The cAMP-dependent Protein Kinase Site (Ser312) Enhances Dorsal Nuclear Import through Facilitating Nuclear Localization Sequence/Importin Interaction

Lyndall J Briggs,David Stein,Jason Goltz,Vanessa C Corrigan,Athina Efthymiadis,Stefan Hübner,David A Jans

doi:10.1074/jbc.273.35.22745

Abstract

Control over the nuclear import of transcription factors (TFs) represents a level of gene regulation integral to cellular processes such as differentiation and transformation. The Drosophila TF Dorsal shares with other rel TF family members the fact that it contains a phosphorylation site for the cAMP-dependent protein kinase (PKA) 22 amino acids N-terminal to the nuclear localization signal (NLS) at amino acids 335-340. This study examines for the first time the nuclear import kinetics of Dorsal fusion proteins in rat hepatoma cells in vivo and in vitro. Nuclear uptake was found to be not only NLS-dependent, but also strongly dependent on the PKA site, whereby substitution of Ser312 by either Ala or Glu using site-directed mutagenesis severely reduced nuclear accumulation. Exogenous cAMP or PKA catalytic subunit significantly enhanced the nuclear import of wild-type proteins both in vivo and in vitro. Using a direct binding assay, the molecular basis of PKA site enhancement of Dorsal fusion protein nuclear import was determined to be PKA site-mediated modulation of NLS recognition by the importin 58/97 complex. The physiological relevance of these results is supported by the observation that Drosophila embryos expressing PKA site Dorsal mutant variants were impaired in development. We conclude that the Dorsal NLS and PKA site constitute a phosphorylation-regulated NLS essential to Dorsal function and able to function in heterologous mammalian cell systems, where phosphorylation modulates the affinity of NLS recognition by importin.

Highlights

Scheduled nuclear import of transcription factors (TFs)1 is a key factor in eukaryotic cell function [1,2,3]
In the case of the tumor antigen (T-ag) CcN motif, we have shown that substitution of one kinase site by a consensus site for another can alter the cellular signals able to regulate the nuclear import of proteins carrying the modified phosphorylation-regulated NLSs (prNLSs) [19]
This study represents the first determination of the nuclear import kinetics of the Drosophila TF Dorsal, defining its prNLS as being functional in nuclear targeting in cAMP-responsive fashion in higher mammals

Summary

Introduction

Scheduled nuclear import of transcription factors (TFs) is a key factor in eukaryotic cell function [1,2,3] While proteins such as histones appear to be constitutively targeted to the nucleus, TFs such as those of the rel family (1, 4 –7), the nuclear factor of activated T-cells [8], SWI5 from yeast [9, 10], and the cytokine responsive signal transducers and activators of transcription (STATs) [11, 12] are translocated to the nucleus only under specific conditions, being otherwise cytoplasmic and thereby directly accessible to cytoplasmic signal-transducing systems [1]. In identical fashion to other rel family members, Dorsal possesses a consensus site for PKA 22 amino acids N-terminal to a 6- amino acid NLS within the ϳ300-amino acid rel homology domain (see Fig. 1), where the enhancing role of PKA in terms of nuclear localization has been qualitatively described, using transfection systems for Dorsal [23] and c-rel [7], and implicated for NF-␬B p65 [5, 6, 23]

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Results

Conclusion