[Cell-penetrating peptide-based functional study of p38 MAPK].
OBJECTIVE
To construct a p38 MAPK protein delivery system based on TAT protein and study its functions in eukaryotic cells.


METHODS
Recombinant vectors pHis-TAT-p38 and pHis-TAT-p38(AF) were constructed, and two recombinant proteins, His-TAT-p38 and His-TAT-p38(AF), were expressed and purified in E. coli. The two fusion proteins were then incubated with ECV304 cells, respectively. The phosphorylation of ATF2 was detected to assay the effect of His-TAT-p38 on endogeneious p38 activity after the cells were stimulated by sorbitol.


RESULTS
The results of restriction enzyme digestion and DNA sequencing showed that the two recombinant vectors were correctly constructed. The recombinant proteins of His-TAT-p38 and His-TAT-p38(AF) were isolated and purified by SDS-PAGE, and Western blotting suggested that His-TAT-p38 and its mutant with dual phosphorylation sites could enter the cells efficiently in a time- and concentration-dependent manner. His-TAT-p38 was found capable of increasing the activity of endogenous p38 in ECV304 cells, but His-TAT-p38(AF) inhibited the phosphorylation of ATF2 so as to block the transduction of p38 signal pathway when the cells were stimulated with sorbitol.


CONCLUSIONS
p38 MAPK protein delivery system based on TAT protein has been constructed successfully. It is confirmed that TAT can transfer the proteins into the cells in a time- and dose-depended manner. TAT-p38 and its dominant negative form possess high biological activity after transduction into ECV304 cells by TAT protein delivery system, and the former can increase the activity of endogenous ATF2, but the latter inhibits the transduction of endogeneious p38 signal pathway in ECV304 cells with high osmotic stress.
Yang
Li-ping
LP
Department of Pathophysiology and Guangdong Provincial Key Laboratory of Functional Proteomics, Southern Medical University, Guangzhou 510515, China.
Liu
Zhi-feng
ZF
Li
Yong-ming
YM
Li
Zhi-jie
ZJ
Jiang
Yong
Y
D015153:Blotting, Western; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D005057:Eukaryotic Cells / Q000166:cytology / Q000378:metabolism; D015696:Gene Products, tat / Q000235:genetics / Q000378:metabolism; D006801:Humans; D010455:Peptides / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D048051:p38 Mitogen-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism*
D015696:Gene Products, tat; D010455:Peptides; D011993:Recombinant Fusion Proteins; D048051:p38 Mitogen-Activated Protein Kinases