Phosphorylation of Ran-binding Protein-1 by Polo-like Kinase-1 Is Required for Interaction with Ran and Early Mitotic Progression

Abstract

Polo-like kinase-1 (Plk1) is essential for progression of mitosis and localizes to centrosomes, central spindles, midbody, and kinetochore. Ran, a small GTPase of the Ras superfamily, plays a role in microtubule dynamics and chromosome segregation during mitosis. Although Ran-binding protein-1 (RanBP1) has been reported as a regulator of RanGTPase for its mitotic functions, the action mechanism between Ran and RanBP1 during mitosis is still unknown. Here, we demonstrated in vitro and in vivo phosphorylation of RanBP1 by Plk1 as well as the importance of phosphorylation of RanBP1 in the interaction between Plk1 and Ran during early mitosis. Both phosphorylation-defective and N-terminal deletion mutant constructs of RanBP1 disrupted the interaction with Ran, and depletion of Plk1 also disrupted the formation of a complex between Ran and RanBP1. In addition, the results from both ectopic expression of phosphorylation-defective mutant construct and a functional complementation on RanBP1 deficiency with this mutant indicated that phosphorylation of RanBP1 by Plk1 might be crucial to microtubule nucleation and spindle assembly during mitosis.

RanBP1 Governs Spindle Assembly by Defining Mitotic Ran-GTP Production

“…RanBP1 was immunopredipiated from anaphase cycling XEE and analyzed by mass spectrometry (Figure S3A). The major phosphorylated residue in this sample was Serine 60 (Ser 60) of RanBP1, which had previously been implicated as a potential site of mitotic phosphorylation in HeLa cells (Hwang et al, 2011). To further validate the Ser 60 phosphorylation on xRanBP1, a phospho-specific antibody was raised against xRanBP1 pSer60.…”

“…Particularly, RCC1 accumulates on chromatin at anaphase onset in cycling XEE (Arnaoutov and Dasso, 2003), and we wondered whether RRR complex-dependent partitioning of RCC1 between the chromatin-bound and -unbound pools might play a role in this phenomenon. Earlier studies have demonstrated mitotic regulation of mammalian RanBP1 through degradation and phosphorylation (Ciciarello et al, 2010; Hwang et al, 2011). We did not find any evidence that RanBP1 was subject to degradation during multiple cell cycles within cycling XEE (Figure 4A, upper panel).…”

The functional landscape of the human phosphoproteome

“…Independently, we sought to validate as a proof-of-principle a candidate regulatory phosphosite in a protein interface. The PLK1-regulated S60 is the best scoring phosphosite (0.65, Figure 3a) of the RAN Binding Protein 1 (RANBP1) 31 . This site is upregulated under okadaic acid and it is near the Ran interaction interface and the transmembrane nuclear transporter Ran GTPase Activating Protein 1 (RanGAP) ( Figure 3b, PDB:1k5g).…”

The functional landscape of the human phosphoproteome

“…Independently, we sought to validate as a proof-of-principle a candidate regulatory phosphosite in a protein-protein interface with no previous experimental evidence. The PLK1-regulated S60 is the best scoring phosphosite (0.65, Figure 3a) of the RAN Binding Protein 1 (RANBP1) (Hwang, Ji, and Jang 2011) . Some of the most relevant features of pS60 are its strong upregulation (logFC 4.44) under okadaic acid treatment and its proximity to the Ran interaction interface and the transmembrane nuclear transporter Ran GTPase Activating Protein 1 (RanGAP) ( Figure 3b , PDB:1k5g).…”