where it associates with and phosphorylates a pool of NPM molecules at Thr199. As this phosphorylation event is required for NPM ubiquitination and proteasomal degradation, TPL2 appears to participate in the maintenance of physiological levels of NPM."
rlimsp
"Thr199-phosphorylated NPM1 (pT199-NPM1) is recruited to nuclear DNA damage foci induced by ionizing radiation (IR). Foci formation is impaired by depletion of the E3 ubiquitin ligases RNF8 and RNF168 or the E2 Ubc13, and pT199-NPM1 binds to Lys63-linked ubiquitin polymers in vitro. Thus, phosphorylated NPM1 may interact with RNF8-dependent ubiquitin conjugates at sites of DNA damage. The interaction was found to rely on T199 phosphorylation, an acidic tract, and an adjacent ubiquitin-interacting motif-like domain."
rlimsp
"Considering the essential role of LANA in KSHV latency and suppression of lytic viral transcription, as well as the observation that v-cyclin promotes LANA-NPM interaction, we sought to address whether there is a correlation between NPM Thr199 phosphorylation, v-cyclin expression, and the extent of spontaneous lytic replication in four different patient-derived KSHV-infected PEL lines (BC-3, BCBL-1, BC-1, JSC-1), IHH (a KSHV-positive lymphoblastoid cell line), and IHE (a KSHV-negative cell line)."
rlimsp
"We have previously identified Thr(199) as the major phosphorylation site of NPM mediated by CDK2/cyclin E (and A), and this phosphorylation is involved in the regulation of centrosome duplication. In this study, we further examined the effect of CDK2-mediated phosphorylation of NPM by using the antibody that specifically recognizes NPM phosphorylated on Thr(199)."
rlimsp
"We have previously identified Thr(199) as the major phosphorylation site of NPM mediated by CDK2/cyclin E (and A), and this phosphorylation is involved in the regulation of centrosome duplication. In this study, we further examined the effect of CDK2-mediated phosphorylation of NPM by using the antibody that specifically recognizes NPM phosphorylated on Thr(199). We found that the phospho-Thr(199) NPM localized to dynamic sub-nuclear structures known as nuclear speckles, which are believed to be the sites of storage and/or assembly of pre-mRNA splicing factors. Phosphorylation on Thr(199) by CDK2/cyclin E (and A) targets NPM to nuclear speckles, and enhances the RNA-binding activity of NPM. Moreover, phospho-Thr(199) NPM, but not unphosphorylated NPM, effectively represses pre-mRNA splicing. These findings indicate the involvement of NPM in the regulation of pre-mRNA processing, and its activity is controlled by CDK2-mediated phosphorylation on Thr(199)."
rlimsp
"To obtain further evidence about the correlation between, v-cyclin expression levels, extent of NPM phosphorylation on Thr199, and spontaneous viral reactivation we over-expressed v-cyclin in BCBL-1 and JSC-1 cells using retroviruses expressing v-cyclin and GFP (KpBMN) or GFP (pBMN) as a control."
sparser
"To determine whether or not phosphorylation of murine NPM-Thr 198 is a cyclin E–cdk2-specific event within the context of cell cycle progression, TKO MEFs were serum-starved and synchronized in G 0 , evidenced by the cells’ low expression levels of cyclin D1 protein ( xref , lane 2)."
sparser
"Given that cyclin D1 protein expression levels were maximal at approximately 8 h after the cells’ release into serum, yet abundant levels of phospho-T198 NPM were already evident by 4-h post-stimulation, this result suggests that cyclin E–cdk2 is not the sole kinase which phosphorylates NPM-Thr 198 within the cell ( xref )."
rlimsp
"We then examined the requirement of NPM phosphorylation on Thr199 for NPM-LANA interaction, by performing anti-LANA immunoprecipitation from cell extracts of U2OS cells expressing LANA and transfected with expression vectors for Myc-v-cyclin, empty vector control, and the eGFP-tagged NPM wt or its phosphosite mutant constructs used in Figure S1D."
rlimsp
"Overexpression of NPMc causes increased phosphorylation of NPM on T199 and, to a lesser degree, S4. T199 phosphorylation is dependent on cdk2 but activators of cdk2 were not elevated. Upon inhibition of cdk2, NPMc-overexpressing cells demonstrate a greater G2/M phase arrest than wtNPM or GFP counterparts. However, the number of cells with 2 centrosomes did not increase concordantly. This suggests that the arrest was caused by a delay in centrosome duplication, most likely due to the inhibition of centrosome duplication caused by unphosphorylated NPMc. Overall, these results suggest that the phosphorylation of T199 is important in the mitotic progression of NPMc-expressing cells."
rlimsp
"Cytoplasmic nucleophosmin has elevated T199 phosphorylation upon which G2/M phase progression is dependent. The cytoplasmic mutant of nucleophosmin (NPMc) is found approximately in one-third of acute myeloid leukemia (AML) cases and is highly associated with normal karyotype. Whereas previous studies have focused on wtNPM in centrosome duplication, we further elucidate the role of NPM in the cell cycle by utilizing the increased cytoplasmic load of NPMc. Overexpression of NPMc causes increased phosphorylation of NPM on T199 and, to a lesser degree, S4."
sparser
"Interactions between RanGTP and Crm1 recruit a fraction of Crm1 to the centrosomes; xref Crm1 in turn interacts with the two NESs of NPM1, thereby recruiting NPM1 to centrosomes of nonmitotic cells. xref NPM1 is phosphorylated on Thr199 by Cdk2/cyclin E late in G 1 phase, xref which causes dissociation of the majority of NPM1 from the centrosomes, a step required for centrosome duplication to occur. xref , xref Following centrosome duplication, Thr199-phosphorylated NPM1 reassociates with the duplicated centrosomes, ensuring that reduplication does not occur."
rlimsp
"In line with this observation, we found that the phosphorylation levels of NPM on Thr199 and Thr234/237 were significantly enhanced in cells treated with PP1/PP2A phosphatase inhibitor OA or calyculin A (Supplemental Figure S2, A and B), thus providing further support to the notion that phosphorylation of this protein is counterbalanced by the action of phosphatase under normal growth condition."
sparser
"We also show that avrainvillamide treatment influences the localization of Thr199-phosphorylated NPM1 during mitosis, causes deregulation of centrosome duplication, and leads to an accumulation of Thr199-phosphorylated NPM1 by inhibiting its dephosphorylation by the beta isoform of protein phosphatase 1 (PP1 β )."
sparser
"These observations are consistent with the hypothesis that displacement of Thr199-phosphorylated NPM1 from duplicated centrosomes enables centrosome reduplication, and suggest that avrainvillamide alters essential cellular processes by preventing NPM1 and Crm1 from interacting with their native binding partners."
rlimsp
"Threonine-199 is one of the most important and well-characterized NPM1 phosphorylation sites. Therefore, we analyzed the phosphorylation at this site within 24 hours after irradiation with 8 Gy in A549, HeLa, and HNSCCUM-02T cells (Figure 1C). Representative blots are shown in Figure S3 in the supplementary data. A549 as well as HeLa cells bear wild-type p53, while HNSCCUM-02T cells bear a p53 mutation which results in unconditionally high p53 expression [58]. Hela cells represent a special case concerning their p53 level; they are affected by a mutation-independent reduction in p53 protein level due to the expression of the viral E6 protein [59]. Thus, the analysis covers different entities as well as differences in the p53 status. We found that NPM1 is dephosphorylated at threonine-199 shortly after irradiation in all three cell lines."
rlimsp
"We found that NPM1 is dephosphorylated at threonine-199 shortly after irradiation in all three cell lines. In A549 cells, threonine-199 was completely dephosphorylated, and the dephosphorylation persisted for 24 hours. In HNSCCUM-02T cells, the dephosphorylation was less pronounced, and 24 hours after irradiation, the phosphorylation at threonine-199 returned to the basal level."
sparser
"NPM1 is phosphorylated on Thr199 by several kinases, including Cdk1, xref Cdk2, xref Cdk4, xref and Cdk6, xref and is dephosphorylated by PP1 β . xref Increases in NPM1 thr 199 phosphorylation are known to repress pre-mRNA splicing, xref reduce NPM1-mediated DNA repair activity, xref and target NPM1 to nuclear speckles. xref Over the course of our studies, we observed an apparent increase in the amount of NPM1pThr199 in avrainvillamide-treated cells ( xref ); this finding was confirmed by subsequent immunoblotting experiments ( xref )."
sparser
"In combination with the earlier result showing that NPM-Thr 198 is constitutively phosphorylated, these data indicate that this particular NPM phosphorylation site is not subject to either positive (that is, cdk-mediated) or negative (that is, ARF-directed) regulation throughout the cell cycle, but is instead constantly being phosphorylated as total levels of NPM rise in the cell."
sparser
"Although this current study demonstrates that ARF induction does not influence NPM-Thr 198 phosphorylation ( xref ), our previously published findings have shown that ARF effectively blocks NPM nucleocytoplasmic shuttling, a critical function of NPM that is essential for cellular growth and proliferation ( xref ; xref )."
sparser
"The RNA-binding activity of NPM1 is diminished after cdc2-mediated phosphorylation of Thr199 of NPM1 during mitosis, and this is suggested to link to the disassembly of nucleolus by disrupting the RNA-protein binding interaction of NPM1 (Hisaoka et al. xref ; Okuwaki et al. xref )."
| PMC
sparser
"We additionally observe that the ability of avrainvillamide to bind to Crm1 and prevent its binding to NES-containing proteins appears to disrupt the interaction between Crm1 and Thr199-phosphorylated NPM1 at key points in the cell-cycle, resulting in unregulated centrosome duplication."
sparser
"Thus, the observed effects of avrainvillamide on the subcellular localization of NPMc+ variants may represent a sublethal phenotype, while the effects of avrainvillamide on the relative levels of Thr199-phosphorylated NPM1 and on the localization of Thr199-phosphorylated NPM1 during mitosis may help explain avrainvillamide-induced antiproliferation in human cancer cells."
rlimsp
"Therefore we conclude that, depletion of 14-3-3γ 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 (Fig. 7a) 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 (Fig. 7b)."
rlimsp
"Our results demonstrate a mechanism by which 14-3-3γ restricts centrosome duplication to once per cell cycle, by inhibiting cdc25C function, thus preventing premature activation of cdk1 during interphase and resulting in a decrease in phosphorylation of T199 residue of the centriolar linker protein NPM1."
sparser
"Following UV-induced damage, NPM is phosphorylated at Thr199 and Thr 234/237, leading to an increase of E2F1 mRNA. xref NPM then associates with pRB and interfers with its repression of E2F1 transcription as seen by an increase of E2F1 at the promoters as well as increased expression of known E2F1 targets in DNA repair, XPC and DDB2. xref "
sparser
"In both lines, prexasertib induced a number of changes, including i) diminished CHK1 activity as manifested by reduced autophosphorylation at Ser 296 ; ii) decreased CHK1-mediated CDC25A phosphorylation, which led to increased CDC25A levels; and iii) and diminished CDK2 inhibitory phosphorylation, which resulted in increased CDK2 activity that was manifested by increased phosphorylation of the CDK2 substrate NPM1 on Thr 199 [ xref ] as shown in Fig. xref C and D ( lanes 2 and 3 vs. lane 1)."
sparser
"To investigate the role of NPM1 T199 phosphorylation in γH2AX foci formation and disassembly, we transiently transfected NPM1-null MEFs with plasmids expressing Myc-tagged wild type NPM1 or a T199 non-phosphorylatable NPM1 mutant (Myc-tagged T199A, xref ), administered 1 Gy and assayed γH2AX foci 1 hr later."
rlimsp
"When Thr¹⁹⁹ hyperphosphorylation of NPM was inhibited by OLO or when NPM was knocked down, we found that radiation-induced apoptosis was more pronounced and polyploidy formation was reduced as compared with negative control while the magnitude of these changes in TK6 was obviously higher than that in WTK1, indicating that NPM has an antagonistic interaction with wt p53."
rlimsp
"Upon phosphorylation on Thr(199) by cyclin-dependent kinase 2 (CDK2)/cyclin E, the majority of centrosomal NPM/B23 dissociates from centrosomes, but some NPM/B23 phosphorylated on Thr(199) remains at centrosomes. It has been shown that Thr(199) phosphorylation of NPM/B23 is critical for the physical separation of the paired centrioles, an initial event of the centrosome duplication process. Here, we identified ROCK II kinase, an effector of Rho small GTPase, as a protein that localizes to centrosomes and physically interacts with NPM/B23. Expression of the constitutively active form of ROCK II promotes centrosome duplication, while down-regulation of ROCK II expression results in the suppression of centrosome duplication, especially delaying the initiation of centrosome duplication during the cell cycle. Moreover, ROCK II regulates centrosome duplication in its kinase and centrosome localization activity-dependent manner. We further found that ROCK II kinase activity is significantly enhanced by binding to NPM/B23 and that NPM/B23 acquires a higher binding affinity to ROCK II upon phosphorylation on Thr(199)."
sparser
"Increased expression of NPM1 phosphorylated on Thr199 in tumor tissues from BRAF mutant colon cancer patients could be thus related to anomalies in the centrosome cycle leading to centrosome amplification, which is a common event in colon cancer linked with mutations in several cancer-associated genes including BRAF [ xref ]."
rlimsp
"NPM is a substrate of cyclin E/A-Cdk2 whose phosphorylation at T199 leads to its dissociation from centrosomes [9],[39], thereby enabling centriole separation and duplication [10]. Aberrantly high NPM-T199 phosphorylation induces unchecked centrosome reduplication during the cell cycle [33]."
rlimsp
"Antibodies were used to RABL6A (rabbit polyclonal made against an internal peptide [NH2-MVAGFQDDVDIEDQC-COOH], 1.5 µg/ml), mouse p19ARF (Novus, rat monoclonal, 1 µg/ml), NPM (Zymed, mouse monoclonal, 1:1,000), phosphorylated NPM-T199 (Cell signaling, Rabbit polyclonal, 1:1000 ), Aurora B (BD Transduction Laboratories, mouse monoclonal, 1:500), FLAG epitope (Sigma-Aldrich, M2 mouse monoclonal, 2 µg/ml), γ-tubulin (Sigma-Aldrich, clone GTU-88 mouse monoclonal, 1∶10,000), and GAPDH (Abcam, Ab8245 mouse monoclonal, 1∶10,000)."
sparser
"It is well established that controlled NPM-T199 phosphorylation by Cdk2-cyclin E/A kinases normally promotes one round of centrosome duplication per cell cycle, while Cdk2 hyper-activation causes unchecked NPM-T199 phosphorylation and extra rounds of centrosome duplication xref , xref , xref ."
sparser
"We then examined the requirement of NPM phosphorylation on Thr199 for NPM-LANA interaction, by performing anti-LANA immunoprecipitation from cell extracts of U2OS cells expressing LANA and transfected with expression vectors for Myc-v-cyclin, empty vector control, and the eGFP-tagged NPM wt or its phosphosite mutant constructs used in xref ."
sparser
"Moreover, phosphorylation of NPM at T199 was lower in drug-treated resistant cells when compared to the parental, indicating that CDK2 activity may be reduced in prexasertib-resistant cells potentially accounting for the strong attenuation in markers of RS (pRPA2) and DNA damage (γH2AX) in drug-treated cultures ( xref )."
rlimsp
"Licensing is modulated by G1 phase Cdks through phosphorylation and inactivation of NPM, as expression of NPM/B23 mutants whose phosphorylation sites were either deleted (NPM∆186-239) or replaced with a non-phosphorylatable residue (NPM T199A) resulted in suppression of centrosome duplication."