Novel properties of the protein kinase CK2-site-regulated nuclear- localization sequence of the interferon-induced nuclear factor IFI 16

2000 ◽  
Vol 353 (1) ◽  
pp. 69-77 ◽  
Author(s):  
Lyndall J. BRIGGS ◽  
Ricky W. JOHNSTONE ◽  
Rachel M. ELLIOT ◽  
Chong-Yun XIAO ◽  
Michelle DAWSON ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Nuclear Localization ◽  
Nuclear Import ◽  
Protein Kinase Ck2 ◽  
Protein Import ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Nuclear Localization Sequence ◽  
Localization Sequence ◽  
Kinase Ck2

Members of the interferon-induced class of nuclear factors possess a putative CcN motif, comparable with that within proteins such as the simian virus 40 large tumour antigen (T-ag), which confers phosphorylation-mediated regulation of nuclear-localization sequence (NLS)-dependent nuclear import. Here we examine the functionality of the interferon-induced factor 16 (IFI 16) CcN motif, demonstrating its ability to target a heterologous protein to the nucleus, and to be phosphorylated specifically by the CcN-motif-phosphorylating protein kinase CK2 (CK2). The IFI 16 NLS, however, has novel properties, conferring ATP-dependent nuclear import completely independent of cytosolic factors, as well as binding to nuclear components. The IFI 16 NLS is not recognized with high affinity by the NLS-binding importin heterodimer, and transport mediated by it is insensitive to non-hydrolysable GTP analogues. The IFI 16 NLS thus mediates nuclear import through a pathway completely distinct from that of conventional NLSs, such as that of T-ag, but intriguingly resembling that of the NLS of the HIV-1 transactivator protein Tat. Since the IFI 16 CK2 site enhances nuclear import through facilitating binding to nuclear components, this represents a novel mechanism by which the site regulates nuclear-protein import, and constitutes a difference between the IFI 16 and Tat NLSs that may be of importance in the immune response.

Novel properties of the protein kinase CK2-site-regulated nuclear- localization sequence of the interferon-induced nuclear factor IFI 16

2000 ◽  
Vol 353 (1) ◽  
pp. 69 ◽  
Author(s):  
Lyndall J. BRIGGS ◽  
Ricky W. JOHNSTONE ◽  
Rachel M. ELLIOT ◽  
Chong-Yun XIAO ◽  
Michelle DAWSON ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Nuclear Localization ◽  
Protein Kinase Ck2 ◽  
Nuclear Localization Sequence ◽  
Nuclear Factor ◽  
Localization Sequence ◽  
Kinase Ck2

scholarly journals Normal Telomere Length Maintenance in Saccharomyces cerevisiae Requires Nuclear Import of the Ever Shorter Telomeres 1 (Est1) Protein via the Importin Alpha Pathway

2014 ◽  
Vol 13 (8) ◽  
pp. 1036-1050 ◽  
Author(s):  
Charlene Hawkins ◽  
Katherine L. Friedman
Keyword(s):  
Telomere Length ◽  
Nuclear Localization ◽  
Nuclear Import ◽  
Protein Import ◽  
Repetitive Sequences ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Sv40 Large T Antigen ◽  
Importin Α

ABSTRACT The Est1 (ever shorter telomeres 1) protein is an essential component of yeast telomerase, a ribonucleoprotein complex that restores the repetitive sequences at chromosome ends (telomeres) that would otherwise be lost during DNA replication. Previous work has shown that the telomerase RNA component ( TLC1 ) transits through the cytoplasm during telomerase biogenesis, but mechanisms of protein import have not been addressed. Here we identify three nuclear localization sequences (NLSs) in Est1p. Mutation of the most N-terminal NLS in the context of full-length Est1p reduces Est1p nuclear localization and causes telomere shortening—phenotypes that are rescued by fusion with the NLS from the simian virus 40 (SV40) large-T antigen. In contrast to that of the TLC1 RNA, Est1p nuclear import is facilitated by Srp1p, the yeast homolog of importin α. The reduction in telomere length observed at the semipermissive temperature in a srp1 mutant strain is rescued by increased Est1p expression, consistent with a defect in Est1p nuclear import. These studies suggest that at least two nuclear import pathways are required to achieve normal telomere length homeostasis in yeast.

scholarly journals Stimulation of nuclear import by simian virus 40-transformed cell extracts is dependent on protein kinase activity.

1995 ◽  
Vol 15 (12) ◽  
pp. 7043-7049 ◽  
Author(s):  
C Feldherr ◽  
D Akin
Keyword(s):  
Protein Kinase ◽  
Nuclear Localization ◽  
Nuclear Import ◽  
Kinase Activity ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Protein Kinase Activity ◽  
Transport Capacity ◽  
3T3 Cells ◽  
Gold Particles

We previously reported that both the nuclear import rate of large karyophilic gold particles and the functional size of the pores are significantly greater in simian virus 40-transformed fibroblasts (the SV-T2 cell line) than in nontransformed BALB/c 3T3 cells. In this study, we found that cytosolic fractions obtained from SV-T2 cultures can increase nuclear transport capacity (both import rate and pore size) when microinjected into BALB/c 3T3 cells. The transport-enhancing function of the extracts can be abolished by the protein kinase inhibitors staurosporine and K252a as well as 5'-p-fluorosulfonylbenzoyladenosine and protein phosphatase 2A, which, although less specific, also interfere with kinase activity. Increases in transport capacity of the same magnitude as that produced by the SV-T2 extracts were obtained by microinjecting protein kinase A or C or recombinant mitogen-activated protein kinase. These data provide further support for the interpretation that the enhancer is a protein kinase. From experiments performed with specific kinase inhibitor peptides, it appears likely that protein kinase C is the active factor in the SV-T2 cytosolic fractions; however, this will require further verification. It was also determined, by using gold particles coated with bovine serum albumin conjugated to synthetic nuclear localization signal peptides that lacked phosphorylation sites, that the enhancer affects the transport machinery rather than the activity of the nuclear localization signals.

scholarly journals Context affects nuclear protein localization in Saccharomyces cerevisiae.

1989 ◽  
Vol 9 (2) ◽  
pp. 384-389 ◽  
Author(s):  
M Nelson ◽  
P Silver
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Nuclear Localization ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Single Amino Acid ◽  
Nuclear Localization Sequence ◽  
Localization Sequence ◽  
Beta Galactosidase

Proteins destined for the nucleus contain nuclear localization sequences, short stretches of amino acids responsible for targeting them to the nucleus. We show that the first 29 amino acids of GAL4, a yeast DNA-binding protein, function efficiently as a nuclear localization sequence when fused to normally cytoplasmic invertase, but not when fused to Escherichia coli beta-galactosidase. Moreover, the nuclear localization sequence from simian virus 40 T antigen functions better when fused to invertase than when fused to beta-galactosidase. A single amino acid change in the T-antigen nuclear localization sequence inhibits the nuclear localization of simian virus 40-invertase and simian virus 40-beta-galactosidase in Saccharomyces cerevisiae. From these results, we conclude that the relative ability of a nuclear localization sequence to act depends on the protein to which it is linked.

Context affects nuclear protein localization in Saccharomyces cerevisiae

1989 ◽  
Vol 9 (2) ◽  
pp. 384-389
Author(s):  
M Nelson ◽  
P Silver
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Nuclear Localization ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Single Amino Acid ◽  
Nuclear Localization Sequence ◽  
Localization Sequence ◽  
Beta Galactosidase

Proteins destined for the nucleus contain nuclear localization sequences, short stretches of amino acids responsible for targeting them to the nucleus. We show that the first 29 amino acids of GAL4, a yeast DNA-binding protein, function efficiently as a nuclear localization sequence when fused to normally cytoplasmic invertase, but not when fused to Escherichia coli beta-galactosidase. Moreover, the nuclear localization sequence from simian virus 40 T antigen functions better when fused to invertase than when fused to beta-galactosidase. A single amino acid change in the T-antigen nuclear localization sequence inhibits the nuclear localization of simian virus 40-invertase and simian virus 40-beta-galactosidase in Saccharomyces cerevisiae. From these results, we conclude that the relative ability of a nuclear localization sequence to act depends on the protein to which it is linked.

Visualization and molecular analysis of nuclear import of protein kinase CK2 subunits in living cells

2001 ◽  
pp. 81-90
Author(s):  
Véronique Martel ◽  
Odile Filhol ◽  
Arsenio Nueda ◽  
Delphine Gerber ◽  
Maria Jose Benitez ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Molecular Analysis ◽  
Nuclear Import ◽  
Protein Kinase Ck2 ◽  
Living Cells ◽  
Kinase Ck2

scholarly journals Live-Cell Fluorescence Imaging Reveals the Dynamics of Protein Kinase CK2 Individual Subunits

2003 ◽  
Vol 23 (3) ◽  
pp. 975-987 ◽  
Author(s):  
Odile Filhol ◽  
Arsenio Nueda ◽  
Véronique Martel ◽  
Delphine Gerber-Scokaert ◽  
Maria José Benitez ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Nuclear Localization ◽  
Protein Kinase Ck2 ◽  
Fluorescent Protein ◽  
Nuclear Translocation ◽  
Nuclear Accumulation ◽  
Nucleocytoplasmic Trafficking ◽  
Green Fluorescent ◽  
Nucleocytoplasmic Distribution ◽  
Kinase Ck2

ABSTRACT Protein kinase CK2 is a multifunctional enzyme which has long been described as a stable heterotetrameric complex resulting from the association of two catalytic (α or α′) and two regulatory (β) subunits. To track the spatiotemporal dynamics of CK2 in living cells, we fused its catalytic α and regulatory β subunits with green fluorescent protein (GFP). Both CK2 subunits contain nuclear localization domains that target them independently to the nucleus. Imaging of stable cell lines expressing low levels of GFP-CK2α or GFP-CK2β revealed the existence of CK2 subunit subpopulations exhibiting differential dynamics. Once in the nucleus, they diffuse randomly at different rates. Unlike CK2β, CK2α can shuttle, showing the dynamic nature of the nucleocytoplasmic trafficking of the kinase. When microinjected in the cytoplasm, the isolated CK2 subunits are rapidly translocated into the nucleus, whereas the holoenzyme complex remains in this cell compartment, suggesting an intramolecular masking of the nuclear localization sequences that suppresses nuclear accumulation. However, binding of FGF-2 to the holoenzyme triggers its nuclear translocation. Since the substrate specificity of CK2α is dramatically changed by its association with CK2β, the control of the nucleocytoplasmic distribution of each subunit may represent a unique potential regulatory mechanism for CK2 activity.

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