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

Abstract

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.

VRK1 interacts with p53 forming a basal complex that is activated by UV‐induced DNA damage

“…This C-terminal region has been described to interact with several proteins and to play a regulatory role [28,29,31]. For these reasons, next we analysed if this VRK1 region was binding to p53 by performing pull-down assays and with two different GST-VRK1 constructs [44]; full-length VRK1 (residues 1-396) and VRK1DN comprising the C-terminus (residues 267-396) (Fig. 1B).…”

“…Endogenous VRK1 protein was immunoprecipitated with monoclonal antibody 1F6 as previously reported [44,45] and the immunoprecipitate was used for an in vitro kinase assay [46] showing either endogenous VRK1 autophosphorylation or phosphorylation of GST-p53 (1-85) used as substrate [7,[46][47][48]. The kinase assay was performed at 30°C for 30 min as previously described [47,48].…”

“…Cloning of human full-length VRK1 (1-396) and VRK1DN (267-396) into mammalian expression vector pCEFL-GST have been described previously [44]. VRK1 mutants were generated using the Quick-Mutagenesis system (Stratagene, San Diego, CA) and specific primers (indicated in Table S1) following the manufacturer's instructions and using plasmid pCEFL-HA-VRK1 as template.…”

Vaccinia-related Kinase 1 (VRK1) Is an Upstream Nucleosomal Kinase Required for the Assembly of 53BP1 Foci in Response to Ionizing Radiation-induced DNA Damage

“…F, mapping the interaction region of 53BP1 with VRK1. Six GST-53BP1 fragments spanning the full-length 53BP1 protein as indicated were incubated with purified VRK1-His and subjected to GST pulldown (25,74). Proteins were separated by SDS-PAGE followed by immunoblotting with anti-VRK1 antibody (1F6 mAb).…”

Photothermal treatment of liver cancer with albumin-conjugated gold nanoparticles initiates Golgi Apparatus–ER dysfunction and caspase-3 apoptotic pathway activation by selective targeting of Gp60 receptor

“…30,31 Research data have shown that when apoptosis occurs in malignant cells, GA is being disassembled in a manner similar to that which occurs in mitosis, a process that is mediated by Polo-like kinase-3 (Plk3) that also plays a crucial role in the DNA damage checkpoints activation. 32 On the other hand, a pool of death receptors that populate the GA in eukaryotic cells may also be responsible for the initiation of cellular apoptosis. 33 Because the Golgi system plays an important role in the induction of apoptosis, triggering it holds great hopes in the development of novel anticancer therapies.…”