Mdm2-Mediated Downmodulation of GRK2 Restricts Centrosome Separation for Proper Chromosome Congression.

Clara Reglero,Federico Mayor,Verónica Rivas,Petronila Penela,Belén Ortiz Del Castillo

doi:10.3390/cells10040729

Abstract

The timing of centrosome separation and the distance moved apart influence the formation of the bipolar spindle, affecting chromosome stability. Epidermal growth factor receptor (EGFR) signaling induces early centrosome separation through downstream G protein-coupled receptor kinase GRK2, which phosphorylates the Hippo pathway component MST2 (Mammalian STE20-like protein kinase 2), in turn allowing NIMA kinase Nek2A activation for centrosomal linker disassembly. However, the mechanisms that counterbalance centrosome disjunction and separation remain poorly understood. We unveil that timely degradation of GRK2 by the E3 ligase Mdm2 limits centrosome separation in the G2. Both knockout expression and catalytic inhibition of Mdm2 result in GRK2 accumulation and enhanced centrosome separation before mitosis onset. Phosphorylation of GRK2 on residue S670 enables a complex pattern of non-K48-linked polyubiquitin chains assembled by Mdm2, which correlate with kinase protein degradation. Remarkably, GRK2-S670A protein fails to phosphorylate MST2 despite overcoming Mdm2-dependent degradation, which results in defective centrosome separation, shorter spindles, and abnormal chromosome congression. Conversely, extra levels of wild-type kinase in the G2 cause increased inter-centrosome distances with longer spindles, also converging in congression issues. Our findings show that the signals enabling activity of the GRK2/MST2/Nek2A axis for separation also switches on Mdm2 degradation of GRK2 to ensure accurate centrosome dynamics and proper mitotic spindle functionality.

Highlights

Abnormal numbers, attachment or movement of chromosomes are all causes of defective chromosome distribution, eventually leading to aneuploidy and cancer [1,2]
During G2, phosphorylation of GRK2 at Ser670 by the CDK2–cyclin A kinase promotes its interaction with the prolyl-isomerase Pin1 and proteasomal degradation of GRK2
We have described that the E3 ligase Mdm2 is involved in the ubiquitination and degradation of GRK2 upon GPCR stimulation [33], and, more interestingly, expression of a mutant unable to be phosphorylated at S670 (GRK2-S670A) completely prevents GRK2 down-modulation by Mdm2 in such context [34]

Summary

Introduction

Attachment or movement of chromosomes are all causes of defective chromosome distribution, eventually leading to aneuploidy and cancer [1,2]. Two different types of connections between centrioles regulate centrosome duplication and separation, which are events coordinated with the cell cycle that occurs once in every cell cycle as part of the centrosome cycle [6]. The S-M linker prevents centriole reduplication, and its removal at mitosis/G1 transition (“centrosome disengagement”) licenses the centrosome for duplication in the S phase of the cell cycle [7,8]. During G2, this linker disassembles (“centrosome disjunction”) in a process governed by cell cycle kinases CDK1, aurora A, PLK1 and NIMArelated kinase Nek, being this latter responsible for phosphorylation of C-Nap and rootletin to allow centrosomes separation and subsequent movement in a kinesin (Eg5)mediated manner, allowing the formation of the two spindle poles [9,11]

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Conclusion