scholarly journals Phosphorylation is required for PMA- and cell-cycle-induced degradation of protein kinase Cδ

2002 ◽  
Vol 368 (1) ◽  
pp. 349-355 ◽  
Author(s):  
Jyoti SRIVASTAVA ◽  
Katarzyna J. PROCYK ◽  
Xavier ITURRIOZ ◽  
Peter J. PARKER
Keyword(s):  
Cell Cycle ◽  
Protein Kinase ◽  
Phosphorylation Site ◽  
Culture Conditions ◽  
Calyculin A ◽  
Down Regulation ◽  
Pkc Isoforms ◽  
Tumour Promoters ◽  
Cycle Dependent ◽  
Nih 3T3

Classical and novel protein kinase C (PKC) isoforms are down-regulated as a result of chronic activation by certain tumour promoters and physiological stimuli; however, the mechanisms leading to down-regulation are not fully understood. In the present study, we have studied the PMA ('TPA')-induced degradation of PKCΔ in NIH 3T3 cells under culture conditions where PKCΔ displays cell-cycle-dependent down-regulation. In contrast with previous studies, a hyperphosphorylated form of this PKC isoform, promoted by calyculin A, was rapidly degraded in PMA-treated cells. Similarly, the presence of calyculin A enhanced the down-regulation of PKCΔ observed on G1/S-phase progression through the cell cycle. Analysis of phosphorylation-site mutants indicated that the T-loop Thr505 phosphorylation site was critical for induced degradation.

scholarly journals Cell Cycle-Dependent Phosphorylation of C/EBPβ Mediates Oncogenic Cooperativity between C/EBPβ and H-RasV12

2004 ◽  
Vol 24 (17) ◽  
pp. 7380-7391 ◽  
Author(s):  
Jon D. Shuman ◽  
Thomas Sebastian ◽  
Philipp Kaldis ◽  
Terry D. Copeland ◽  
Songyun Zhu ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Phosphorylation Site ◽  
Mouse Skin ◽  
Ras Signaling ◽  
Transactivation Domain ◽  
3T3 Cells ◽  
Focus Formation ◽  
Cycle Dependent ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

ABSTRACT CCAAT/enhancer binding protein β (C/EBPβ) is a widely expressed transcription factor whose activity is regulated by oncogenic Ha-RasV12 signaling. C/EBPβ is essential for the development of mouse skin tumors containing Ras mutations and can cooperate with RasV12 to transform NIH 3T3 cells. Here we have investigated Ras-induced phosphorylation of C/EBPβ in fibroblasts and report a novel proline-directed phosphoacceptor site at Ser64 within the transactivation domain. Ser64 phosphorylation was induced by activated Ras and Raf but was not blocked by chemical inhibitors of MEK1/2, phosphatidylinositol 3-kinase, JNK, or p38 mitogen-activated protein kinases. Ser64 was efficiently phosphorylated in vitro by the cyclin-dependent kinases Cdk2 and Cdc2. Thr189, previously identified as an ERK1/2 phosphorylation site that regulates C/EBPβ activity, was also a substrate for Cdk phosphorylation. Ser64 and Thr189 phosphorylation was low in serum-starved (G0) cells but was strongly increased in mid-G1 cells and in cells arrested in S or M phase. In addition, phosphorylation on both sites was blocked by treating cells with the Cdk inhibitor roscovitine. In contrast to wild-type C/EBPβ, which enhances transformation of NIH 3T3 cells, mutants bearing alanine substitutions at Ser64 and/or Thr189 inhibited RasV12-induced focus formation. Our findings support a role for C/EBPβ as a nuclear effector of Ras signaling and transformation, and they indicate that cell cycle-dependent phosphorylation of C/EBPβ on Ser64 and Thr189 is required to promote Ras-induced transformation of NIH 3T3 cells.

The Xenopus protein kinase pEg2 associates with the centrosome in a cell cycle-dependent manner, binds to the spindle microtubules and is involved in bipolar mitotic spindle assembly

1998 ◽  
Vol 111 (5) ◽  
pp. 557-572 ◽  
Author(s):  
C. Roghi ◽  
R. Giet ◽  
R. Uzbekov ◽  
N. Morin ◽  
I. Chartrain ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Protein Kinase ◽  
Mitotic Spindle ◽  
Cultured Cells ◽  
Dependent Manner ◽  
Egg Extracts ◽  
Pericentriolar Material ◽  
Spindle Microtubules ◽  
Cycle Dependent

By differential screening of a Xenopus laevis egg cDNA library, we have isolated a 2,111 bp cDNA which corresponds to a maternal mRNA specifically deadenylated after fertilisation. This cDNA, called Eg2, encodes a 407 amino acid protein kinase. The pEg2 sequence shows significant identity with members of a new protein kinase sub-family which includes Aurora from Drosophila and Ipl1 (increase in ploidy-1) from budding yeast, enzymes involved in centrosome migration and chromosome segregation, respectively. A single 46 kDa polypeptide, which corresponds to the deduced molecular mass of pEg2, is immunodetected in Xenopus oocyte and egg extracts, as well as in lysates of Xenopus XL2 cultured cells. In XL2 cells, pEg2 is immunodetected only in S, G2 and M phases of the cell cycle, where it always localises to the centrosomal region of the cell. In addition, pEg2 ‘invades’ the microtubules at the poles of the mitotic spindle in metaphase and anaphase. Immunoelectron microscopy experiments show that pEg2 is located precisely around the pericentriolar material in prophase and on the spindle microtubules in anaphase. We also demonstrate that pEg2 binds directly to taxol stabilised microtubules in vitro. In addition, we show that the presence of microtubules during mitosis is not necessary for an association between pEg2 and the centrosome. Finally we show that a catalytically inactive pEg2 kinase stops the assembly of bipolar mitotic spindles in Xenopus egg extracts.

scholarly journals Invariant Leu Preceding Turn Motif Phosphorylation Site Controls the Interaction of Protein Kinase C with Hsp70

2006 ◽  
Vol 281 (43) ◽  
pp. 32461-32468 ◽  
Author(s):  
Tianyan Gao ◽  
Alexandra C. Newton
Keyword(s):  
Protein Kinase C ◽  
Heat Shock ◽  
Protein Kinase ◽  
Phosphorylation Site ◽  
Insoluble Fraction ◽  
Down Regulation ◽  
Kinase C ◽  
Shock Proteins ◽  
Invariant Residue ◽  
Autophosphorylation Site

Heat shock proteins play important roles in regulating signal transduction in cells by associating with, and stabilizing, diverse signaling molecules, including protein kinases. Previously, we have shown that heat shock protein Hsp70 associates with protein kinase C (PKC) via an interaction that is triggered by dephosphorylation at the turn phosphorylation motif. Here we have identified an invariant residue in the carboxyl terminus of PKC that mediates the binding to Hsp70. Specifically, we show that Hsp70 binds to Leu (Leu-640) immediately preceding the conserved turn motif autophosphorylation site (Thr-641) in PKC βII. Co-immunoprecipitation experiments reveal that mutation of Leu-640 to Gly decreases the interaction of Hsp70 with PKC βII. This weakened interaction between Hsp70 and the mutant PKCs results in accumulation of dephosphorylated PKC in the detergent-insoluble fraction of cells. In addition, the Hsp70-binding mutant is considerably more sensitive to down-regulation compared with WT PKC: disruption of Hsp70 binding leads to accelerated dephosphorylation and enhanced ubiquitination of mutant PKC upon phorbol ester treatment. Last, pulse-chase experiments demonstrate that Hsp70 preferentially binds the species of mature PKC that has become dephosphorylated compared with the newly synthesized protein that has yet to be phosphorylated. Thus, Hsp70 binds a hydrophobic residue preceding the turn motif, protecting PKC from down-regulation and sustaining the signaling lifetime of the kinase.

scholarly journals Cell cycle-dependent regulation of the DNA-dependent protein kinase

1999 ◽  
Vol 32 (4) ◽  
pp. 239-248 ◽  
Author(s):  
A. Nilsson ◽  
F. Sirzén ◽  
R. Lewensohn ◽  
N. Wang ◽  
S. Skog
Keyword(s):  
Cell Cycle ◽  
Protein Kinase ◽  
Dependent Protein Kinase ◽  
Dependent Protein ◽  
Cycle Dependent

scholarly journals Protein Kinase C Regulates Late Cell Cycle-Dependent Gene Expression

2012 ◽  
Vol 32 (22) ◽  
pp. 4651-4661 ◽  
Author(s):  
Z. Darieva ◽  
N. Han ◽  
S. Warwood ◽  
K. S. Doris ◽  
B. A. Morgan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Kinase C ◽  
Cycle Dependent

Periodic biosynthesis of the human M-phase promoting factor catalytic subunit p34 during the cell cycle

1990 ◽  
Vol 10 (7) ◽  
pp. 3847-3851
Author(s):  
C H McGowan ◽  
P Russell ◽  
S I Reed
Keyword(s):  
Cell Cycle ◽  
Protein Kinase ◽  
Hela Cells ◽  
Catalytic Subunit ◽  
Dependent Manner ◽  
Reversible Phosphorylation ◽  
M Phase ◽  
A Cell ◽  
Cycle Dependent

The product of the CDC2Hs gene is the protein kinase subunit of the M-phase promoting factor, which is required for entry into mitosis. The activity of this kinase is regulated in a cell cycle-dependent manner by reversible phosphorylation and through association with other proteins. We report here that in HeLa cells, the abundance of the CDC2Hs mRNA and the rate of synthesis of the encoded protein, p34, vary in a cell cycle-dependent manner.

A Role for NIPA in DNA Damage Response.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1265-1265
Author(s):  
Christine von Klitzing ◽  
Florian Bassermann ◽  
Stephan W. Morris ◽  
Christian Peschel ◽  
Justus Duyster
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Dna Damage Response ◽  
Phosphorylation Site ◽  
Genotoxic Stress ◽  
S Phase ◽  
Damage Response ◽  
A Cell ◽  
Cycle Dependent

Abstract The nuclear interaction partner of ALK (NIPA) is a nuclear protein identified by our group in a screen for NPM-ALK interaction partners. We recently reported that NIPA is an F-box protein that assembles with SKP1, Cul1 and Roc1 to establish a novel SCF-type E3 ubiquitin ligase. The formation of the SCFNIPA complex is regulated by cell cycle-dependent phosphorylation of NIPA that restricts SCFNIPA assembly from G1- to late S-phase, thus allowing its substrates to be active from late S-phase throughout mitosis. Proteins involved in cell cycle regulation frequently play a role in DNA damage checkpoints. We therefore sought to determine whether NIPA has a function in the cellular response to genotoxic stress. For this reason we treated NIH/3T3 cells with various DNA-damaging agents. Surprisingly, we observed phosphorylation of NIPA in response to some of these agents, including UV radiation. This phosphorylation was cell cycle phase independent and thus independent of the physiological cell cycle dependent phosphorylation of NIPA. The relevant phosphorylation site is identical to the respective site in the course of cell cycle-dependent phosphorylation of NIPA. Thus, phosphorylation of NIPA upon genotoxic stress would inactivate the SCFNIPA complex in a cell cycle independent manner. Interestingly, this phosphorylation site lies within a consensus site of the Chk1/Chk2 checkpoint kinases. These kinases are central to DNA damage checkpoint signaling. Chk1 is activated by ATR in response to blocked replication forks as they occur after treatment with UV. We performed experiments using the ATM/ATR inhibitor caffeine and the Chk1 inhibitor SB218078 to investigate a potential role of Chk1 in NIPA phosphorylation. Indeed, we found both inhibitors to prevent UV-induced phosphorylation of NIPA. Current experiments applying Chk1 knock-out cells will unravel the role of Chk1 in NIPA phosphorylation. Additional experiments were performed to investigate a function for NIPA in DNA-damage induced apoptosis. In this regard, we observed overexpression of NIPA WT to induce apoptosis in response to UV, whereas no proapoptotic effect was seen with the phosphorylation deficient NIPA mutant. Therefore, the phosphorylated form of NIPA may be involved in apoptotic signaling pathways. In summary, we present data suggesting a cell cycle independent function for NIPA. This activity is involved in DNA damage response and may be involved in regulating apoptosis upon genotoxic stress.

Effects of PKC activation on the meiotic maturation, fertilization and early embryonic development of mouse oocytes

Zygote ◽  
2003 ◽  
Vol 11 (4) ◽  
pp. 329-337 ◽  
Author(s):  
He-Mei Quan ◽  
Heng-Yu Fan ◽  
Xiao-Qian Meng ◽  
Li-Jun Huo ◽  
Da-Yuan Chen ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Protein Kinase ◽  
Subcellular Localization ◽  
Cell Cycle Progression ◽  
Meiotic Maturation ◽  
Cycle Progression ◽  
Mouse Oocytes ◽  
Early Embryos ◽  
Pkc Isoforms ◽  
Pkc Activation

Protein kinase C (PKC) is a family of Ser/Thr protein kinase widely distributed in eukaryotes. There is evidence that PKC plays key roles in the meiotic maturation and activation of mammalian oocytes. However, the mechanism of PKC's actions and the PKC isoforms responsible for these actions are poorly understood. In this study, we reveal in mouse eggs and early embryos: (1) the effects of PKC on the meiotic and mitotic cell cycle progression during oocyte maturation, egg activation and embryonic cleavages; (2) the functional importance of classical PKC subclasses in these processes; and (3) the subcellular localization of the PKCα isoform during development from GV stage oocytes to the blastocyst stage embryos. The results indicate that the PKC activator phorbol 12-myristate 13-acetate (PMA) inhibits the meiotic resumption of cumulus-free mouse oocytes by a mechanism dependent not only on classical PKC activity but also on other PKC isoforms. PKC activation after germinal vesicle breakdown leads to the inhibition of mitogen-activated protein kinase phosphorylation and the arrest of cell cycle at MI stage. The second polar body emission and the cleavages of early embryos are blocked after prolonged PKC activation. The subcellular localization of PKCα isoform in mouse oocytes and embryos is developmental-stage associated. All these results suggest that PKC has multiple functional roles in the cell cycle progression of mouse oocytes and embryos.

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