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 amino-terminal regulatory region of PKN as a bait, a clone encoding a neuron-specific basic Helix-Loop-Helix (bHLH) transcription factor, NDRF/NeuroD2 was isolated. NDRF/NeuroD2 was co-precipitated with PKN from the lysate of COS-7 cells transfected with both expression constructs for NDRF/NeuroD2 and PKN. In vitro binding studies using the deletion mutants of NDRF/NeuroD2 synthesized in a rabbit reticulocyte lysate indicated that the internal region containing the bHLH domain of NDRF/NeuroD2 was necessary and sufficient for the interaction with PKN. In addition, recombinant NDRF/NeuroD2 purified from Escherichia coli could bind PKN, suggesting the direct interaction between NDRF/NeuroD2 and PKN. Transient transfection assays using P19 cells revealed that expression of NDRF/NeuroD2 increased the transactivation of the rat insulin promoter element 3 (RIPE3) enhancer up to approximately 12-fold and that co-expression of catalytically active form of PKN, but not kinase-deficient derivative, resulted in a further threefold increase of NDRF/NeuroD2-mediated transcription. These findings suggest that PKN may contribute to transcriptional responses through the post-translational modification of the NDRF/NeuroD2-dependent transcriptional machinery.
Shibata
H
H
Department of Biology, Faculty of Science, Kobe University, Kobe, Japan.
Oda
H
H
Mukai
H
H
Oishi
K
K
Misaki
K
K
Ohkubo
H
H
Ono
Y
Y
D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins; D019556:COS Cells; D002384:Catalysis; D002460:Cell Line; D005786:Gene Expression Regulation; D018257:Helix-Loop-Helix Motifs; D006801:Humans; D009154:Mutation; D009479:Neuropeptides / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D011493:Protein Kinase C; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D011817:Rabbits; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured; D020798:Two-Hybrid System Techniques
D051792:Basic Helix-Loop-Helix Proteins; D009479:Neuropeptides; D011493:Protein Kinase C; D017346:Protein Serine-Threonine Kinases; D011505:Protein-Tyrosine Kinases; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; C490925:NEUROD2 protein, human; C490924:Neurod2 protein, rat; C061570:protein kinase N