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: 4.6). 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.

  • [Show abstract] [Hide abstract]
    ABSTRACT: The mammalian GRASP proteins are Golgi-localized homotypic membrane tethers that organize Golgi stacks into a long, contiguous ribbon-like structure. It is unknown how GRASPs undergo trans pairing given that cis interactions between the proteins in the plane of the membrane are intrinsically favored. To test the hypothesis that myristoylation of the self-interacting GRASP domain restricts its orientation on the membrane to favor trans pairing we established an in vitro assay that recapitulates GRASP-dependent membrane tethering and used neutron reflection under similar conditions to determine the orientation of the GRASP domain. In vivo, the membrane association of GRASP proteins is conferred by the simultaneous insertion of an N-terminal myristic acid and binding to a Golgi-associated binding partner. In our assay, the latter contact was replaced using a C-terminal hexa-His moiety, which bound to Ni2+-conjugated lipids incorporated into a substrate-supported bilayer lipid membrane. Non-myristoylated protein lacked a fixed orientation on the membrane and inefficiently tethered liposomes. In contrast, myristoylated GRASP promoted tethering and exhibited a unique membrane complex compatible with trans but not cis interactions. Thus, myristoylation restricts the membrane orientation of the GRASP domain favoring interactions in trans for membrane tethering.
    Journal of Biological Chemistry 02/2014; 289(14). DOI:10.1074/jbc.M113.543561 · 4.60 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Protein-protein interactions (PPIs) are essential events to play important roles in a series of biological processes. There are probably more ways of PPIs than we currently realized. Structural and functional investigations of weak PPIs have lagged behind those of strong PPIs due to technical difficulties. Weak PPIs are often short-lived, which may result in more dynamic signals with important biological roles within and/or between cells. For example, the characteristics of PSD-95/Dlg/ZO-1 (PDZ) domain binding to internal sequences, which are primarily weak interactions, have not yet been systematically explored. In the present study, we constructed a nearly random octapeptide yeast two-hybrid library. A total of 24 PDZ domains were used as baits for screening the library. Fourteen of these domains were able to bind internal PDZ-domain binding motifs (PBMs), and PBMs screened for nine PDZ domains exhibited strong preferences. Among 11 PDZ domains that have not been reported their internal PBM binding ability, six were confirmed to bind internal PBMs. The first PDZ domain of LNX2, which has not been reported to bind C-terminal PBMs, was found to bind internal PBMs. These results suggest that the internal PBMs binding ability of PDZ domains may have been underestimated. The data provided diverse internal binding properties for several PDZ domains that may help identify their novel binding partners.
    PLoS ONE 02/2014; 9(2):e88286. DOI:10.1371/journal.pone.0088286 · 3.53 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The Golgi complex of mammalian cells is composed of interconnected stacks of flattened cisternae that form a continuous membrane system in the pericentriolar region of the cell. At the onset of mitosis, this so-called Golgi ribbon is converted into small tubular-vesicular clusters in a tightly regulated fragmentation process, which leads to a temporary loss of the physical Golgi-centrosome proximity. Mitotic Golgi breakdown is required for Golgi partitioning into the two daughter cells, cell cycle progression and may contribute to the dispersal of Golgi-associated signaling molecules. Here, we review our current understanding of the mechanisms that control mitotic Golgi reorganization, its biological significance, and assays that are used to study this process.
    Methods in cell biology 01/2013; 118:383-400. DOI:10.1016/B978-0-12-417164-0.00023-9 · 1.44 Impact Factor