A consensus cAMP-dependent protein kinase (PK-A) site in place of the CcN motif casein kinase II site simian virus 40 large T-antigen confers PK-A-mediated regulation of nuclear import.
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.
Xiao
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CY
Division for Biochemistry and Molecular Biology, John Curtin School of Medical Research, Canberra, ACT Australia.
Hübner
S
S
Elliot
R M
RM
Caon
A
A
Jans
D A
DA
D000595:Amino Acid Sequence; D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D001693:Biological Transport, Active; D047390:Casein Kinase II; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D016384:Consensus Sequence; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D066298:In Vitro Techniques; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins / Q000378:metabolism; D056489:Nucleoplasmins; D010750:Phosphoproteins; D010766:Phosphorylation; D015202:Protein Engineering; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014981:Xenopus
D000952:Antigens, Polyomavirus Transforming; D009687:Nuclear Proteins; D056489:Nucleoplasmins; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases; D017868:Cyclic AMP-Dependent Protein Kinases