Characterization of the REST/NRSF-interacting LIM domain protein (RILP): localization and interaction with REST/NRSF

J Neurochem. 2006 Feb;96(4):1130-8. doi: 10.1111/j.1471-4159.2005.03608.x. Epub 2006 Jan 17.

Abstract

We previously identified a nuclear envelope protein repressor element-1 silencing transcription factor (REST)/neuron-restrictive silencer factor (NRSF)-interacting Lin-11, Isl-1 and Mec-3 (LIM) domain protein (RILP) that we proposed functions in the nuclear translocation of the transcriptional repressor REST/NRSF. In this study we assessed the functionality of the prenylation motif, protein kinase A (PKA) phosphorylation sites and nuclear localization sequences (NLSs) of RILP. [(3)H]-mevalonolactone labeled endogenous RILP, showing that RILP is indeed prenylated, while phosphorylation analysis showed that the two PKA sites are phosphorylated. Blocking RILP prenylation, mutating the NLSs or mutating the PKA phosphorylation sites caused RILP to mislocalize to the cytosol. Concurrent with this mislocalization of RILP, REST/NRSF and REST4, which are normally found in the nucleus, co-localized in the cytosol with the RILP mutants. This provides additional evidence that RILP interacts with REST/NRSF and REST4 in vivo, and is involved in the nuclear localization of REST/NRSF and REST4. Reporter gene analysis using the promoter region of the human cholinergic gene locus revealed that these RILP mutants prevented repression of the reporter gene. By trapping REST/NRSF in the cytosol, the RILP mutants prevented translocation to the nucleus where REST/NRSF binds to an RE-1/NRSE element to repress gene transcription. These results show that RILP is required for REST/NRSF nuclear targeting and function.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Adaptor Proteins, Signal Transducing
  • Carrier Proteins / genetics
  • Carrier Proteins / metabolism*
  • Cyclic AMP-Dependent Protein Kinases / genetics
  • Cyclic AMP-Dependent Protein Kinases / metabolism
  • DNA Primers
  • HeLa Cells
  • Humans
  • Mutagenesis
  • Phosphorylation
  • Protein Prenylation
  • Recombinant Proteins / metabolism
  • Repressor Proteins / genetics
  • Repressor Proteins / metabolism*
  • Transcription Factors / genetics
  • Transcription Factors / metabolism*

Substances

  • Adaptor Proteins, Signal Transducing
  • Carrier Proteins
  • DNA Primers
  • RE1-silencing transcription factor
  • RILP protein, human
  • Recombinant Proteins
  • Repressor Proteins
  • Transcription Factors
  • Cyclic AMP-Dependent Protein Kinases