Distinct mechanisms of nuclear accumulation regulate the functional consequence of E2F transcription factors

1997 ◽  
Vol 110 (22) ◽  
pp. 2819-2831 ◽  
Author(s):  
K.E. Allen ◽  
S. de la Luna ◽  
R.M. Kerkhoven ◽  
R. Bernards ◽  
N.B. La Thangue
Keyword(s):  
Cell Cycle ◽  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Cell Cycle Progression ◽  
Nuclear Accumulation ◽  
Cycle Progression ◽  
Pocket Proteins ◽  
Functional Consequence ◽  
Pocket Protein ◽  
Localization Signal

Transcription factor E2F plays an important role in coordinating and integrating early cell cycle progression with the transcription apparatus. It is known that physiological E2F arises when a member of two families of proteins, E2F and DP, interact as E2F/DP heterodimers and that transcriptional activity is regulated through the physical association of pocket proteins such as pRb. However, little information is available regarding the mechanisms which control the levels of functional E2F. In this study, we have characterised one such mechanism which regulates the nuclear accumulation and activity of E2F. Specifically, we show that E2F proteins fall into two distinct categories according to their ability to accumulate in nuclei, one being exemplified by E2F-1 and the other by E2F-4 and -5. Thus, E2F-1 possesses an intrinsic nuclear localization signal whereas E2F-4 and -5 are devoid of such a signal. Furthermore, we find for E2F-4 and -5 that two distinct processes govern their nuclear accumulation whereby the nuclear localization signal is supplied in trans from either a DP heterodimer partner or a physically associated pocket protein. It is consistent with the role of pocket proteins in regulating nuclear accumulation that we find E2F-5 to be nuclear during early cell cycle progression with an increased cytoplasmic concentration in cycling cells. Our data show that the mechanism of nuclear accumulation determines the functional consequence of E2F on cell cycle progression: pocket protein-mediated accumulation impedes cell cycle progression, whereas DP-regulated nuclear accumulation promotes cell cycle progression. Moreover, the inactivation of pocket proteins by the adenovirus Ela protein, and subsequent release of E2F, failed to displace nuclear E2F. Our study identifies a new level of regulation in the control of E2F activity exerted at the level of nuclear accumulation where subunit composition and interaction with pocket proteins dictates the functional consequence on cell cycle progression.

scholarly journals The cell cycle-dependent nuclear import of v-Jun is regulated by phosphorylation of a serine adjacent to the nuclear localization signal.

1995 ◽  
Vol 130 (2) ◽  
pp. 255-263 ◽  
Author(s):  
T Tagawa ◽  
T Kuroki ◽  
P K Vogt ◽  
K Chida
Keyword(s):  
Cell Cycle ◽  
Nuclear Localization ◽  
Nuclear Import ◽  
Nuclear Localization Signal ◽  
Protein Kinase Inhibitors ◽  
Nuclear Accumulation ◽  
Dependent Manner ◽  
Localization Signal ◽  
Cycle Dependent

Cell cycle-dependent phosphorylation and nuclear import of the tumorigenic transcription factor viral Jun (v-Jun) were investigated in chicken embryo fibroblasts. Nuclear accumulation of v-Jun but not of cellular Jun (c-Jun) is cell cycle dependent, decreasing in G1 and increasing in G2. The cell cycle-dependent regulation of v-Jun was mapped to a single serine residue at position 248 (Ser248), adjacent to the nuclear localization signal (NLS). Ser248 of v-Jun represents an amino acid substitution, replacing cysteine of c-Jun. It was shown by peptidase digestion and immunoprecipitation with antibody to the NLS that v-Jun is phosphorylated at Ser248 in the cytoplasm but not in the nucleus. This phosphorylation is high in G1 and low in G2. Nuclear accumulation of v-Jun is correlated with underphosphorylation at Ser248. The regulation of nuclear import by phosphorylation was also examined using NLS peptides with Ser248 of v-Jun. Phosphorylation of the serine inhibited nuclear import mediated by the NLS peptide in vivo and in vitro. The protein kinase inhibitors staurosporine and H7 stimulated but the phosphatase inhibitor okadaic acid inhibited nuclear import mediated by the NLS peptide. The cytosolic activity of protein kinases phosphorylating Ser248 increased in G0 and decreased during cell cycle progression, reaching a minimum in G2, whereas phosphatase activity dephosphorylating Ser248 was not changed. These results show that nuclear import of v-Jun is negatively regulated by phosphorylation at Ser248 in the cytoplasm in a cell cycle-dependent manner.

Nuclear accumulation of the E2F heterodimer regulated by subunit composition and alternative splicing of a nuclear localization signal

1996 ◽  
Vol 109 (10) ◽  
pp. 2443-2452 ◽  
Author(s):  
S. de la Luna ◽  
M.J. Burden ◽  
C.W. Lee ◽  
N.B. La Thangue
Keyword(s):  
Cell Cycle ◽  
Transcription Factor ◽  
Alternative Splicing ◽  
Dna Binding ◽  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Binding Activity ◽  
Nuclear Accumulation ◽  
Dna Binding Activity ◽  
Localization Signal

The cellular transcription factor E2F plays a critical role in integrating cell cycle progression with the transcription apparatus by virtue of a physical interaction and control by key regulators of the cell cycle, such as pRb, cyclins and cyclin-dependent kinases. Generic E2F DNA binding activity arises when a member of two families of proteins, E2F and DP, form heterodimeric complexes, an interaction which results in co-operative transcriptional and DNA binding activity. Here, we characterise a new and hitherto unexpected mechanism of control influencing the activity of E2F which is mediated at the level of intracellular location through a dependence on heterodimer formation for nuclear translocation. Nuclear accumulation is dramatically influenced by two distinct processes: alternative splicing of a nuclear localization signal and subunit composition of the E2F heterodimer. These data define a new level of control in the E2F transcription factor whereby interplay between subunits dictates the levels of nuclear DNA binding activity.

scholarly journals Mutant Parathyroid Hormone-Related Protein, Devoid of the Nuclear Localization Signal, Markedly Inhibits Arterial Smooth Muscle Cell Cycle and Neointima Formation by Coordinate Up-Regulation of p15Ink4b and p27kip1

Endocrinology ◽  
2008 ◽  
Vol 150 (3) ◽  
pp. 1429-1439 ◽  
Author(s):  
Nathalie Fiaschi-Taesch ◽  
Brian Sicari ◽  
Kiran Ubriani ◽  
Irene Cozar-Castellano ◽  
Karen K. Takane ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Nuclear Localization Signal ◽  
Cell Cycle Progression ◽  
Related Protein ◽  
Cycle Progression ◽  
Vsmc Proliferation ◽  
Adenoviral Delivery ◽  
Arterial Restenosis ◽  
Localization Signal

Arterial expression of PTH-related protein is markedly induced by angioplasty. PTH-related protein contains a nuclear localization signal (NLS). PTH-related protein mutants lacking the NLS (ΔNLS-PTH-related protein) are potent inhibitors of arterial vascular smooth muscle cell (VSMC) proliferation in vitro. This is of clinical relevance because adenoviral delivery of ΔNLS-PTH-related protein at angioplasty completely inhibits arterial restenosis in rats. In this study we explored the cellular mechanisms through which ΔNLS-PTH-related protein arrests the cell cycle. In vivo, adenoviral delivery of ΔNLS-PTH-related protein at angioplasty markedly inhibited VSMC proliferation as compared with angioplastied carotids infected with control adenovirus (Ad.LacZ). In vitro, ΔNLS-PTH-related protein overexpression was associated with a decrease in phospho-pRb, and a G0/G1 arrest. This pRb underphosphorylation was associated with stable levels of cdks 2, 4, and 6, the D and E cyclins, p16, p18, p19, and p21, but was associated with a dramatic decrease in cdk-2 and cdk4 kinase activities. Cyclin A was reduced, but restoring cyclin A adenovirally to normal did not promote cell cycle progression in ΔNLS-PTH-related protein VSMC. More importantly, p15INK4 and p27kip1, two critical inhibitors of the G1/S progression, were markedly increased. Normalization of both p15INK4b and p27kip1 by small interfering RNA knockdown normalized cell cycle progression. These data indicate that the changes in p15INK4b and p27kip1 fully account for the marked cell cycle slowing induced by ΔNLS-PTH-related protein in VSMCs. Finally, ΔNLS-PTH-related protein is able to induce p15INK4 and p27kip1 expression when delivered adenovirally to primary murine VSMCs. These studies provide a mechanistic understanding of ΔNLS-PTH-related protein actions, and suggest that ΔNLS-PTH-related protein may have particular efficacy for the prevention of arterial restenosis. This study provides the mechanistic underpinnings for understanding how Δ-NLS-PTHrP functions, and suggests that Δ-NLS-PTHrP may have particular efficacy for the prevention of arterial re-stenosis.

scholarly journals Ebolavirus VP24 Binding to Karyopherins Is Required for Inhibition of Interferon Signaling

2009 ◽  
Vol 84 (2) ◽  
pp. 1169-1175 ◽  
Author(s):  
Mathieu Mateo ◽  
St. Patrick Reid ◽  
Lawrence W. Leung ◽  
Christopher F. Basler ◽  
Viktor E. Volchkov
Keyword(s):  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Nuclear Accumulation ◽  
Interferon Signaling ◽  
Loss Of Function ◽  
Proposed Model ◽  
Localization Signal ◽  
Ifn Γ ◽  
Alpha Beta ◽  
Signal Receptors

ABSTRACT The Ebolavirus VP24 protein counteracts alpha/beta interferon (IFN-α/β) and IFN-γ signaling by blocking the nuclear accumulation of tyrosine-phosphorylated STAT1 (PY-STAT1). According to the proposed model, VP24 binding to members of the NPI-1 subfamily of karyopherin alpha (KPNα) nuclear localization signal receptors prevents their binding to PY-STAT1, thereby preventing PY-STAT1 nuclear accumulation. This study now identifies two domains of VP24 required for inhibition of IFN-β-induced gene expression and PY-STAT1 nuclear accumulation. We demonstrate that loss of function correlates with loss of binding to KPNα proteins. Thus, the VP24 IFN antagonist function requires the ability of VP24 to interact with KPNα.

Xenopus nuclear factor 7 (xnf7) possesses an NLS that functions efficiently in both oocytes and embryos

1993 ◽  
Vol 105 (2) ◽  
pp. 389-395
Author(s):  
X. Li ◽  
L.D. Etkin
Keyword(s):  
Transcription Factor ◽  
Zinc Finger ◽  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Nuclear Accumulation ◽  
Nuclear Factor ◽  
Zinc Finger Gene ◽  
Early Embryos ◽  
Localization Signal ◽  
Nuclear Phosphoprotein

Xenopus nuclear factor 7 (xnf7) is a nuclear phosphoprotein that is encoded by a member of a novel zinc finger gene family and likely functions as a transcription factor. It possesses a nuclear localization signal (NLS) similar to the bipartite basic NLS of nucleoplasmin, but unlike nucleoplasmin, which re-enters nuclei immediately after fertilization, xnf7 remains cytoplasmic until the mid-blastula transition (MBT). We have measured the accumulation of injected labeled xnf7 protein or protein produced from synthetic xnf7 transcripts in the oocyte nuclei (GV). The data show that the NLS of xnf7 functions efficiently in oocytes. Mutations in either of the bipartite basic domains of the xnf7 NLS inhibit nuclear accumulation, while mutations in the spacer sequences have no effect. The xnf7 NLS linked to pyruvate kinase directs the efficient accumulation of this protein into nuclei of early embryos prior to the MBT. These data suggest that retention of the xnf7 protein during development is the result of a mechanism that interferes with the xnf7 NLS function.

scholarly journals Phosphorylation adjacent to the nuclear localization signal of human dUTPase abolishes nuclear import: structural and mechanistic insights

2013 ◽  
Vol 69 (12) ◽  
pp. 2495-2505 ◽  
Author(s):  
Gergely Róna ◽  
Mary Marfori ◽  
Máté Borsos ◽  
Ildikó Scheer ◽  
Enikő Takács ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Nucleocytoplasmic Transport ◽  
Wild Type ◽  
Post Translational Modification ◽  
Nuclear Localization Signals ◽  
M Phase ◽  
Importin Α ◽  
Localization Signal

Phosphorylation adjacent to nuclear localization signals (NLSs) is involved in the regulation of nucleocytoplasmic transport. The nuclear isoform of human dUTPase, an enzyme that is essential for genomic integrity, has been shown to be phosphorylated on a serine residue (Ser11) in the vicinity of its nuclear localization signal; however, the effect of this phosphorylation is not yet known. To investigate this issue, an integrated set of structural, molecular and cell biological methods were employed. It is shown that NLS-adjacent phosphorylation of dUTPase occurs during the M phase of the cell cycle. Comparison of the cellular distribution of wild-type dUTPase with those of hyperphosphorylation- and hypophosphorylation-mimicking mutants suggests that phosphorylation at Ser11 leads to the exclusion of dUTPase from the nucleus. Isothermal titration microcalorimetry and additional independent biophysical techniques show that the interaction between dUTPase and importin-α, the karyopherin molecule responsible for `classical' NLS binding, is weakened significantly in the case of the S11E hyperphosphorylation-mimicking mutant. The structures of the importin-α–wild-type and the importin-α–hyperphosphorylation-mimicking dUTPase NLS complexes provide structural insights into the molecular details of this regulation. The data indicate that the post-translational modification of dUTPase during the cell cycle may modulate the nuclear availability of this enzyme.

scholarly journals Mechanisms Directing the Nuclear Localization of the CtBP Family Proteins

2006 ◽  
Vol 26 (13) ◽  
pp. 4882-4894 ◽  
Author(s):  
Alexis Verger ◽  
Kate G. R. Quinlan ◽  
Linda A. Crofts ◽  
Stefania Spanò ◽  
Daniela Corda ◽  
...  
Keyword(s):  
Nuclear Localization ◽  
Nuclear Import ◽  
Nuclear Localization Signal ◽  
Transcriptional Repression ◽  
Intracellular Trafficking ◽  
Nuclear Accumulation ◽  
Splice Isoforms ◽  
Localization Signal ◽  
Splice Isoform ◽  
Partner Proteins

ABSTRACT The C-terminal binding protein (CtBP) family includes four proteins (CtBP1 [CtBP1-L], CtBP3/BARS [CtBP1-S], CtBP2, and RIBEYE) which are implicated both in transcriptional repression and in intracellular trafficking. However, the precise mechanisms by which different CtBP proteins are targeted to different subcellular regions remains unknown. Here, we report that the nuclear import of the various CtBP proteins and splice isoforms is differentially regulated. We show that CtBP2 contains a unique nuclear localization signal (NLS) located within its N-terminal region, which contributes to its nuclear accumulation. Using heterokaryon assays, we show that CtBP2 is capable of shuttling between the nucleus and cytoplasm of the cell. Moreover, CtBP2 can heterodimerize with CtBP1-L and CtBP1-S and direct them to the nucleus. This effect strongly depends on the CtBP2 NLS. PXDLS motif-containing transcription factors, such as BKLF, that bind CtBP proteins can also direct them to the nucleus. We also report the identification of a splice isoform of CtBP2, CtBP2-S, that lacks the N-terminal NLS and localizes to the cytoplasm. Finally, we show that mutation of the CtBP NADH binding site impairs the ability of the proteins to dimerize and to associate with BKLF. This reduces the nuclear accumulation of CtBP1. Our results suggest a model in which the nuclear localization of CtBP proteins is influenced by the CtBP2 NLS, by binding to PXDLS motif partner proteins, and through the effect of NADH on CtBP dimerization.

scholarly journals Glycogen Synthase Kinase 3 Phosphorylates RBL2/p130 during Quiescence

2004 ◽  
Vol 24 (20) ◽  
pp. 8970-8980 ◽  
Author(s):  
Larisa Litovchick ◽  
Anton Chestukhin ◽  
James A. DeCaprio
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Glycogen Synthase ◽  
Glycogen Synthase Kinase ◽  
Glycogen Synthase Kinase 3 ◽  
Growth Factor Signaling ◽  
Cycle Progression ◽  
Pocket Proteins ◽  
Cyclin Dependent Kinases ◽  
The Stability

ABSTRACT Phosphorylation of the retinoblastoma-related or pocket proteins RB1/pRb, RBL1/p107, and RBL2/p130 regulates cell cycle progression and exit. While all pocket proteins are phosphorylated by cyclin-dependent kinases (CDKs) during the G1/S-phase transition, p130 is also specifically phosphorylated in G0-arrested cells. We have previously identified several phosphorylated residues that match the consensus site for glycogen synthase kinase 3 (GSK3) in the G0 form of p130. Using small-molecule inhibitors of GSK3, site-specific mutants of p130, and phospho-specific antibodies, we demonstrate here that GSK3 phosphorylates p130 during G0. Phosphorylation of p130 by GSK3 contributes to the stability of p130 but does not affect its ability to interact with E2F4 or cyclins. Regulation of p130 by GSK3 provides a novel link between growth factor signaling and regulation of the cell cycle progression and exit.

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