TY - JOUR
T1 - A novel nuclear localization signal in the human single-minded proteins SIM1 and SIM2
AU - Yamaki, Akiko
AU - Kudoh, Jun
AU - Shimizu, Nobuyoshi
AU - Shimizu, Yoshiko
N1 - Funding Information:
The authors thank N. Kawahara for her contribution at the initial stage of this work. This work was supported in part by a Grant-in-Aid for Encouragement of Young Scientists (to A.Y.), a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan (to Y.S.), a Fund for ‘Research for the Future’ Program from the Japan Society for the Promotion of Science and MEXT (to N.S.), and ‘Gakujyutsu-Frontier’ program and Project Research Grant of Kyorin University (to Y.S.).
PY - 2004/1/16
Y1 - 2004/1/16
N2 - Human Single-minded 1 (SIM1) and SIM2 genes were found as homologs of Drosophila sim gene which plays a key role in the midline cell lineage of the central nervous system. SIM proteins belong to a family of transcription factors, called bHLH/PAS. Here we examined the intracellular localization of SIM proteins using the expression constructs of whole SIM2 or SIM1 protein fused with enhanced green fluorescent protein (EGFP). The transient expression analysis revealed the nuclear localization of SIM proteins in the cultured cells. To identify the nuclear localization signal, we made expression constructs of EGFP-fusion protein consisting of various portions of SIM proteins. Transfection assay showed the presence of NLS activity in the small region of 23 and 21 amino acid residues at the central part of SIM2 and SIM1 proteins, respectively. Further analysis with amino acid substitution of this small region of SIM2 protein revealed the critical role of five amino acid residues (Arg367, Lys373, Pro385, Tyr386, and Gln389) in NLS activity. The consensus sequence of RKxxKx[K/R]xxxxKxKxRxxPY was estimated as a presumptive NLS in SIM proteins from various species. Thus, the NLS consisting of a cluster of basic amino acids with Pro and Tyr at the C-terminal end is novel and well conserved in the SIM proteins during evolution.
AB - Human Single-minded 1 (SIM1) and SIM2 genes were found as homologs of Drosophila sim gene which plays a key role in the midline cell lineage of the central nervous system. SIM proteins belong to a family of transcription factors, called bHLH/PAS. Here we examined the intracellular localization of SIM proteins using the expression constructs of whole SIM2 or SIM1 protein fused with enhanced green fluorescent protein (EGFP). The transient expression analysis revealed the nuclear localization of SIM proteins in the cultured cells. To identify the nuclear localization signal, we made expression constructs of EGFP-fusion protein consisting of various portions of SIM proteins. Transfection assay showed the presence of NLS activity in the small region of 23 and 21 amino acid residues at the central part of SIM2 and SIM1 proteins, respectively. Further analysis with amino acid substitution of this small region of SIM2 protein revealed the critical role of five amino acid residues (Arg367, Lys373, Pro385, Tyr386, and Gln389) in NLS activity. The consensus sequence of RKxxKx[K/R]xxxxKxKxRxxPY was estimated as a presumptive NLS in SIM proteins from various species. Thus, the NLS consisting of a cluster of basic amino acids with Pro and Tyr at the C-terminal end is novel and well conserved in the SIM proteins during evolution.
KW - Amino acid substitution
KW - Deletion constructs
KW - Enhanced green fluorescent protein
KW - Nuclear localization signal
KW - SIM2
KW - Transfection
KW - single-minded 1
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U2 - 10.1016/j.bbrc.2003.11.168
DO - 10.1016/j.bbrc.2003.11.168
M3 - Article
C2 - 14697214
AN - SCOPUS:0346433889
VL - 313
SP - 482
EP - 488
JO - Biochemical and Biophysical Research Communications
JF - Biochemical and Biophysical Research Communications
SN - 0006-291X
IS - 3
ER -