Phosphorylation of nucleoporin Tpr governs its differential localization and is required for its mitotic function

Abstract

A major constituent of the nuclear basket region of the NPC, nucleoporin Tpr plays a multi-dimensional role in regulating important processes. We have previously established that Tpr is phosphorylated in both, MAP kinase dependent and independent manner, and found that Tpr acts as both a substrate and as a scaffold for ERK2. Here, we report the identification of S2059 and S2094 as the major novel ERK-independent phosphorylation sites and T1677, S2020, S2023 and S2034 as the minor ERK independent phosphorylation sites found in the Tpr protein in vivo. Our results suggest that Protein Kinase A phosphorylates the S2094 residue, and the site is hyperphosphorylated during mitosis. Further, we find that Tpr is phosphorylated at the S2059 residue by CDK1 and the phosphorylated form distinctly localizes with chromatin during the telophase. Abrogation of S2059 phosphorylation abolishes its interaction with Mad1, thus compromising the localization of both Mad1 and Mad2 proteins, and results in cell cycle defects. The identification of novel phosphorylation sites on Tpr and the observations presented in this study open fresh avenues for the better understanding of Tpr functions.