Ubiquitin-activating enzyme, E1, is phosphorylated in mammalian cells by the protein kinase Cdc2

1995 ◽  
Vol 108 (6) ◽  
pp. 2145-2152
Author(s):  
Y. Nagai ◽  
S. Kaneda ◽  
K. Nomura ◽  
H. Yasuda ◽  
T. Seno ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Line ◽  
Mammalian Cells ◽  
Peptide Mapping ◽  
Mutant Cell ◽  
Phosphorylation Site ◽  
Cdc2 Kinase ◽  
Key Enzyme

The ubiquitin-activating enzyme (E1) is the first enzyme in the pathway leading to formation of ubiquitin-protein conjugates. E1 was found to be phosphorylated in cells of a mouse mammary carcinoma cell line, FM3A. Peptide mapping of trypsin digests of labeled E1 indicated that two oligopeptides were mainly phosphorylated in vivo. The same oligopeptides were also labeled in vitro on Cdc2 kinase-mediated phosphorylation of E1, affinity-purified from the same cell line. The Cdc2 kinase is a key enzyme playing a pivotal role in G2/M transition in the cell cycle. The phosphorylation of one of the two oligopeptides was prominent at the G2/M phase of the cell cycle, and dependent upon the Cdc2 kinase activity in vivo since it was significantly reduced in tsFT210, a mutant cell line deficient in Cdc2 kinase. Mutation analysis indicated that the serine residue at the fourth position of the E1 enzyme was a phosphorylation site of Cdc2 kinase. These findings suggest that E1 is a target of Cdc2 kinase in the cell, implying that the ubiquitin system may be dynamically involved in cell cycle control through phosphorylation of this key enzyme.

An Orally Bioavailable Inhibitor of FLT3 and Syk Kinases Prevents Tumor Growth in Subcutaneously Implanted Human Tumor Xenografts and Promotes Cell Death of FLT3 Mutant AML Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 243-243 ◽  
Author(s):  
Polly R. Pine ◽  
Rena Bahjat ◽  
Betty Chang ◽  
Vanessa Taylor ◽  
Vadim Markovstov ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Tumor Growth ◽  
Cell Line ◽  
Mutant Cell ◽  
Dependent Manner ◽  
Tumor Xenografts ◽  
Orally Bioavailable ◽  
Dose Dependent

Abstract Background. Phase 1 clinical studies have shown that an orally bioavailable syk kinase inhibitor, R788/406, is very well tolerated in human volunteers for up to 7 days (and in primates for up to 28 days) at doses achieving concentrations in excess of 5 micromolar (ED50 for in vivo biomarker of syk-inhibition in humans is 1 micromolar). In biochemical kinase assays, R788/406 inhibits syk and FLT3 at less than 10 nM, and in cell-based assays at less than 100 nM. The demonstration of biological activity and excellent tolerability in humans, and the equipotent inhibition of FLT3 and syk kinases in biochemical assays led us to explore the potential for use of R788/406 in a xenograft of a human AML FLT-3ITD mutant cell line. Objective: To evaluate the in vitro and in vivo activity of R788/406 in a human AML FLT3-ITD mutant cell line, MV411, a model system for examination of FLT3 mutant AML. Methods: MV411 cells were treated with R788/406 and evaluated for cell viability and markers of apoptosis (Annexin-V/PI and caspase) by FACS. Cell cycle analysis was performed on cells stained with PI. 5 X 106 MV411 cells harvested in logarithmic phase growth were injected with Matrigel SC in NOD/SCID mice. Treatment with R788/406 began when tumors reached a predetermined size (mean volume of 100 mm3) and continued for 26 days. At sacrifice, tumor xenografts were lysed, immunoprecipitated with anti-FLT-3, and probed with anti-phosphotyrosine 4G10 or anti-FLT-3. Results: R788/406 potently and selectively induced dose-dependent cytotoxicity of MV-411 AML cells in vitro with an ED50 of 20nM. Pretreatment of cells with R788/406 promoted dephosphorylation of constitutively active pFLT3, as well as a reduction of pStat5 and pErk1/2 in the FLT3 signaling cascade. Moreover, R788/406 induced cell cycle arrest in the G1 phase and subsequent apoptosis in MV411 cells in a dose-dependent manner. Twice daily administration of R788/406 to NOD/SCID mice bearing SC MV411 tumors reduced tumor growth significantly in a dose dependent manner. When compared to vehicle controls, daily doses of 40 and 80mg/kg R788/406 resulted in 45% and 82% inhibition of mean tumor volumes, respectively. At study termination mean tumor volumes were 686.90 ± 115.56 and 224.45 ± 49.80 for 40 and 80 mg/kg R788/406-treated animals compared to 1255.48 ± 182.94 for vehicle controls with a final %T/C of -0.4 (range of %T/C throughout study was −7.9 to −0.4). During the study, no significant body weight loss was observed in any of the animals in this study. Ex vivo analyses of subcutaneous tumors from MV411 tumor-bearing mice showed that R788/406 completely inhibited constitutive FLT3 activation and downstream signaling events. Conclusions: R788/406 is well tolerated in humans (and primates) at concentrations well in excess of those that inhibit syk in vivo. Given the equipotent inhibition of syk and FLT3, the in vivo activity against human syk, and the xenograft data reported here, R788/406 may be a promising agent for FLT-3 AML.

scholarly journals Dynamics of human keratin 18 phosphorylation: polarized distribution of phosphorylated keratins in simple epithelial tissues.

1995 ◽  
Vol 131 (5) ◽  
pp. 1291-1301 ◽  
Author(s):  
J Liao ◽  
L A Lowthert ◽  
N O Ku ◽  
R Fernandez ◽  
M B Omary
Keyword(s):  
Cell Cycle ◽  
Peptide Mapping ◽  
Phosphorylation Site ◽  
Tissue Type ◽  
Intermediate Filament Proteins ◽  
Liver And Pancreas ◽  
Preferential Binding ◽  
Keratin 18

Phosphorylation of keratin polypeptides 8 and 18 (K8/18) and other intermediate filament proteins results in their reorganization in vitro and in vivo. In order to study functional aspects of human K18 phosphorylation, we generated and purified a polyclonal antibody (termed 3055) that specifically recognizes a major phosphorylation site (ser52) of human K18 but not dephosphorylated K18 or a ser52-->ala K18 mutant. Pulse-chase experiments followed by immunoprecipitation and peptide mapping of in vivo 32PO4-labeled K8/18 indicated that the overall phosphorylation turnover rate is faster for K18 versus K8, and that ser52 of K18 is a highly dynamic phosphorylation site. Isoelectric focusing of 32PO4 labeled K18 followed by immunoblotting with 3055 showed that the major phosphorylated K18 species contain ser52 phosphorylation but that some K18 molecules exist that are preferentially phosphorylated on K18 sites other than ser52. Immunoblotting of total cell lysates obtained from cells at different stages of the cell cycle showed that ser52 phosphorylation increases three to fourfold during the S and G2/M phases of the cell cycle. Immunofluorescence staining of cells at different stages of mitosis, using 3055 or other antibodies that recognize the total keratin pool, resulted in preferential binding of the 3055 antibody to the reorganized keratin fraction. Staining of human tissues or tissues from transgenic mice that express human K18 showed that the phospho-ser52 K18 species are located preferentially in the basolateral and apical domains in the liver and pancreas, respectively, but no preferential localization was noted in other simple epithelial organs examined. Our results support a model whereby phosphorylated intermediate filaments are localized in specific cellular domains depending on the tissue type and site(s) of phosphorylation. In addition, ser52 of human K18 is a highly dynamic phosphorylation site that undergoes modulation during the S and G2/M phases of the cell cycle in association with filament reorganization.

scholarly journals Akt kinase LANCL2 functions as a key driver in EGFR-mutant lung adenocarcinoma tumorigenesis

2021 ◽  
Vol 12 (2) ◽  
Author(s):  
Yuqing Lou ◽  
Jianlin Xu ◽  
Yanwei Zhang ◽  
Wei Zhang ◽  
Xueyan Zhang ◽  
...  
Keyword(s):  
Lung Adenocarcinoma ◽  
Cell Line ◽  
Quantitative Polymerase Chain Reaction ◽  
Mutant Cell ◽  
A549 Cells ◽  
The Cancer Genome Atlas ◽  
Akt Kinase ◽  
Egfr Expression

AbstractEpidermal growth factor receptor (EGFR) is a key oncogene in lung adenocarcinoma (LUAD). Resistance to EGFR tyrosine kinase inhibitors is a major obstacle for EGFR-mutant LUAD patients. Our gene chip array, quantitative polymerase chain reaction validation, and shRNA-based high-content screening identified the Akt kinase lanthionine synthetase C-like protein 2 (LANCL2) as a pro-proliferative gene in the EGFR-mutant LUAD cell line PC9. Therefore, we investigated whether LANCL2 plays a role in promoting cell proliferation and drug resistance in EGFR-mutant LUAD. In silico clinical correlation analysis using the Cancer Genome Atlas Lung Adenocarcinoma dataset revealed a positive correlation between LANCL2 and EGFR expression and an inverse relationship between LANCL2 gain-of-function and survival in LUAD patients. The EGFR-mutant LUAD cell lines PC9 and HCC827 displayed higher LANCL2 expression than the non-EGFR-mutant cell line A549. In addition, LANCL2 was downregulated following gefitinib+pemetrexed combination therapy in PC9 cells. LANCL2 knockdown reduced proliferation and enhanced apoptosis in PC9, HCC827, and A549 cells in vitro and suppressed murine PC9 xenograft tumor growth in vivo. Notably, LANCL2 overexpression rescued these effects and promoted gefitinib + pemetrexed resistance in PC9 and HCC827 cells. Pathway analysis and co-immunoprecipitation followed by mass spectrometry of differentially-expressed genes in LANCL2 knockdown cells revealed enrichment of several cancer signaling pathways. In addition, Filamin A and glutathione S-transferase Mu 3 were identified as two novel protein interactors of LANCL2. In conclusion, LANCL2 promotes tumorigenic proliferation, suppresses apoptosis, and promotes gefitinib+pemetrexed resistance in EGFR-mutant LUAD cells. Based on the positive association between LANCL2, EGFR, and downstream Akt signaling, LANCL2 may be a promising new therapeutic target for EGFR-mutant LUAD.

scholarly journals Functional Interactions between Herpesvirus Oncoprotein MEQ and Cell Cycle Regulator CDK2

1999 ◽  
Vol 73 (5) ◽  
pp. 4208-4219 ◽  
Author(s):  
Juinn-Lin Liu ◽  
Ying Ye ◽  
Zheng Qian ◽  
Yongyi Qian ◽  
Dennis J. Templeton ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Phosphorylation Site ◽  
Fibroblast Cell ◽  
S Phase ◽  
Morphological Transformation ◽  
Necrosis Factor Alpha ◽  
Coiled Bodies ◽  
Cdk Phosphorylation

ABSTRACT Marek’s disease virus, an avian alphaherpesvirus, has been used as an excellent model to study herpesvirus oncogenesis. One of its potential oncogenes, MEQ, has been demonstrated to transform a rodent fibroblast cell line, Rat-2, in vitro by inducing morphological transformation and anchorage- and serum-independent growth and by protecting cells from apoptosis induced by tumor necrosis factor alpha, C2-ceramide, UV irradiation, or serum deprivation. In this report, we show that there is a cell cycle-dependent colocalization of MEQ protein and cyclin-dependent kinase 2 (CDK2) in coiled bodies and the nucleolar periphery during the G1/S boundary and early S phase. To our knowledge, this is the first demonstration that CDK2 is found to localize to coiled bodies. Such an in vivo association and possibly subsequent phosphorylation may result in the cytoplasmic translocation of MEQ protein. Indeed, MEQ is expressed in both the nucleus and the cytoplasm during the G1/S boundary and early S phase. In addition, we were able to show in vitro phosphorylation of MEQ by CDKs. We have mapped the CDK phosphorylation site of MEQ to be serine 42, a residue in the proximity of the bZIP domain. An indirect-immunofluorescence study of the MEQ S42D mutant, in which the CDK phosphorylation site was mutated to a charged residue, reveals more prominent cytoplasmic localization. This lends further support to the notion that the translocation of MEQ is regulated by phosphorylation. Furthermore, phosphorylation of MEQ by CDKs drastically reduces the DNA binding activity of MEQ, which may in part account for the lack of retention of MEQ oncoprotein in the nucleus. Interestingly, the localization of CDK2 in coiled bodies and the nucleolar periphery is observed only in MEQ-transformed Rat-2 cells, implicating MEQ in modifying the subcellular localization of CDK2. Taken together, our data suggest that there is a novel reciprocal modulation between the herpesvirus oncoprotein MEQ and CDK2.

scholarly journals Raddeanin A Induces Apoptosis and Cycle Arrest in Human HCT116 Cells through PI3K/AKT Pathway Regulation In Vitro and In Vivo

2019 ◽  
Vol 2019 ◽  
pp. 1-11
Author(s):  
Chunqin Meng ◽  
Yuhao Teng ◽  
Xiaodong Jiang
Keyword(s):  
Cell Cycle ◽  
Cell Line ◽  
Akt Signaling ◽  
Akt Signaling Pathway ◽  
Cycle Arrest ◽  
Colorectal Cell ◽  
Pathway Regulation ◽  
Hct116 Cells

This study aimed to investigate the in vitro and in vivo effects of Raddeanin A on apoptosis and the cell cycle in the human colorectal cell line, HCT116, and to explore the possible underlying mechanisms of action. We found the growth inhibition rate gradually increased as the drug concentration increased via the 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide (MTT) assay, which indicated that Raddeanin A significantly inhibited the growth of HCT116 cells. Flow cytometry (FCM) showed that Raddeanin A concentration-dependently induced apoptosis in HCT116 cells. In addition, the percentage of cells in the G0/G1phase was noticeably increased, which indicated that Raddeanin A blocked cell cycle progression in HCT116 cells and caused arrest in the G0/G1phase. Moreover, the expression of proteins involved in the PI3K/AKT signaling pathway (e.g., p-PI3K and p-AKT) was decreased. The results showed that in vivo revealed that Raddeanin A significantly inhibited tumor growth in an HCT116-xenografted mouse model; apoptotic cells were also detected in the tumor tissue. The expression of the tissue proteins cyclinD1, cyclinE, p-PI3K, and p-AKT was decreased. The above results show that the Raddeanin A exerted a strong antitumor effect in the human colorectal cell line HCT116 both in vitro and in vivo. This effect may be caused by the induction of apoptosis and cycle arrest achieved through PI3K/AKT signaling pathway regulation.

PI3K/Akt/FOXO3a Pathway Contributes to Thrombopoietin-Induced Proliferation of Primary Megakaryocytes In Vitro and In Vivo Via Modulation of p27Kip1.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 202-202
Author(s):  
Takafumi Nakao ◽  
Amy E Geddis ◽  
Norma E. Fox ◽  
Kenneth Kaushansky
Keyword(s):  
Cell Cycle ◽  
Cell Line ◽  
Cell Cycle Progression ◽  
Wild Type ◽  
Cycle Progression ◽  
P27kip1 Expression ◽  
Cell Counts ◽  
Deficient Mice

Abstract Thrombopoietin (TPO), the primary regulator of megakaryocyte (MK) and platelet formation, modulates the activity of multiple signal transduction molecules, including those in the Jak/STAT, p42/p44 MAPK, and phosphatidylinositol 3-kinase (PI3K)/Akt pathways. In the previous study, we reported that PI3K and Akt are necessary for TPO-induced cell cycle progression of primary MK progenitors. The absence of PI3K activity results in a block of transition from G1 to S phase in these cells (Geddis AE et al. JBC2001276:34473–34479). However, the molecular events secondary to the activation of PI3K/Akt responsible for MK proliferation remain unclear. In this study we show that FOXO3a and its downstream target p27Kip1 play an important role in TPO-induced proliferation of MK progenitors. TPO induces phosphorylation of Akt and FOXO3a in both UT-7/TPO, a megakaryocytic cell line, and primary murine MKs in a PI3K dependent fashion. Cell cycle progression of UT-7/TPO cells is blocked in G1 phase by inhibition of PI3K. We found that TPO down-modulates p27Kip1 expression at both the mRNA and protein levels in UT-7/TPO cells and primary MKs in a PI3K dependent fashion. UT-7/TPO stably expressing constitutively active Akt or a dominant-negative form of FOXO3a failed to induce p27Kip1 expression after TPO withdrawal. Induced expression of an active form of FOXO3a resulted in increased p27Kip1 expression in this cell line. In an attempt to assess whether FOXO3a has an effect of MK proliferation in vivo, we compared the number of MKs in Foxo3a-deficient mice and in wild type controls. Although peripheral blood cell counts of erythrocytes, neutrophils, monocytes and platelets were normal in the Foxo3a-deficient mice, total nucleated marrow cell count of Foxo3a-deficient mice were 60% increased compared with wild type controls. In addition, the increase of MKs was more profound than that of total nucleated marrow cells; CD41+ MKs from Foxo3a-deficient mice increased 2.1-fold, and mature MKs with 8N and greater ploidy increased 2.5-fold, compared with wild type controls. Taken together with the previous observation that p27Kip1-deficient mice also display increased numbers of MK progenitors, our findings strongly suggest that the effect of TPO on MK proliferation is mediated by PI3K/Akt-induced FOXO3a inactivation and subsequent p27Kip1 down-regulation in vitro and in vivo.

The Novel Kinesin Spindle Protein Inhibitor SB-743921 Exhibits Marked Activity In In Vivo and In Vitro In Models of Aggressive Diffuse Large B-Cell Lymphoma

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 118-118
Author(s):  
Danielle C Bongero ◽  
Luca Paoluzzi ◽  
Enrica Marchi ◽  
Neisa Roberto ◽  
Rafael Escandon ◽  
...  
Keyword(s):  
Cell Cycle ◽  
B Cell ◽  
Cell Line ◽  
Germinal Center ◽  
Mitotic Spindle ◽  
Tumor Volume ◽  
P Value ◽  
Kinesin Spindle Protein

Abstract Abstract 118 A mitotic spindle target that has emerged as unique and potentially restricted to the mitotic spindle is Eg5, also known as the kinesin spindle protein (KSP). SB-743921 induces mitotic spindle dysfunction and cell cycle arrest by inhibiting Eg5. Preliminary Phase 1 studies of SB-743921 have demonstrated that this compound is not associated with any neuropathy like other anti-mitotic agents. These studies have also demonstrated a potential signal in patients with relapsed and refractory lymphoma. We investigated the efficacy of SB-743921 in aggressive B-cell lymphomas to evaluate effectiveness and tolerability in germinal center (GCB) and post germinal center (ABC) diffuse large B-cell lymphomas (DLBCL). For cytotoxicity assays, luminescent cell viability was performed using CellTiter-Glo™ followed by acquisition with Biotek Synergy HT. The IC50s were calculated using the Calcusyn software (Biosoft). Cell Cycle was assessed by staining with Vybrant DyeCycle Green (Invitrogen) followed by FACSCalibur acquisition. Whole cell lysate proteins were extracted and quantified according to Bradford assay. After electrophoresis on a gradient 4–20% SDS-PAGE gels the proteins were transferred to nitrocellulose membrane. After blocking and incubation with the primary and the secondary antibodies, the chemiluminescent agent was added and the x-ray films were exposed to the membranes. In vivo experiments were performed with five to 7-week-old severe combined immunodeficiency (SCID) beige mice (Taconic Laboratories, Germantown, NY) injected with 1 × 107 Ly1-DLBCL cells on the flank via a subcutaneous (SQ) route. When tumor volumes approached 80 mm3, mice were separated into cohorts of ten mice each. Tumors were assessed using the two largest perpendicular axes (l, length; w, width) as measured with standard calipers. Tumor volume was calculated using the formula 4/3 r3, where r=(l + w) / 4. Tumor-bearing mice were assessed for weight loss and tumor volume at least twice weekly. The IC50 values for SB-743921 across a panel of different DLBCL lines are listed in table 1. Cell cycle analysis showed that compared to the untreated group, after treatment with 100nM of SB 743921 the percentage of GCB cells in G2/M phase increased from 17.6% to 40.3% (+129%) in Ly7, 23.9% to 40.7 % (+70%) in Sudhl6 and from 17.55% to 32.4% (+85%) in Ly1. In comparison, the percent increase of cells in G2/M for the ABC lines was statistically less (p-value 0.001). For example, Ly10 increased from 15% to 27.6% (+45%), Riva from 29.3% to 36.95% (+26%) and Sudhl2 from 22.6% to 27.6% (+22%). Immunoblot analysis of DLBCL cells treated with SB-743921 probed for Eg5, CyclinB1, and phosphorylated BubR1 revealed that although all cells demonstrated a measurable increase in Eg5, the total Eg5 present varied from cell line to cell line. The In vivo xenograft experiment was conducted with the GCB Ly1 cell line and consisted of 4 cohorts; one control and 3 treatments with doses of 2.5 mg/kg, 5 mg/kg and 10 mg/kg. SB-743921 was administered by the intraperitoneal route on days 1, 5, and 9 on a 23 day cycle for 2 cycles. The graph below displays the inhibition of tumor growth in the cohorts after treatment with SB-74321. All 3 cohorts had a p-value of <0.001 relative to the control. In conclusion, SB-743921 is promising as a single agent for treatment of DLBCL. Future studies exploring the specific cell cycle features of different cell lines with respect to their check-point control will afford new opportunities to better understand the mechanisms of increased resistance in ABC compared to GCB. The data suggests SB 743921 overall is effective in the treatment of DLBCL both in vitro and in vivo. Further studies exploring potential synergistic interactions with conventional chemotherapeutic agents as well as establishing the most effective treatment schedules for the agent may provide a new approach to treating these diseases. Disclosures: Escandon: Cytokinetics: Employment. Wood:Cytokinetics: Employment. O'Connor:Millennium Pharmaceuticals, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Preclinical Rationale for the Use of the Combined Treatment with the AKT Inhibitor Perifosine and the Multikinase Inhibitor Sorafenib in Hodgkin Lymphoma

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1653-1653
Author(s):  
Silvia Locatelli ◽  
Arianna Giacomini ◽  
Anna Guidetti ◽  
Loredana Cleris ◽  
Michele Magni ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Growth ◽  
Cell Line ◽  
Cell Lines ◽  
Drug Combination ◽  
Akt Inhibitor ◽  
Sorafenib Treatment ◽  
Responsive Cell

Abstract Abstract 1653 Introduction: A significant proportion of Hodgkin lymphoma (HL) patients refractory to first-line chemotherapy or relapsing after autologous transplantation are not cured with currently available treatments and require new treatments. The PI3K/AKT and RAF/MEK/ERK pathways are constitutively activated in the majority of HL. These pathways can be targeted using the AKT inhibitor perifosine (Æterna Zentaris GmBH, Germany, EU), and the RAF/MEK/ERK inhibitor sorafenib (Nexavar®, Bayer, Germany, EU). We hypothesized that perifosine in combination with sorafenib might have a therapeutic activity in HL by overcoming the cytoprotective and anti-apoptotic effects of PI3K/Akt and RAF/MEK/ERK pathways. Since preclinical evidence supporting the anti-lymphoma effects of the perifosine/sorafenib combination are still lacking, the present study aimed at investigating in vitro and in vivo the activity and mechanism(s) of action of this two-drug combination. METHODS: Three HL cell lines (HD-MyZ, L-540 and HDLM-2) were used to investigate the effects of perifosine and sorafenib using in vitro assays analyzing cell growth, cell cycle distribution, gene expression profiling (GEP), and apoptosis. Western blotting (WB) experiments were performed to determine whether the two-drug combination affected MAPK and PI3K/AKT pathways as well as apoptosis. Additionally, the antitumor efficacy and mechanism of action of perifosine/sorafenib combination were investigated in vivo in nonobese diabetic/severe combined immune-deficient (NOD/SCID) mice. RESULTS: While perifosine and sorafenib as single agents exerted a limited activity against HL cells, exposure of HD-MyZ and L-540 cell lines, but not HDLM-2 cells, to perifosine/sorafenib combination resulted in synergistic cell growth inhibition (40% to 80%) and cell cycle arrest. Upon perifosine/sorafenib exposure, L-540 cell line showed significant levels of apoptosis (up to 70%, P ≤.0001) associated with severe mitochondrial dysfunction (cytochrome c, apoptosis-inducing factor release and marked conformational change of Bax accompanied by membrane translocation). Apoptosis induced by perifosine/sorafenib combination did not result in processing of caspase-8, -9, -3, or cleavage of PARP, and was not reversed by the pan-caspase inhibitor Z-VADfmk, supporting a caspase-independent mechanism of apoptosis. In responsive cell lines, WB analysis showed that anti-proliferative events were associated with dephosphorylation of MAPK and PI3K/Akt pathways. GEP analysis of HD-MyZ and L-540 cell lines, but not HDLM-2 cells indicated that perifosine/sorafenib treatment induced upregulation of genes involved in amino acid metabolism and downregulation of genes regulating cell cycle, DNA replication and cell death. In addition, in responsive cell lines, perifosine/sorafenib combination strikingly induced the expression of tribbles homologues 3 (TRIB3) both in vitro and in vivo. Silencing of TRIB3 prevented cell growth reduction induced by perifosine/sorafenib treatment. In vivo, the combined perifosine/sorafenib treatment significantly increased the median survival of NOD/SCID mice xenografted with HD-MyZ cell line as compared to controls (81 vs 45 days, P ≤.0001) as well as mice receiving perifosine alone (49 days, P ≤.03) or sorafenib alone (54 days, P ≤.007). In mice bearing subcutaneous nodules generated by HD-MyZ and L-540 cell lines but not HDLM-2 cell line, perifosine/sorafenib treatment induced significantly increased levels of apoptosis (2- to 2.5-fold, P ≤.0001) and necrosis (2- to 8-fold, P ≤.0001), as compared to controls or treatment with single agents. CONCLUSIONS: Perifosine/sorafenib combination resulted in potent anti-HL activity both in vitro and in vivo. These results warrant clinical evaluation in HL patients. Disclosures: No relevant conflicts of interest to declare.

scholarly journals A dominant-negative cyclin D1 mutant prevents nuclear import of cyclin-dependent kinase 4 (CDK4) and its phosphorylation by CDK-activating kinase.

1997 ◽  
Vol 17 (12) ◽  
pp. 7362-7374 ◽  
Author(s):  
J A Diehl ◽  
C J Sherr
Keyword(s):  
Cyclin D1 ◽  
Nuclear Localization ◽  
Mammalian Cells ◽  
Phosphorylation Site ◽  
Dominant Negative ◽  
Cyclin Dependent Kinase ◽  
Linker Peptide ◽  
Cdk Activating Kinase

Cyclins contain two characteristic cyclin folds, each consisting of five alpha-helical bundles, which are connected to one another by a short linker peptide. The first repeat makes direct contact with cyclin-dependent kinase (CDK) subunits in assembled holoenzyme complexes, whereas the second does not contribute directly to the CDK interface. Although threonine 156 in mouse cyclin D1 is predicted to lie at the carboxyl terminus of the linker peptide that separates the two cyclin folds and is buried within the cyclin subunit, mutation of this residue to alanine has profound effects on the behavior of the derived cyclin D1-CDK4 complexes. CDK4 in complexes with mutant cyclin D1 (T156A or T156E but not T156S) is not phosphorylated by recombinant CDK-activating kinase (CAK) in vitro, fails to undergo activating T-loop phosphorylation in vivo, and remains catalytically inactive and unable to phosphorylate the retinoblastoma protein. Moreover, when it is ectopically overexpressed in mammalian cells, cyclin D1 (T156A) assembles with CDK4 in the cytoplasm but is not imported into the cell nucleus. CAK phosphorylation is not required for nuclear transport of cyclin D1-CDK4 complexes, because complexes containing wild-type cyclin D1 and a CDK4 (T172A) mutant lacking the CAK phosphorylation site are efficiently imported. In contrast, enforced overexpression of the CDK inhibitor p21Cip1 together with mutant cyclin D1 (T156A)-CDK4 complexes enhanced their nuclear localization. These results suggest that cyclin D1 (T156A or T156E) forms abortive complexes with CDK4 that prevent recognition by CAK and by other cellular factors that are required for their nuclear localization. These properties enable ectopically overexpressed cyclin D1 (T156A), or a more stable T156A/T286A double mutant that is resistant to ubiquitination, to compete with endogenous cyclin D1 in mammalian cells, thereby mobilizing CDK4 into cytoplasmic, catalytically inactive complexes and dominantly inhibiting the ability of transfected NIH 3T3 fibroblasts to enter S phase.

scholarly journals BRCA1 Is Phosphorylated at Serine 1497 In Vivo at a Cyclin-Dependent Kinase 2 Phosphorylation Site

1999 ◽  
Vol 19 (7) ◽  
pp. 4843-4854 ◽  
Author(s):  
Heinz Ruffner ◽  
Wei Jiang ◽  
A. Grey Craig ◽  
Tony Hunter ◽  
Inder M. Verma
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Kinase Activity ◽  
Phosphorylation Site ◽  
Cyclin A ◽  
Dominant Negative ◽  
Protein Kinase Activity ◽  
Cyclin Dependent Kinase

ABSTRACT BRCA1 is a cell cycle-regulated nuclear protein that is phosphorylated mainly on serine and to a lesser extent on threonine residues. Changes in phosphorylation occur in response to cell cycle progression and DNA damage. Specifically, BRCA1 undergoes hyperphosphorylation during late G1 and S phases of the cell cycle. Here we report that BRCA1 is phosphorylated in vivo at serine 1497 (S1497), which is part of a cyclin-dependent kinase (CDK) consensus site. S1497 can be phosphorylated in vitro by CDK2-cyclin A or E. BRCA1 coimmunoprecipitates with an endogenous serine-threonine protein kinase activity that phosphorylates S1497 in vitro. This cellular kinase activity is sensitive to transfection of a dominant negative form of CDK2 as well as the application of the CDK inhibitors p21 and butyrolactone I but not p16. Furthermore, BRCA1 coimmunoprecipitates with CDK2 and cyclin A. These results suggest that the endogenous kinase activity is composed of CDK2-cyclin complexes, at least in part, concordant with the G1/S-specific increase in BRCA1 phosphorylation.

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