Peripheral Golgi protein GRASP65 is a target of mitotic polo-like kinase (Plk) and Cdc2

Abstract

Cell division is characterized by orchestrated events of chromosome segregation, distribution of cellular organelles, and the eventual partitioning and separation of the two daughter cells. Mitotic kinases, including polo-like kinases (Plk), influence multiple events in mitosis. In yeast two-hybrid screens using mammalian Plk C-terminal domain baits, we have identified Golgi peripheral protein GRASP65 (Golgi reassembly stacking protein of 65 kDa) as a Plk-binding protein. GRASP65 appears to function in the postmitotic reassembly of Golgi stacks. In this report we demonstrate binding between Plk and GRASP65 and provide in vitro and in vivo evidence that Plk is a GRASP65 kinase. Moreover, we show that Cdc2 can also phosphorylate GRASP65. In addition, we present data which support the observation that the conserved C terminus of Plk is important for its function. Deletion or frameshift mutations in the conserved C-terminal domain of Plk greatly diminish its ability to phosphorylate GRASP65. These and previous findings suggest that phosphorylation of Golgi components by mitotic kinases may regulate mechanisms of Golgi inheritance during cell division.

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

“…However, despite the fact that ERK phosphorylates GRASP65 in interphase in response to EGF stimulation, GRASP65 does not seem to be mitotically phosphorylated by MEK1/ERK. 46,48 As mentioned above, GRASP65 is also a Plk1 substrate, [41][42]45 although the site has not been identified. 46,48 Nevertheless, Plk1 was shown to dock on CDK1-phosphorylated GRASP65, suggesting it could be recruited on Golgi membrane and drive the stack vesiculation or control the progression of mitosis 46 (see below).…”

“…22,25 This effect is likely due to the cessation of ER-Golgi and intra-Golgi transport that is observed in metaphase leading partly to an accumulation of COPI vesicles. [35][36][37] Several CDK1-phosphorylated substrates functioning in the early secretory pathway, such as Rab1, 38 GM130, [39][40] GRASP65 [41][42][43] and p47, 44 could contribute to this.…”

“…Moreover, Plk1 does not seem to localize on the Golgi membrane at the onset of mitosis. 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 …”

“…52 GRASP65 is a major mitotically-phosphorylated Golgi protein. [41][42]49 Detailed mapping by mass spectrometry has revealed 4 CDK1-cyclinB phosphorylation sites and two other potential mitotically-regulated phosphorylation sites by nonidentified kinases. 46 All these sites are located at the serine/proline-rich C-terminal portion.…”

Getting in and out of mitosis with Polo-like kinase-1

“…Both Plk1 and Plk3 were suggested to regulate partitioning of Golgi-stacks during cell division (Sutterlin et al, 2001;Ruan et al, 2004). In addition, Plk1 was shown to interact and phosphorylate the Golgi-associated proteins Grasp65 and Nir2 (Lin et al, 2000;Litvak et al, 2004) and Plk3 colocalizes to Golgimarkers Giantin. Finally, overexpression of Plk3 results in premature Golgi-partitioning (Ruan et al, 2004).…”