Mitotic Inhibition of GRASP65 Organelle Tethering Involves Polo-like Kinase 1 (PLK1) Phosphorylation Proximate to an Internal PDZ Ligand

Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 10/2010; 285(51):39994-40003. DOI: 10.1074/jbc.M110.189449
Source: PubMed


GRASP65 links cis-Golgi cisternae via a homotypic, N-terminal PDZ interaction, and its mitotic phosphorylation disrupts this
activity. Neither the identity of the PDZ ligand involved in the GRASP65 self-interaction nor the mechanism by which phosphorylation
inhibits its interaction is known. Phospho-mimetic mutation of known cyclin-dependent kinase 1/cyclin B sites, all of which
are in the C-terminal “regulatory domain” of the molecule, failed to block organelle tethering. However, we identified a site
phosphorylated by Polo-like kinase 1 (PLK1) in the GRASP65 N-terminal domain for which mutation to aspartic acid blocked tethering
and alanine substitution prevented mitotic Golgi unlinking. Further, using interaction assays, we discovered an internal PDZ
ligand adjacent to the PLK phosphorylation site that was required for tethering. These results reveal the mechanism of phosphoinhibition
as direct inhibition by PLK1 of the PDZ ligand underlying the GRASP65 self-interaction.

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    • "Since mitotically phosphorylated GRASP65 binds PLK1 (Preisinger et al., 2005), PLK1 may be recruited to GRASP65 by CDK1/ERK phosphorylation so that PLK1 can phosphorylate Ser189 to unlink the Golgi. Consistent with this, HeLa cells expressing a version of GRASP65 that cannot be phosphorylated at Ser189 fail to unlink their Golgi ribbons at the onset of mitosis (Sengupta and Linstedt, 2010; Figure 2A). Accordingly, this prevents/delays cells to enter mitosis (Sütterlin et al., 2002;Duran et al., 2008). "
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    ABSTRACT: Originally identified as Golgi stacking factors in vitro, the Golgi reassembly stacking protein (GRASP) family has been shown to act as membrane tethers with multiple cellular roles. As an update to previous comprehensive reviews of the GRASP family (Vinke et al., 2011) (Giuliani et al., 2011;Jarvela and Linstedt, 2012), we outline here the latest findings concerning their diverse roles. New insights into the mechanics of GRASP-mediated tethering come from recent crystal structures. The models of how GRASP65 and GRASP55 tether membranes relate directly to their role in Golgi ribbon formation in mammalian cells and the unlinking of the ribbon at the onset of mitosis. However, it is also clear that GRASPs act outside the Golgi with roles at the ER and ER exit sites (ERES). Furthermore, the proteins of this family display other roles upon cellular stress, especially in mediating unconventional secretion of both transmembrane proteins (Golgi bypass) and cytoplasmic proteins (through secretory autophagosomes).
    Full-text · Article · Jan 2016 · Frontiers in Cell and Developmental Biology
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    • "The role of S376 phosphorylation is the least well understood to date. In addition to the three phosphorylation sites characterized in this study, GRASP65 is also modified by kinases on S189, S216, S217 and S367 at least in vitro (Preisinger et al., 2005; Sengupta and Linstedt, 2010). Characterization of these sites and their relationships may provide further insight in understanding the function of GRASP65 and Golgi biogenesis during cell division. "
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    ABSTRACT: GRASP65 phosphorylation during mitosis and dephosphorylation after mitosis are required for Golgi disassembly and reassembly during the cell cycle. At least eight phosphorylation sites on GRASP65 have been identified, but whether they are modified in a coordinated fashion during mitosis is so far unknown. In this study, we raised phospho-specific antibodies that recognize phosphorylated T220/T224, S277 and S376 residues of GRASP65, respectively. Biochemical analysis showed that cdc2 phosphorylates all three sites, while plk1 enhances the phosphorylation. Microscopic studies using these antibodies for double and triple labeling demonstrate sequential phosphorylation and dephosphorylation during the cell cycle. S277 and S376 are phosphorylated from late G2 phase through metaphase until telophase when the new Golgi is reassembled. T220/224 is not modified until prophase, but is highly modified from prometaphase to anaphase. In metaphase, phospho-T220/224 signal localizes on both Golgi haze and mitotic Golgi clusters that represent dispersed Golgi vesicles and Golgi remnants, respectively, while phospho-S277 and S376 labeling is more concentrated on mitotic Golgi clusters. Expression of a phosphorylation-resistant GRASP65 mutant T220A/T224A inhibited mitotic Golgi fragmentation to a much larger extent than the expression of the S277A and S376A mutants. In cytokinesis, T220/224 dephosphorylation occurs prior to that of S277, but after S376. This study provides evidence that GRASP65 is sequentially phosphorylated and dephosphorylated during mitosis at different sites to orchestrate Golgi disassembly and reassembly during cell division, with phosphorylation of the T220/224 site being most critical in the process.
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