Requirements for Protein Phosphorylation and the Kinase Activity of Polo-like Kinase 1 (Plk1) for the Kinetochore Function of Mitotic Arrest Deficiency Protein 1 (Mad1)

Ya-Hui Chi,Kerstin Haller,Michael D Ward,O John Semmes,Yan Li,Kuan-Teh Jeang

doi:10.1074/jbc.m804967200

Abstract

Mitotic arrest deficiency protein 1 (Mad1) is associated with microtubule-unattached kinetochores in mitotic cells and is a component of the spindle assembly checkpoint (SAC). Here, we have studied the phosphorylation of Mad1 and mapped using liquid chromatography-tandem mass spectrometry several phosphorylated amino acids in this protein. One phosphorylated residue, Thr680, was characterized to be important for the kinetochore localization of Mad1 and its SAC function. We also found that in mitotic cells Mad1 co-immunoprecipitated with Plk1. Depletion of cellular Plk1 using small interfering RNAs and inhibition of the kinase activity of Plk1 using a kinase-dead mutant or a small molecule inhibitor attenuated Mad1 phosphorylation and its association with kinetochores. Collectively, these findings indicate mechanistic roles contributed by protein phosphorylation and Plk1 to the SAC activity of Mad1.

Highlights

Faithful chromosome partitioning from a mother cell to her daughter cells is essential for maintaining euploidy
We demonstrate that Mitotic arrest deficiency protein 1 (Mad1) is hyperphosphorylated when Plk1 is overexpressed and that the kinase activity of Plk1 is required for Mad1 to locate to microtubule-unattached kinetochores
Cell Cycle-dependent Phosphorylation of Mad1—Mad1 was first reported to be phosphorylated upon spindle assembly checkpoint (SAC) activation in Saccharomyces cerevisiae [6]

Summary

Introduction

Faithful chromosome partitioning from a mother cell to her daughter cells is essential for maintaining euploidy. Thr680, was characterized to be important for the kinetochore localization of Mad1 and its SAC function. An association between cell endogenous Mad1 and Plk1 in M phase was demonstrated by immunoprecipitating the former using a mixture of ␣Mad1-N and ␣Mad1-C antibodies (Fig. 2B).

Results

Conclusion