A Consensus cAMP-dependent Protein Kinase (PK-A) Site in Place of the CcN Motif Casein Kinase II Site of Simian Virus 40 Large T-antigen Confers PK-A-mediated Regulation of Nuclear Import

Abstract

The regulation of nuclear protein transport by phosphorylation plays a central role in gene expression in eukaryotic cells. We previously showed that nuclear import of SV40 large tumor antigen (T-ag) fusion proteins is regulated by the CcN motif, comprising phosphorylation sites for casein kinase II and the cyclin-dependent kinase cdc2, together with the nuclear localization signal. Regulation of nuclear uptake by CcN motif kinase sites also holds true for the yeast transcription factor SWI5 and the Xenopus nuclear phosphoprotein nucleoplasmin. To test directly whether a kinase site other than those of the CcN motif could regulate nuclear import of T-ag, the CcN motif casein kinase II site, which markedly increases the rate of T-ag nuclear import, was replaced by a consensus site for the cAMP-dependent protein kinase (PK-A) using site-directed mutagenesis. The resultant fusion protein could be specifically phosphorylated by PK-A in vitro and in cell extracts. Nuclear import of the fluorescently labeled protein was analyzed in the HTC rat hepatoma cell line both in vivo (microinjected cells) and in vitro (mechanically perforated cells) in the presence and the absence of cAMP and/or PK-A catalytic subunit using confocal laser scanning microscopy. In vitro PK-A-prephosphorylated protein was also tested. All results indicated that the rate of nuclear import was increased by phosphorylation at the PK-A site (2-5-fold), demonstrating that kinases other than those of the CcN motif can regulate nuclear import in response to stimulatory signals. The phosphorylation-regulated nuclear localization signal derived here represents an important first step toward developing a signal conferring inducible nuclear targeting of molecules of interest.

Supramolecular structure and nuclear targeting efficiency determine the enhancement of transfection by modified polylysines

“…12 To determine nuclear import kinetics, the labelled pLy derivatives, subsequent to precomplexation with or without DNA, were added to mechanically perforated HTC cells together with cytosolic extracts (untreated reticulocyte lysate) and an ATP-regenerating system. 12,29,30 CLSM imaging, analysis and curve fitting was carried out as previously. 12,28,30 EM and AFM Plasmid DNA and its complexes with pLy and pLyP101 at various Ly/Nu ratios were prepared for EM and rotary shadowing as previously.…”

“…12,29,30 CLSM imaging, analysis and curve fitting was carried out as previously. 12,28,30 EM and AFM Plasmid DNA and its complexes with pLy and pLyP101 at various Ly/Nu ratios were prepared for EM and rotary shadowing as previously. 12,31 For AFM, pLy and pLyP101-plasmid DNA complexes at various Ly/Nu ratios were diluted with HBS to a final DNA concentration of 10 ng/l.…”

Mutual exclusivity of DNA binding and nuclear localization signal recognition by the yeast transcription factor GAL4: implications for nonviral DNA delivery

“…40,41 Mammalian expression vectors A 44 bp oligonucleotide (5Ј-GATCGGAAGACTCTCCTC CGAGCGCTCGGAAGACTCTCCTCCGC-3Ј), containing tandem repeats of the 17mer shown to be specifically recognised by the GAL4 DNA binding domain, 25,42 was annealed with the complementary oligonucleotide (5Ј-GATCGCGGAGGAGAGTCTTCCGAGCGCTCGGAGG AGAGTCTTCC-3Ј) to yield a double stranded oligomer (17m) which was then ligated into the unique BamHI sites of the reporter plasmids pSV2neo 43 and pCH110. 44 These confer SV40 promoter directed expression of the neomycin resistance gene that confers resistance to the antibiotic G418, and the E. coli enzyme ␤-galactosidase, respectively.…”

“…The dried gel was either exposed to radiographic Biomax film (Eastman Kodak, Rochester, NY, USA), or a phosphorimager (Molecular Dynamics, Sunnyvale, CA, USA) was used to quantify the levels of bound and free radiolabelled oligomer. 33,40 For competition experiments, labelled 17m was incubated with HisGAL4(1-147) in the presence of increasing amounts of unlabelled 17m or of a sequence-unrelated oligomer (22mer: 5Ј-GATCTGATTGGGTTTCTCCCAG-TAGCGCTTGATTGGGTTTCTCCCAGT-3Ј), or with plasmids with (pSV2neo-17m and pCH110-17m) or without the 17m sequence. The latter included plasmids containing the 46-mer SOS (5Ј-GATCTGCTGTATATATA TACAGCGCTACTGTATATACACCCAGGGC-3Ј which contains specific binding sites in tandem for the DNAbinding protein lexA) inserted into the BamHI site of plasmids pSV2neo or pCH110 as for the 17m and complimentary oligomers above.…”

Perforin-dependent nuclear entry of granzyme B precedes apoptosis, and is not a consequence of nuclear membrane dysfunction

“…The validity of this approach was confirmed by TUNEL analysis (compare Figures 2A and 5), and propidium iodide staining and electron microscopy (data not shown). Visualization and quantitation of the cellular and nuclear uptake of FITC-grB and the other FITC-labeled compounds was carried out using CLSM as described previously (Jans et al, 1991;Walaschewski et al, 1995;Xiao et al, 1996), and in particular for FITClabeled grB Jans et al, 1996). Image analysis and curve fitting was carried out as described Ymer and Jans, 1995).…”