Molecular cloning and characterization of a novel protein kinase C-interacting protein with structural motifs related to RBCC family proteins.
A novel protein kinase C (PKC)-interacting protein was identified by the yeast two-hybrid screening using the regulatory domain of PKC beta I as a bait. The protein contained several structural motifs such as two putative coiled-coil regions, a RING-finger, a B-box, and a B-box-like motif in the order from NH2- to COOH-terminals. The molecular organization of the protein resembles the structure of the RBCC protein family proteins which usually have a RING-finger, a B-box, and a coiled-coil region. Therefore, the protein identified was designated as RBCK1 (RBCC protein interacting with PKC 1). Northern blot analysis showed that RBCK1 gene is expressed ubiquitously among rat tissues. RBCK1 protein associated with PKC beta I and PKC zeta when coexpressed in cultured mammalian cells. By the polymerase chain reaction-assisted DNA-binding site selection and the electrophoretic mobility shift assay, RBCK1 protein was shown to bind to several DNA fragments containing TGG-rich sequences. When the yeast GAL4 DNA-binding domain fused RBCK1 protein was expressed in COS-7 cells harboring the luciferase gene placed under a synthetic promoter containing GAL4-binding sites, the fusion protein showed enhanced transcriptional activity comparing with the GAL4 DNA-binding domain. These results suggest that RBCK1 protein might be a transcription factor that has a role in the signaling pathway through PKC.
Tokunaga
C
C
Biosignal Research Center, Faculty of Science, Kobe University, Japan.
Kuroda
S
S
Tatematsu
K
K
Nakagawa
N
N
Ono
Y
Y
Kikkawa
U
U
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D007527:Isoenzymes / Q000378:metabolism*; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011493:Protein Kinase C / Q000378:metabolism*; D064546:Protein Kinase C beta; D051381:Rats; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015533:Transcriptional Activation
D017931:DNA Primers; D018076:DNA, Complementary; D007527:Isoenzymes; D009419:Nerve Tissue Proteins; D014157:Transcription Factors; C489276:protein kinase C-binding protein Beta15, rat; D011493:Protein Kinase C; D064546:Protein Kinase C beta