Plk3 Interacts with and Specifically Phosphorylates VRK1 in Ser342, a Downstream Target in a Pathway That Induces Golgi Fragmentation

Golgi fragmentation is a process that is necessary to allow its redistribution into daughter cells during mitosis, a process controlled by serine-threonine kinases. This Golgi fragmentation is activated by MEK1 and Plk3. Plk3 is a kinase that is a downstream target in the Golgi fragmentation pathway induced by MEK1 or by nocodazole. In this work, we have identified that Plk3 and VRK1 are two consecutive steps in this signaling pathway. Plk3 interacts with VRK1, forming a stable complex detected by reciprocal immunoprecipitations and pull-down assays; VRK1 colocalizes with giantin in the Golgi apparatus, as Plk3 also does, forming clearly detectable granules. VRK1 does not phosphorylate Plk3, but Plk3 phosphorylates the C-terminal region of VRK1 in Ser342. VRK1 with substitutions in S342 is catalytically active but blocks Golgi fragmentation, indicating that its specific phosphorylation is necessary for this process. The induction of Golgi fragmentation by MEK1 and Plk3 can be inhibited by kinase-dead VRK1, the knockdown of VRK1 by siVRK1, kinase-dead Plk3, or PD98059, a MEK1 inhibitor. The Plk3-VRK1 kinase module might represent two consecutive steps of a signaling cascade that participates in the regulation of Golgi fragmentation.The Golgi apparatus in mammalian cells is formed by cistern stacks, tubules, and small vesicles, which undergo extensive and sequential fragmentation in mitosis (33). The reorganization of the Golgi apparatus, involving fragmentation, dispersal, and reassembly, is tightly regulated during mitosis (1, 27, 30), and reversible phosphorylation plays a critical role (1, 21), although the components and their sequential organization in the context of the initiation or execution of the signal required for Golgi fragmentation are only partially known.Many signaling pathways are composed of consecutive kinases. Characterization of new signaling pathways requires the identification of their components, the connections between them, and the order in which they are organized. Human VRK1 is a novel serine-threonine kinase that phosphorylates several proteins implicated in cellular responses to stress and DNA damage, such as p53 (5, 20, 40), c-Jun (31), and ATF2 (32), as well as proteins needed for nuclear envelope assembly required at the end of mitosis, such as Baf (25). In addition, VRK1 kinase activity is inhibited by interaction with RanGDP, and this inhibition is relieved by RanGTP, suggesting an asymmetric distribution of its activity within the nucleus and in mitosis (29). These properties suggest that the VRK1 gene plays a role in the regulation of cell cycle initiation and/or progression, consistent with its requirement for entry into the cell cycle, where it behaves as an immediate-early response gene like c-MYC and FOS (36). The loss of VRK1 by use of small interfering RNA (siRNA) induces an early G₁ block, before cyclin D1 expression (36), which is accompanied by a reduction in the phospho-retinoblastoma level and an accumulation of cycle inhibitors, such as p27 (36), resulting in a stop in cell cycle progression (36, 40).Several kinases are implicated in the control of cell proliferation and in different mitotic checkpoints; among them are the polo-like kinase (Plk) family, which is a group composed of four proteins (14, 39, 46). One of them, Plk3, contributes as a mediator of DNA damage checkpoint responses, since its kinase activity increases after oxidative stress (43) and induction of DNA damage by ionizing radiomimetic drugs (45). Plk3 physically interacts with and phosphorylates p53 in Ser20, and this interaction increases in response to DNA damage and induces either cell cycle arrest or apoptosis (44) so that genetic stability can be maintained by the prevention of the accumulation of genetic damage. Furthermore, Plk3 interacts with Chk2 (2, 45), an important mediator of DNA damage responses (6, 16), and there is a functional connection between them since Plk3 phosphorylates Chk2 in Ser62 and Ser73, which are necessary for full Chk2 activation by ATM (4). In mitotic cells, Plk3 is localized associated with the spindle poles and mitotic spindles, and deregulated expression of Plk3 induces cell cycle arrest and apoptosis by the perturbation of microtubule integrity (41). In addition, Plk3 expression is induced after mitogenic stimulation, and it is required for mitotic (28) and S-phase (48) entry. Plk3 also regulates Cdc25C (3, 23, 26) and the NF-κB signaling pathway (19). VRK1 phosphorylates p53 in Thr18 (20, 40), a residue phosphorylated in response to taxol, an inhibitor of microtubule polymerization (34).There is a possibility that VRK1 and Plk3 might be connected in some way, since subpopulations of both VRK1 (37) and Plk3 (28) have been detected in the Golgi apparatus near the centrosome, where they colocalize with Golgi markers such as giantin or GM130 (33). Golgi fragmentation can be induced by MEK1 (1, 15), and this signal is partly mediated by Plk3 (28, 42). Moreover, Golgi fragmentation is a required step during mitosis, occurring late in the G₂/M phase of the cell cycle (11), and MEK1 is implicated in the activation of this process (1, 15).The common biological aspects of VRK1 and Plk3 proteins and the association of VRK1 and Plk3 subpopulations in the Golgi apparatus led us to think that there might be a functional connection between these two kinases and thus that they might be components in a common signaling pathway. In this work, we explored the possible connection between VRK1 and Plk3 and determined if they were functionally related in a biological process, Golgi fragmentation, in which one of them, Plk3, is already known to participate. This work demonstrates that Plk3 and VRK1 are consecutive components in the signaling pathway that induces Golgi fragmentation in mitosis.