Recombinant C-terminal fragment of SM22 induces cytoskeleton reorganization.
- Author:
Jian-Hong SHI
1
;
Bin ZHENG
;
Fang MENG
;
Jin-Kun WEN
;
Mei HAN
Author Information
- Publication Type:Journal Article
- MeSH: Actins; metabolism; Amino Acid Sequence; Animals; Cell Line; Cytoskeleton; metabolism; physiology; Microfilament Proteins; genetics; Molecular Sequence Data; Muscle Proteins; genetics; Muscle, Smooth, Vascular; cytology; metabolism; Myocytes, Smooth Muscle; metabolism; Protein Binding; Rats; Rats, Sprague-Dawley; Recombinant Proteins; genetics
- From: Chinese Journal of Applied Physiology 2007;23(3):370-374
- CountryChina
- Language:Chinese
-
Abstract:
AIMTo investigate the interaction between C-terminal domains of SM22alpha and cytoskeleton F-actin.
METHODSProkaryotic expression vector containing SM22alpha cDNA and GST sequence was constructed. The induction conditions were optimized to increase the product of soluble GST-SM22alpha fusion protein in E coli. Expression products were purified and rabbit anti-GST-SM22alpha polyclonal antibody was produced by the purified fusion protein. In order to explore the effect of SM22alpha on cytoskeleton reorganization, VSMCs were treated with serum withdrawal and then serum stimulation to induce contractile/synthetic phenotypic modulation. SM22alpha protein distribution in F-actin/G-actin fractions was detected by Western blotting. The interaction between SM22alpha and actin was examined by GST pull down assay and coimmunoprecipitation. Colocalization of endogenous SM22alpha with F-actin was observed by immunofluorescence.
RESULTSThe results showed that the expression of soluble GST-SM22alpha protein was the highest under condition induced by 30 degrees C, 0.5 mmol/L IPTG for 6 h. Immunofluorescence and Western blotting of protein extracts from F-actin/G-actin fractions revealed that SM22alpha colocalized with F-actin during VSMC redifferentiation. GST pull down assay and coimmunoprecipitation showed that SM22alpha interacted with F-actin by C-terminal domains to participate in cytoskeleton reorganization.
CONCLUSIONThe recombinant SM22alpha C-terminal domains have the ability to bind F-actin, by which SM22alpha interacts with actin and participates in cytoskeleton reorganization.
