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.
Highlights
Proteins such as histones appear to be constitutively targeted to the nucleus, others are only translocated to the nucleus under specific conditions, otherwise being predominantly cytoplasmic (Nigg et al, 1991; Jans, 1995)
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
We have demonstrated that nuclear import of SV40 large tumor antigen (T-ag) fusion proteins is regulated by the CcN motif (Jans et al, 1991), comprising phosphorylation sites for casein kinase II (CKII) and the cyclin-dependent kinase cdc2 together with the nuclear localization signal (NLS)
Summary
Chemicals and Reagents—Isopropyl--thiogalactoside, recombinant human CKII (EC 2.7.1.3), the CKII-specific peptide substrate Arg-ArgArg-Asp-Asp-Asp-Ser-Asp-Asp-Asp, -galactosidase (EC 3.2.1.23.37), and polyethylene glycol were from Boehringer Mannheim, 5-iodacetamido-fluorescein was from Molecular Probes, and kemptide (Leu-ArgArg-Ala-Ser-Leu-Gly) and PK-A (EC 2.7.1.37) C-subunit (bovine heart) were from Sigma. Phosphorylation in cytosolic extract from HTC cells and reticulocyte lysate (the cytosol routinely used in the in vitro nuclear transport assay) was performed as described (Jans and Jans, 1994), except that where indicated, cAMP (25 M) and/or PK-I 5–24 peptide (2.5 M) was included in the incubation (1 h, 30 °C). Prior to SDS gel electrophoresis, phosphorylated fusion proteins were separated from components of the cytosolic extracts using affinity chromatography performed in batch. Quantitation of the stoichiometry of phosphorylation in cell extracts was performed using a Molecular Dynamics PhosphorImager, where exposure values for fusion protein bands were converted to absolute values through identical analysis of in vitro phosphorylated samples of predetermined stoichiometry of phosphorylation (Botterell et al, 1987; Jans and Jans, 1994)
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