Nuclear Import Of Myopodin Mediated By Importin 13

Nucleocytoplasmic transport of macromolecules is mediated by the soluble transportreceptors collectively named importinβsuper family proteins.To investigate molecularmechanisms of lung organogenesis,we found a novel importinβhomolog,namedimportin13.Importin13 is a novel mediator of nuclear import and export,the onlybi-directional transportor in mammalian importins.Recent research reports discribed thedemonstrated developmental and glucocorticoid regulation of importin 13nucleocytoplasmic shuttling in fetal Lung,this dual regulative mechanisms was not foundin other importinβmembranes by now.Importin13 was enriched in lung,brain,heart of fetal rat and human in Northernanalysis.To investigate the function of importin13 in heart,we used importin13 proteinas bait to screen a human normal heart cDNA library using the yeast two-hybrid systemand found a cDNA containing the C-terminal 359-698 amno acids of human Myopodin.Recently some paper describe the characteristics of myopodin protein including:(1)structural protein (actin-bundling protein);(2) participating in a signaling pathway(nucleocytoplasmic shuttling between Z-disc and nucleus);(3) suppression of prostateand bladder cancer growth and metastasis.Most importantly,nuclear myopodinexpression was a tumor suppressor.An article explained the importinαbinding and the subsequent nuclear import ofmyopodin are regulated by phosphorylation-dependent binding of myopodin to 14-3-3.However importinαproteins can not import into nucleus by themselves,they aretransported by importinβas adaptor.Therefore the real nuclear import machinery ofmyopodin remains to be established.Because of the direct relationship betweennucleocytoplasmic shuttle of myopodin and its tumor suppression activity,our researchwill focus the interaction of myopodin and importin13 and the nucleocytoplasmictransport mechanism of myopodin.The results of these studies include:1.We found myopodin was a novel imp13-binding protein by using the yeasttwo-hybrid system.To research the biological function of myopodin protein,wecloned the full length of myopodin open reading form gene from mRNA of Chinese skeleton muscle by RT-PCR。2.Interaction of full length myopodin and importin 13 was demonstrated in vitro byGST pul ldown and in vivo by coimmunoprecipitation.3.We observed the subcellular localization of endogenous myopodin protein,recombinant protein containing different region of myopodin and YFP and myopodinNLS mutations.It was possible that myopodin had other NLSs besides NLS1 andNLS2,and the nuclear import activity ofNLS1 was much weaker than NLS2.4.Myopodin was proved to be a shuttling protein by heterokaryon nucleocytoplasmicshuttling assay.5.Importin13 is the nuclear import receptor of myopodin:(1) N-myopodin can notinteract with importin13,which indirectly approved that importin13 binded toC-myopodin;(2) The mutation of two potential NLSs did not affect the interaction ofmyopodin mutation and importin13,it was possible there were other no-classicalNLSs in myopodin protein;(3) Results of Ran binding assay showed that importin13was nuclear transport receptor of myopodin;(4) Nuclear entry of endogenousmyopodin is blocked by the C-terminal IPO13;(5) There were no or weak interactionbetween myopodin and importin7,importin8,importinα2,importinβ2 respectively,which confirmed that importin13 wasthe especial nuclear transportor of myopodin.