Investigation Of The Interaction Between RCAN1-4 Exon7 And Calcineurin

Calcineurin(CN)is a Ca2+/calmodulin-dependent serine/threonine protein phosphatase.It has been shown that excessive inhibition of CN is a critical factor for Down syndrome and Alzheimer’s disease.Regulator of calcineurin(RCAN)is important endogenous regulating factors of CN,and has complicated relationship with CN.Previous studies have shown that RCAN 1-4 can inhibit the phosphatase activity of CN in vitro,and its C-terminal fragment encoded by RCAN1-4 exon7 has similar inhibitory ability as the full-length protein.In this study,a combination of Nuclear Magnetic Resonance(NMR)and enzyme kinetic analysis were used to study the inhibition of CN phosphatase activity by RCAN1-4 exon7.Firstly,we obtained high-purity of CN,hRCAN1-4 exon7 and mRcan1-4 exon7 by using GST/Ni2+ affinity column and size-exclusion column.NMR titration experiments showed that:1)the catalytic domain(CNA)of CN is suficient for interaction with RCAN1-4 exon7,whereas CNB does not interact with RCAN1-4 exon7;2)there are two separate regions in mRcanl-4 exon7 involving in interactions with mCNA(1-347),which are located at residue 143-166 including EV-motif,and residue 183-192 including TxxP motif,and the binding sites were identified;3)the same results were obtained by truncated mRcan1-4 exon7.Finally,competitive NMR titration experiments show that there may exist an overlapped binding region in CN for mRcan1-4 exon7 and mCN-AID(CN Autoinhibitiory domain),and mRcan1-4 exon7 may bind near to the catalytic center.Through enzyme kinetic analysis,it was found that only full-length exon7 could inhibit the phosphatase activity of mCNA(1-347),and both truncated exon7 could not inhibit the phosphatase activity of mCNA(1-347).