Article

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

ABSTRACT

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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    • "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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