[Identification of RACK1-PER1 protein interaction sites].
OBJECTIVE
By screening the cDNA library of suprachiasmatic nucleus (SCN) region of human hypothalamus, we try to capture the novel proteins interacting with PER1 and investigate the interplaying characteristic of RACK1 and PER1.


METHODS
By yeast two-hybrid system, the new protein in human SCN, which was associating with PER1-PAS domain was obtained. Then five truncated fragments of RACK1 cDNA was cloned into yeast expression vector to form recombinant library plasmid and was co-transformed into yeast strain AH109 with bait plasmid containing PER1-PAS domain. The transformants were selected via nutrition-deficient medium, and the positive clones were identified or obtained by checking the expressin of report gene. At last all the protein interactions were confirmed by co-immunoprecipitation tests.


RESULTS
one of the positive clones in human SCN cDNA library were identified with part of the RACK1 protein sequence. Three positive clones, which contained respectively the fragment of RACK1 (WD1-7), RACK1 (WD4-7) or RACK1 (WD5-7) were obtained through yeast two-hybrid screen with various RACK1 fragments and PER1-PAS domain. The RACK1 and PER1 protein interaction was determined by beta-galactosidase assay and co-immunopreipitation.


CONCLUSIONS
The direct interaction between RACK1 and PER1 has been approved. RACK1 is composed of seven WD40 repeats and the minimal interacting sites are limited in V-VII WD40 domains in the present study,which means C-terminal amino acid of RACK1 may be essential to the interacting.
Lu
Fang
F
Department of Biomedical Engineering, West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu 610041, China.
Hu
Li-juan
LJ
Lu
De-song
DS
Wang
Yu-hui
YH
Liu
Yan-you
YY
Gan
Lu
L
Xue
Jian-xin
JX
Wan
Chao-min
CM
Wang
Zheng-rong
ZR
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015723:Gene Library; D006801:Humans; D047468:Immunoprecipitation; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D056950:Period Circadian Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D000075370:Receptors for Activated C Kinase; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013493:Suprachiasmatic Nucleus / Q000378:metabolism; D020798:Two-Hybrid System Techniques
D009363:Neoplasm Proteins; C539022:PER1 protein, human; D056950:Period Circadian Proteins; C419368:RACK1 protein, human; D000075370:Receptors for Activated C Kinase; D011956:Receptors, Cell Surface; D019204:GTP-Binding Proteins