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.
Highlights
Aspects of the mechanism underlying Golgi unlinking are beginning to emerge in part due to a better understanding of Golgi ribbon formation [8, 9, 13]
Oligomer formation depends on the conserved tandem PDZ-like domains at the N terminus referred to as the GRASP domain, and point mutations in the predicted binding groove of the first PDZ domain of GRASP65, PDZ1, block tethering activity and Golgi ribbon formation [17, 19]
The GRASP65 binding groove is known to be required for Golgi ribbon formation, but the corresponding ligand had not been identified [19]
Summary
Aspects of the mechanism underlying Golgi unlinking are beginning to emerge in part due to a better understanding of Golgi ribbon formation [8, 9, 13]. GRASP55 phosphorylation drives Golgi unlinking by blocking trans complexes involved in membrane tethering This model applies to GRASP65, which is directly phosphorylated at multiple sites by CDK1, ERK, and PLK1, and phosphorylation blocks its homo-oligomerization in vitro (24 –29). The known sites are outside the GRASP domain in a long nonconserved segment referred to as the serine-proline-rich regulatory domain [17, 21, 24, 27] It is unclear how phosphorylation of the C-terminal domain regulates tethering activity of the N-terminal self-interacting domain. Because CDK1 and ERK phosphorylate serine or threonine residues adjacent to proline it could be that GRASP phosphorylation by these kinases creates a binding site in the C-terminal domain for PLK1. The results support a model in which phosphorylation by PLK1 alters the activity of an adjacent internal PDZ ligand so that it can no longer bind the PDZ1 groove of a GRASP65 molecule on an apposing membrane
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