scholarly journals Nuclear Entry Mechanism of Rat PER2 (rPER2): Role of rPER2 in Nuclear Localization of CRY Protein

2001 ◽  
Vol 21 (19) ◽  
pp. 6651-6659 ◽  
Author(s):  
Koyomi Miyazaki ◽  
Miho Mesaki ◽  
Norio Ishida
Keyword(s):  
Nuclear Localization ◽  
Clock Gene ◽  
Clock Genes ◽  
Nuclear Entry ◽  
Cry Protein ◽  
Terminal Domain ◽  
Carboxyl Terminal Domain ◽  
Localization Signal ◽  
Carboxyl Terminal

ABSTRACT Mammalian PERIOD2 protein (PER2) is the product of a clock gene that controls circadian rhythms, because PER2-deficient mice have an arrhythmic phenotype. The nuclear entry regulation of clock gene products is a key step in proper circadian rhythm formation in bothDrosophila and mammals, because the periodic transcription of clock genes is controlled by an intracellular, oscillating, negative feedback loop. The present study used deletion mutants of rat PER2 (rPER2) to identify the functional nuclear localization signal (NLS) in rPER2. The elimination of putative NLS (residues 778 to 794) from the rPER2 fragment resulted in the loss of nuclear entry activity. Adding the NLS to the cytosolic protein (bacterial alkaline phosphatase) translocates the fusion protein to the nuclei. The data indicate the presence of a functional NLS in rPER2. Furthermore, intact rPER2 was preferentially translocated from the cytoplasm to the nucleus when coexpressed with human CRY1 (hCRY1). However, rPER2 mutants lacking a carboxyl-terminal domain could not enter the nucleus even in the presence of hCRY1. In addition, coexpression of the nuclear localization domain (residues 512 to 794) lacking rPER2 and CRY1 changed the subcellular localization of CRY1 from the nucleus to the cytoplasm. In vitro protein interaction studies demonstrated that the carboxyl-terminal domain of rPER2 is essential for binding to CRY1. The data suggested that both the rPER2 NLS and carboxyl-terminal CRY binding domain are essential for nuclear entry of the rPER2-CRY1 complex.

scholarly journals Tyrosine phosphorylation of mammalian RNA polymerase II carboxyl-terminal domain

1993 ◽  
Vol 90 (23) ◽  
pp. 11167-11171 ◽  
Author(s):  
R Baskaran ◽  
M E Dahmus ◽  
J Y Wang
Keyword(s):  
Tyrosine Kinase ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Tandem Repeats ◽  
Consensus Sequence ◽  
Src Tyrosine Kinase ◽  
Terminal Domain ◽  
Carboxyl Terminal Domain ◽  
Carboxyl Terminal

The carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II is composed of tandem repeats of the consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. Phosphorylation of the CTD occurs during formation of the initiation complex and is correlated with the transition from complex assembly to elongation. Previously, serine and threonine residues within the CTD have been shown to be modified by the addition of phosphate and by the addition of O-linked GlcNAc. Our results establish that the CTD is also modified in vivo by phosphorylation on tyrosine. Furthermore, a nuclear tyrosine kinase encoded by the c-abl protooncogene phosphorylates the CTD to a high stoichiometry in vitro. Under conditions of maximum phosphorylation, approximately 30 mol of phosphate are incorporated per mol of CTD. The observation that the CTD is not phosphorylated by c-Src tyrosine kinase under identical conditions indicates that the CTD is not a substrate of all tyrosine kinases. Phosphorylation of tyrosine residues within the CTD may modulate the interaction of RNA polymerase II with the preinitiation complex and, hence, may be important in regulating gene expression.

scholarly journals In vitro reconstitution of a heterotrimeric nucleoporin complex consisting of recombinant Nsp1p, Nup49p, and Nup57p.

1997 ◽  
Vol 8 (1) ◽  
pp. 33-46 ◽  
Author(s):  
N L Schlaich ◽  
M Häner ◽  
A Lustig ◽  
U Aebi ◽  
E C Hurt
Keyword(s):  
Nucleocytoplasmic Transport ◽  
Full Length ◽  
Yeast Cells ◽  
Heptad Repeat ◽  
Nuclear Pores ◽  
Terminal Domain ◽  
Carboxyl Terminal Domain ◽  
Carboxyl Terminal

The yeast nucleoporins Nsp1p, Nup49p, and Nup57p form a complex at the nuclear pores which is involved in nucleocytoplasmic transport. To investigate the molecular basis underlying complex formation, recombinant full-length Nup49p and Nup57p and the carboxyl-terminal domain of Nsp1p, which lacks the FXFG repeat domain, were expressed in Escherichia coli. When the three purified proteins were mixed together, they spontaneously associated to form a 150-kDa complex of 1:1:1 stoichiometry. In this trimeric complex, Nup57p fulfills the role of an organizing center, to which Nup49p and Nsp1p individually bind. For this interaction to occur, only two heptad repeat regions of the Nsp1p carboxyl-terminal domain are required, each region being about 50 amino acids in length. Finally, the reconstituted complex has the capability to bind to full-length Nic96p but not to mutant forms which also do not interact in vivo. When added to permeabilized yeast cells, the complex associates with the nuclear envelope and the nuclear pores. We conclude that Nsp1p, Nup49p, and Nup57p can reconstitute a complex in vitro which is competent for further assembly with other components of nuclear pores.

scholarly journals Interaction of the Vp3 Nuclear Localization Signal with the Importin α2/β Heterodimer Directs Nuclear Entry of Infecting Simian Virus 40

2002 ◽  
Vol 76 (18) ◽  
pp. 9368-9377 ◽  
Author(s):  
Akira Nakanishi ◽  
Dorothy Shum ◽  
Hiroshi Morioka ◽  
Eiko Otsuka ◽  
Harumi Kasamatsu
Keyword(s):  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Selective Exposure ◽  
Nuclear Entry ◽  
New Host ◽  
Localization Signal ◽  
Nucleoprotein Complexes

ABSTRACT For nuclear entry of large nucleoprotein complexes, it is thought that one key nuclear localization signal (NLS) of a protein component becomes exposed to mediate importin recognition. We show that the nuclear entry of simian virus 40 involves a dynamic interplay between two distinct interiorly situated capsid NLSs, the Vp1 NLS and the Vp3 NLS, and the selective exposure and importin recognition of the Vp3 NLS. The Vp3 NLS-null mutants assembled normally into virion-like particles (VLP) in mutant DNA-transfected cells. When used to infect a new host, the null VLP entered the cell normally but was impaired in viral DNA nuclear entry due to a lack of recognition by the importin α2/β heterodimer, leading to reduced viability. Both Vp3 and Vp1 NLSs directed importin interaction in vitro, but the Vp1 NLS, which overlaps the Vp1 DNA binding domain, did not bind importins in the presence of DNA. The results suggest that certain canonical NLSs within a nucleoprotein complex, such as the Vp1 NLS, can be masked from functioning by binding to the nucleic acid component and that the availability of an NLS that is not masked and can become exposed for importin binding, such as the Vp3 NLS, is a general feature of the nuclear entry of the nucleoprotein complexes, including those of other animal viruses.

scholarly journals ADA3, a putative transcriptional adaptor, consists of two separable domains and interacts with ADA2 and GCN5 in a trimeric complex.

1995 ◽  
Vol 15 (3) ◽  
pp. 1203-1209 ◽  
Author(s):  
J Horiuchi ◽  
N Silverman ◽  
G A Marcus ◽  
L Guarente
Keyword(s):  
Activation Domains ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Trimeric Complex ◽  
Nonoverlapping Domains ◽  
Carboxyl Terminal Domain ◽  
Carboxyl Terminal ◽  
Amino Terminal Domain

Mutations in yeast ADA2, ADA3, and GCN5 weaken the activation potential of a subset of acidic activation domains. In this report, we show that their gene products form a heterotrimeric complex in vitro, with ADA2 as the linchpin holding ADA3 and GCN5 together. Further, activation by LexA-ADA3 fusions in vivo are regulated by the levels of ADA2. Combined with a prior observation that LexA-ADA2 fusions are regulated by the levels of ADA3 (N. Silverman, J. Agapite, and L. Guarente, Proc. Natl. Acad. Sci. USA 91:11665-11668, 1994), this finding suggests that these proteins also form a complex in cells. ADA3 can be separated into two nonoverlapping domains, an amino-terminal domain and a carboxyl-terminal domain, which do not separately complement the slow-growth phenotype or transcriptional defect of a delta ada3 strain but together supply full complementation. The carboxyl-terminal domain of ADA3 alone suffices for heterotrimeric complex formation in vitro and activation of LexA-ADA2 in vivo. We present a model depicting the ADA complex as a coactivator in which the ADA3 amino-terminal domain mediates an interaction between activation domains and the ADA complex.

Sign in / Sign up

Export Citation Format

Share Document