Interaction of PKN with a neuron-specific basic Helix-Loop-Helix transcription factor, NDRF/NeuroD2

By the yeast two-hybrid screening of a human brain cDNA library with the am ino-terminal regulatory region of PKN as a bait, a clone encoding a neuron- specific basic Helix-Loop-Helix (bHLH) transcription factor, NDRF/NeuroD2 w as isolated. NDRF/NeuroD2 was co-precipitated with PKN from the lysate of C OS-7 cells transfected with both expression constructs for NDRF/NeuroD2 and PKN. In vitro binding studies using the deletion mutants of NDRF/NeuroD2 s ynthesized in a rabbit reticulocyte lysate indicated that the internal regi on containing the bHLH domain of NDRF/NeuroD2 was necessary and sufficient for the interaction with PKN. In addition, recombinant NDRF/NeuroD2 purifie d from Escherichia coli could bind PKN, suggesting the direct interaction b etween NDRF/NeuroD2 and PKN. Transient transfection assays using P19 cells revealed that expression of NDRF/NeuroD2 increased the transactivation of t he rat insulin promoter element 3 (RIPE3) enhancer up to approximately in-f old and that co-expression of catalytically active form of PKN, but not kin ase-deficient derivative, resulted in a further threefold increase of NDRF/ NeuroD2-mediated transcription. These findings suggest that PKN may contrib ute to transcriptional responses through the post-translational modificatio n of the NDRF/NeuroD2-dependent transcriptional machinery. (C) 1999 Elsevie r Science B.V. All rights reserved.