Identification of protein domains required for makorin-2-mediated neurogenesis inhibition in Xenopus embryos

Makorin-2, consisting of four highly conserved C3H zinc fingers, a Cys-His motif and a C3HC4 RING zinc finger domain, is a putative ribonucleoprotein. We have previously reported that Xenopus makorin-2 (mkrn2) is a neurogenesis inhibitor acting upstream of glycogen synthase kinase-3 beta (GSK-3 beta) in the phosphatidylinositol 3-kinase/Akt pathway. In an effort to identify the functional domains required for its anti-neurogenic activity, we designed and constructed a series of N- and C-terminal truncation mutants of mkrn2. Concurred with the full-length mkrn2, we showed that overexpression of one of the truncation mutants mkrn2(s)-7, which consists of only the third C3H zinc finger, Cys-His motif and C3HC4 RING zinc finger, is essential and sufficient to produce the phenotypical dorso-posterior deficiencies and small-head/short-tail phenotype in tadpoles. In animal cap explant assay, we further demonstrated that mkrn2(s)-7 not only inhibits activin and retinoic acid-induced animal cap neuralization and the expression of a pan-neural marker neural cell adhesion molecule, but also induces GSK-3 beta expression. These results collectively suggest that the third C3H zinc finger, Cys-His motif and C3HC4 RING zinc finger are indispensable for the anti-neurogenic activity of mkrn2. (C) 2010 Elsevier Inc. All rights reserved.