Plk1 docking to GRASP65 phosphorylated by Cdk1 suggests a mechanism for Golgi checkpoint signalling

Abstract

GRASP65, a structural protein of the Golgi apparatus, has been linked to the sensing of Golgi structure and the integration of this information with the control of mitotic entry in the form of a Golgi checkpoint. We show that Cdk1-cyclin B is the major kinase phosphorylating GRASP65 in mitosis, and that phosphorylated GRASP65 interacts with the polo box domain of the polo-like kinase Plk1. GRASP65 is phosphorylated in its C-terminal domain at four consensus sites by Cdk1-cyclin B, and mutation of these residues to alanine essentially abolishes both mitotic phosphorylation and Plk1 binding. Expression of the wildtype GRASP65 C-terminus but not the phosphorylation defective mutant in normal rat kidney cells causes a delay but not the block in mitotic entry expected if this were a true cell cycle checkpoint. These findings identify a Plk1-dependent signalling mechanism potentially linking Golgi structure and cell cycle control, but suggest that this may not be a cell cycle checkpoint in the classical sense.

Mitogen-activated Protein Kinase Kinase 1-dependent Golgi Unlinking Occurs in G2Phase and Promotes the G2/M Cell Cycle Transition

“…That is, MEK inhibition might block a rearrangement in the Golgi apparatus that somehow controls G 2 /M kinetics. Indeed, a remarkable finding by Malhotra and colleagues (Sutterlin et al, 2002), now supported by others (Hidalgo Carcedo et al, 2004;Preisinger et al, 2005;Yoshimura et al, 2005), indicates that Golgi disassembly is required for G 2 /M progression. Antibody or dominant-negative interference of GRASP65 blocks mitotic Golgi disassembly in permeabilized cells and when these agents are introduced into intact cells, the cells fail to exit G 2 (Sutterlin et al, 2002).…”

“…In addition to lateral cross-bridging of Golgi ministacks, GM130/GRASP65 complexes, and possibly golgin45/GRASP55 complexes, also associate with numerous factors on the cytoplasmic face of the Golgi apparatus, including the protein kinases Ysk1, Mst4, and PLK1 (Preisinger et al, 2004;Preisinger et al, 2005). Regulation of these associated factors, rather than Golgi unlinking per se, may be the mechanism facilitating G 2 /M.…”

GRASP55 Regulates Golgi Ribbon Formation

“…There may also be a sequential relationship in the regulation of the two proteins at the onset of mitosis. Evidence suggests that GRASP55 is phosphorylated in late G2 before CDK1 activation, and, therefore, that its regulation occurs upstream of GRASP65 phosphorylation by CDK1 and CDK1-activated PLK1 (Elia et al, 2003a,b;Wang et al, 2003;Preisinger et al, 2005;Feinstein and Linstedt, 2007). If so, GRASP65 phosphorylation might be considered redundant with respect to unlinking the Golgi.…”

Golgi Ribbon Unlinking: An Organelle-Based G2/M Checkpoint

“…45 However, depletion of Plk1 by RNAi was later shown not to affect the first step of mitotic Golgi disassembly, but to be required for full Golgi stack vesiculation. 46 However, as Plk1 depletion causes profound mitotic spindle defects leading to a cell arrest in prometaphase 46 (For a review see Ref. 47), the persistence of Golgi fragments could be an indirect effect of the mitotic block, rather than a direct role of Plk1 on the mitotic Golgi disassembly.…”

“…The only strong link between this kinase and the Golgi architecture is the discovery of the Golgi structural protein GRASP65 as Plk1 target [41][42]45 (see below), although the exact phosphorylation site has yet to be identified. 46,48 …”