The effect of PKA-phosphorylation on the structure of inhibitor-1 studied by NMR spectroscopy.

Inhibitor-1 is an acid- and heat-stable protein. It can be turned into a potent inhibitor of protein phosphatase-1 (PP1) after phosphorylation at Thr35 by c-AMP-dependent protein kinase (PKA). Although it has been known that pre-phosphorylation is essential for inhibition of PP1, the structure-function relationship of Thr(35)-phosphorylated inhibitor-1, such as whether or not PKA-phosphorylation pre-triggers conformational changes in inhibitor-1, remains unclear. In this study, we performed structural characterization of Thr(35)-phosphoroylated inhibitor-1 by using multi-dimensional heternuclear NMR spectroscopy. The result of structural comparison between Thr(35)-phosphoroylated and non-phosphorylated inhibitor-1 indicated that PKA-phosphorylation has no significant effect on the global conformation of free-state inhibitor-1. This finding may support the inference that regulation of the interactions between inhibitor-1 and PP1 through PKA-phosphorylation mainly depends on the phosphate group instead of phosphorylation-induced conformational change.