Author(s)

Kanako Obayashi¹, Kayoko Takada¹, Kazuaki Ohashi², Ayako Ohashi-Kobayashi³, Mayumi Nakanishi-Matsui⁴, Makoto Araki⁵, Masatomo Maeda^5*

¹Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Japan.
²Department of Medical Biochemistry, School of Pharmacy, Iwate Medical University, Shiwa-Gun, Japan.
³Department of Immunobiology, School of Pharmacy, Iwate Medical University, Shiwa-Gun, Japan.
⁴Department of Biochemistry, School of Pharmacy, Iwate Medical University, Shiwa-Gun, Japan.
⁵Department of Molecular Biology, School of Pharmacy, Iwate Medical University, Shiwa-Gun, Japan.

The transcriptional factor GATA-6 gene produces two translational isoforms from a single mRNA through ribosomal leaky scanning. L-type GATA-6 has an extension of 146 amino acid residues at its amino terminus. In the extension, there is a unique PEST sequence (Glu³¹-Cys⁴⁶), which is composed of an amino terminal Pro-rich segment and a carboxyl terminal Ser-cluster. Substitution of either half of the PEST sequence with Ala residues by cassette mutagenesis reduced the apparent molecular size of L-type GATA-6 on SDS-polyacrylamide gel-electrophoresis. However, the effect of substitution of the Pro-rich segment was much more significant; the mobility increase of the Pro-rich segment on the gel was 13% while that of the Ser-cluster was 8%. Substitution of each amino acid residue demonstrated that the effect of Pro substitution is greater than that of the Ser and Thr residues. Such increased mobility of L-type GATA-6 in the presence of a detergent may apparently correlate with the decrease in transcription activity in vivo as determined by means of luciferase reporter gene assay. The activity of ΔAla (with Ala residues instead of the PEST sequence) was reduced to one fifth of that of ΔA (with the PEST sequence). These results suggest that the PEST sequence of L-type GATA-6 does not function as a constitutive protein degradation signal, but rather plays structural and functional roles in the activation of gene expression on the GATA responsive promoter.

Cassette Mutagenesis, Long-Type GATA-6, Mobility on Gel-Electrophoresis, PEST Sequence, Proline-Rich Segment, Transcription Factor

Obayashi, K. , Takada, K. , Ohashi, K. , Ohashi-Kobayashi, A. , Nakanishi-Matsui, M. , Araki, M. and Maeda, M. (2014) Increased Electrophoretic Mobility of Long-Type GATA-6 Transcription Factor upon Substitution of Its PEST Sequence. Advances in Bioscience and Biotechnology, 5, 1032-1042. doi: 10.4236/abb.2014.513118.