Binding of p110 retinoblastoma protein inhibits nuclear import of simian virus SV40 large tumor antigen

Alex J Fulcher, Manisha M Dias, David Andrew Jans

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Abstract

Nuclear import of the simian virus 40 large tumour antigen (T-ag) is dependent on its nuclear localisation signal (NLS) within amino acids 126-132 that is recognised by the importin alpha/beta1 heterodimer, as well as a protein kinase CK2 site at serine 112 upstream of the NLS, which enhances the interaction c. 50-fold. Here we show for the first time that T-ag nuclear import is negatively regulated by N-terminal sequences (amino acids 102-110) which represent the binding site (BS) for the retinoblastoma (Rb) tumour suppressor protein (p110(Rb)). Quantitative confocal laser scanning microscopic analysis of the transport properties of T-ag constructs with or without Rb binding site mutations in living transfected cells or in a reconstituted nuclear transport system indicate that the presence of the RbBS significantly reduces nuclear accumulation of T-ag. A number of approaches, including the analysis of T-ag nuclear import in an isogenic cell pair with and without functional p110(Rb) implicate p110(Rb) binding as being responsible for the reduced nuclear accumulation, with the serine(106) phosphorylation site within the RbBS appearing to enhance the inhibitory effect. Immunoprecipitation experiments confirmed association of T-ag and p110(Rb) and dependence thereof on negative charge at serine(106). The involvement of p110(Rb) in modulating T-ag nuclear transport has implications for the regulation of nuclear import of other proteins from viruses of medical significance that interact with p110(Rb), and how this may relate to transformation.
Original languageEnglish
Pages (from-to)17744 - 17753
Number of pages10
JournalThe Journal of Biological Chemistry
Volume285
Issue number23
DOIs
Publication statusPublished - 2010

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