scholarly journals Specific Phosphorylation of Nucleophosmin on Thr199by Cyclin- dependent Kinase 2-Cyclin E and Its Role in Centrosome Duplication

2001 ◽  
Vol 276 (24) ◽  
pp. 21529-21537 ◽  
Author(s):  
Yukari Tokuyama ◽  
Henning F. Horn ◽  
Kenji Kawamura ◽  
Pheruza Tarapore ◽  
Kenji Fukasawa
Keyword(s):  
Cyclin E ◽  
Centrosome Duplication ◽  
Cyclin Dependent Kinase

scholarly journals Differential Regulation of Retinoblastoma Tumor Suppressor Protein by G1 Cyclin-Dependent Kinase Complexes In Vivo

2001 ◽  
Vol 21 (14) ◽  
pp. 4773-4784 ◽  
Author(s):  
Sergei A. Ezhevsky ◽  
Alan Ho ◽  
Michelle Becker-Hapak ◽  
Penny K. Davis ◽  
Steven F. Dowdy
Keyword(s):  
Cyclin E ◽  
Active Form ◽  
Dominant Negative ◽  
Tumor Suppressor Protein ◽  
Cyclin D ◽  
Cyclin Dependent Kinase ◽  
Retinoblastoma Tumor Suppressor Protein ◽  
Retinoblastoma Tumor Suppressor ◽  
Retinoblastoma Tumor

ABSTRACT The retinoblastoma tumor suppressor protein (pRB) negatively regulates early-G1 cell cycle progression, in part, by sequestering E2F transcription factors and repressing E2F-responsive genes. Although pRB is phosphorylated on up to 16 cyclin-dependent kinase (Cdk) sites by multiple G1 cyclin-Cdk complexes, the active form(s) of pRB in vivo remains unknown. pRB is present as an unphosphorylated protein in G0 quiescent cells and becomes hypophosphorylated (∼2 mol of PO4 to 1 mol of pRB) in early G1 and hyperphosphorylated (∼10 mol of PO4 to 1 mol of pRB) in late G1 phase. Here, we report that hypophosphorylated pRB, present in early G1, represents the biologically active form of pRB in vivo that is assembled with E2Fs and E1A but that both unphosphorylated pRB in G0 and hyperphosphorylated pRB in late G1 fail to become assembled with E2Fs and E1A. Furthermore, using transducible dominant-negative TAT fusion proteins that differentially target cyclin D-Cdk4 or cyclin D-Cdk6 (cyclin D-Cdk4/6) and cyclin E-Cdk2 complexes, namely, TAT-p16 and TAT–dominant-negative Cdk2, respectively, we found that, in vivo, cyclin D-Cdk4/6 complexes hypophosphorylate pRB in early G1 and that cyclin E-Cdk2 complexes inactivate pRB by hyperphosphorylation in late G1. Moreover, we found that cycling human tumor cells expressing deregulated cyclin D-Cdk4/6 complexes, due to deletion of the p16 INK4a gene, contained hypophosphorylated pRB that was bound to E2Fs in early G1and that E2F-responsive genes, including those for dihydrofolate reductase and cyclin E, were transcriptionally repressed. Thus, we conclude that, physiologically, pRB is differentially regulated by G1 cyclin-Cdk complexes.

KARAKTERISTIK SEL KANKER ORAL BARU (Sp-C1) DAN UJI HAMBATAN PERTUMBUHAN SEL Sp-C1 MENGGUNAKAN TERAPI GEN pcDNA3.1 -p27Kip1mutant type in vitro

2014 ◽  
Vol 18 (2) ◽  
pp. 120-125
Author(s):  
Supriatno
Keyword(s):  
Cyclin E ◽  
Cyclin Dependent Kinase ◽  
Empty Vector

Sel primer kanker oral (sel Sp-C1) mempunyai beberapa karakteristik pertumbuhan sel dan ekspresi protein antiapoptosis maupun protein regulator positif siklus sel. Karakteristik sel Sp-C1 penting diketahui untuk memudahkanpeneliti menggunakan model in vitro sel kanker oral. Tujuan penelitian adalah memperkenalkan jenis sel kanker oral baru(sel Sp-C1) dengan karakteristiknya, serta menguji hambatan pertumbuhan sel Sp-C1 mengunakan terapi gen p27Kip1mt secara in vitro. Isolasi sel Sp-C1 berasal dari jaringan limfonodi servikal yang termetastasis kanker lidah. Proses isolasididapatkan fibroblas dan sel primer kanker oral yang diinkubasi pada refrigerator -800C. Hambatan pertumbuhan sel SpC1 diperlakukan dengan terapi gen pcDNA3.1 -p27Kip1 mt dan pcDNA3.1-neo (empty vector) menggunakan uji MTT.Hasil penelitian menunjukkan karakteristik pertumbuhan sel Sp-C1 yang relatif cepat dan ekspresi protein p45Skp2, αtubulin, cyclin-dependent kinase-2 (CDK-2), cyclin-E, metastatic associated protein-1 (MTA-1) dan protein antiapoptosis maspin. Selanjutnya, sel transfektan Sp-C1-pcDNA3.1-p27Kip1 mt mempunyai potensi hambatanpertumbuhan sel yang signifikan dibandingkan pcDNA3.1-neo (P< 0.05). Kesimpulan, sel Sp-C1 mempunyaikarakteristik tertentu dan dapat digunakan sebagai model penelitian in vitro kanker mulut. pcDNA3.1-p27Kip1 mtmempunyai potensi hambatan pertumbuhan sel Sp-C1 yang kuat. Metode transfer gen secara in vitro merupakan proseduryang sederhana dan suatu strategi baru terapi gen terhadap sel kanker oral.

scholarly journals p57Kip2 Stabilizes the MyoD Protein by Inhibiting Cyclin E-Cdk2 Kinase Activity in Growing Myoblasts

1999 ◽  
Vol 19 (11) ◽  
pp. 7621-7629 ◽  
Author(s):  
Emmanuel G. Reynaud ◽  
Karine Pelpel ◽  
Martine Guillier ◽  
Marie Pierre Leibovitch ◽  
Serge A. Leibovitch
Keyword(s):  
Cyclin E ◽  
Tight Binding ◽  
Dominant Negative ◽  
Myoblast Differentiation ◽  
C2c12 Myoblast ◽  
Cdk Inhibitors ◽  
Cyclin Dependent Kinase ◽  
Muscle Creatine Kinase ◽  
Mouse Muscle ◽  
Cdk2 Activity

ABSTRACT We show that expression of p57Kip2, a potent tight-binding inhibitor of several G1cyclin–cyclin-dependent kinase (Cdk) complexes, increases markedly during C2C12 myoblast differentiation. We examined the effect of p57Kip2 on the activity of the transcription factor MyoD. In transient transfection assays, transcriptional transactivation of the mouse muscle creatine kinase promoter by MyoD was enhanced by the Cdk inhibitors. In addition, p57Kip2, p21Cip1, and p27Kip1 but not p16Ink4a induced an increased level of MyoD protein, and we show that MyoD, an unstable nuclear protein, was stabilized by p57Kip2. Forced expression of p57Kip2 correlated with hypophosphorylation of MyoD in C2C12 myoblasts. A dominant-negative Cdk2 mutant arrested cells at the G1 phase transition and induced hypophosphorylation of MyoD. Furthermore, phosphorylation of MyoD by purified cyclin E-Cdk2 complexes was inhibited by p57Kip2. In addition, the NH2 domain of p57Kip2 necessary for inhibition of cyclin E-Cdk2 activity was sufficient to inhibit MyoD phosphorylation and to stabilize it, leading to its accumulation in proliferative myoblasts. Taken together, our data suggest that repression of cyclin E-Cdk2-mediated phosphorylation of MyoD by p57Kip2 could play an important role in the accumulation of MyoD at the onset of myoblast differentiation.

scholarly journals Cyclin-dependent kinase 2 is dispensable for normal centrosome duplication but required for oncogene-induced centrosome overduplication

Oncogene ◽  
2006 ◽  
Vol 25 (20) ◽  
pp. 2943-2949 ◽  
Author(s):  
A Duensing ◽  
Y Liu ◽  
M Tseng ◽  
M Malumbres ◽  
M Barbacid ◽  
...  
Keyword(s):  
Centrosome Duplication ◽  
Cyclin Dependent Kinase

An expansion phase precedes terminal erythroid differentiation of hematopoietic progenitor cells from cord blood in vitro and is associated with up-regulation of cyclin E and cyclin-dependent kinase 2

Blood ◽  
2000 ◽  
Vol 96 (12) ◽  
pp. 3985-3987 ◽  
Author(s):  
Mu-Shui Dai ◽  
Charlie R. Mantel ◽  
Zhen-Biao Xia ◽  
Hal E. Broxmeyer ◽  
Li Lu
Keyword(s):  
Cell Cycle ◽  
Cord Blood ◽  
Cyclin E ◽  
Erythroid Differentiation ◽  
S Phase ◽  
Cyclin D3 ◽  
G1 Phase ◽  
Cyclin Dependent Kinase ◽  
Terminal Erythroid Differentiation

The dynamics of cell cycle regulation were investigated during in vitro erythroid proliferation and differentiation of CD34+cord blood cells. An unusual cell cycle profile with a majority of cells in S phase (70.2%) and minority of cells in G1 phase (27.4%) was observed in burst-forming unit-erythrocytes (BFU-E)–derived erythroblasts from a 7-day culture of CD34+ cells stimulated with interleukin 3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), Steel factor, and Epo. Terminal erythroid differentiation was accompanied by a rapid increase of G0/G1 phase cells. Expression of cyclin E and cyclin-dependent kinase 2 (cdk2) correlated with the proportion of S phase cells. Cyclin D3 was moderately up-regulated during the proliferation phase, and both cyclin E and D3 were rapidly down-regulated during terminal differentiation. This suggests that the high proliferation potential of erythroblasts is associated with temporal up-regulation of cyclin E and cdk2.

scholarly journals Interaction between ROCK II and Nucleophosmin/B23 in the Regulation of Centrosome Duplication

2006 ◽  
Vol 26 (23) ◽  
pp. 9016-9034 ◽  
Author(s):  
Zhiyong Ma ◽  
Masayuki Kanai ◽  
Kenji Kawamura ◽  
Kozo Kaibuchi ◽  
Keqiang Ye ◽  
...  
Keyword(s):  
Cyclin E ◽  
Active Form ◽  
Small Gtpase ◽  
Centrosome Duplication ◽  
Physical Interaction ◽  
Dependent Manner ◽  
Physical Separation ◽  
Initial Event ◽  
Activity Dependent

ABSTRACT Nucleophosmin (NPM)/B23 has been implicated in the regulation of centrosome duplication. NPM/B23 localizes between two centrioles in the unduplicated centrosome. Upon phosphorylation on Thr199 by cyclin-dependent kinase 2 (CDK2)/cyclin E, the majority of centrosomal NPM/B23 dissociates from centrosomes, but some NPM/B23 phosphorylated on Thr199 remains at centrosomes. It has been shown that Thr199 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 Thr199. Moreover, physical interaction between ROCK II and NPM/B23 in vivo occurs in association with CDK2/cyclin E activation and the emergence of Thr199-phosphorylated NPM/B23. All these findings point to ROCK II as the effector of the CDK2/cyclin E-NPM/B23 pathway in the regulation of centrosome duplication.

scholarly journals Phosphorylation of the myristoylated protein kinase C substrate MARCKS by the cyclin E–cyclin-dependent kinase 2 complex in vitro

1999 ◽  
Vol 340 (3) ◽  
pp. 775-782 ◽  
Author(s):  
Stéphane MANENTI ◽  
Emiko YAMAUCHI ◽  
Odile SOROKINE ◽  
Martine KNIBIEHLER ◽  
Alain VAN DORSSELAER ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Cyclin E ◽  
Cyclin Dependent Kinase ◽  
Kinase Substrate ◽  
Cyclin Dependent Kinases ◽  
Kinase C ◽  
Cdk2 Complex

The myristoylated alanine-rich C-kinase substrate (MARCKS) purified from brain was recently characterized as a proline-directed kinase(s) substrate in vivo [Taniguchi, Manenti, Suzuki and Titani (1994) J. Biol. Chem. 269, 18299-18302]. Here we have investigated the phosphorylation of MARCKS by various cyclin-dependent kinases (Cdks) in vitro. We established that Cdk2, Cdk4 and, to a smaller extent, Cdk1 that have been immunoprecipitated from cellular extracts phosphorylate MARCKS. Comparison of MARCKS phosphorylation by protein kinase C (PKC) and by the purified cyclin E-Cdk2 complex suggested that two residues were phosphorylated by Cdk2 under these conditions. To identify these sites, Cdk2-phosphorylated MARCKS was digested with lysyl endoprotease and analysed by electrospray MS. Comparison with the digests obtained from the unphosphorylated protein demonstrated that two peptides, Gly12-Lys30 and Ala138-Lys152, were phosphorylated by cyclin E-Cdk2. The identity of these peptides was confirmed by automatic Edman degradation. On the basis of the consensus phosphorylation sequence described for Cdk2, and on MS/MS analysis of the Ala138-Lys152 peptide, we concluded that Ser27, one of the phosphorylation sites identified in vivo, and Thr150 were the Cdk2 targets in vitro. None of the other sites described in vivo were phosphorylated in these conditions. Interestingly, a preliminary phosphorylation of MARCKS by PKC improved the initial rate of phosphorylation by Cdk2 without modifying the number of sites concerned. In contrast, phosphorylation of MARCKS by Cdk2 did not significantly affect further phosphorylation by PKC.

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