Plk1 Phosphorylates CLIP-170 and Regulates Its Binding to Microtubules for Chromosome Alignment

Abstract

ABSTRACT. The microtubule (MT) cytoskeleton is essential for cellular morphogenesis, cell migration, and cell division. MT organization is primarily mediated by a variety of MT-associated proteins. Among these proteins, plus-end-tracking proteins (+TIPs) are evolutionarily conserved factors that selectively accumulate at growing MT plus ends. Cytoplasmic linker protein (CLIP)-170 is a +TIP that associates with diverse proteins to determine the behavior of MT ends and their linkage to intracellular structures, including mitotic chromosomes. However, how CLIP-170 activity is spatially and temporally controlled is largely unknown. Here, we show that phosphorylation at Ser312 in the third serine-rich region of CLIP-170 is increased during mitosis. Polo-like kinase 1 (Plk1) is responsible for this phosphorylation during the mitotic phase of dividing cells. In vitro analysis using a purified CLIP-170 N-terminal fragment showed that phosphorylation by Plk1 diminishes CLIP-170 binding to the MT ends and lattice without affecting binding to EB3. Furthermore, we demonstrate that during mitosis, stable kinetochore/MT attachment and subsequent chromosome alignment require CLIP-170 and a proper phosphorylation/dephosphorylation cycle at Ser312. We propose that CLIP-170 phosphorylation by Plk1 regulates proper chromosome alignment by modulating the interaction between CLIP-170 and MTs in mitotic cells and that CLIP-170 activity is stringently controlled by its phosphorylation state, which depends on the cellular context.

TTBK2 with EB1/3 regulates microtubule dynamics in migrating cells through KIF2A phosphorylation

“…Recombinant GST-fused proteins were produced in Escherichia coli (XL-1 blue, BL21DE3, or RosettaDE3) using isopropyl-β- d -thiogalactopyranoside and were purified as described previously ( Wang et al, 2012 ; Kakeno et al, 2014 ). To produce GST, GST-EB1, and GST-EB3, we used pGEX (GE Healthcare).…”

“…The end-tracking assay was performed as previously described ( Kakeno et al, 2014 ). Flow chambers (22 mm wide × 1 mm high × 0.15 mm deep) were assembled between a coverslip and a precleaned glass microslide using double-sided tape.…”

Mechanisms of Chromosome Congression during Mitosis

“…However, CLIP-170 inhibition does not seem to affect kinetochore microtubule dynamics or stability, possibly because it is stripped from the kinetochore by Dynein upon the establishment of end-on kinetochore-microtubule attachments [103,104]. Moreover, phosphorylation of CLIP-170 at S312 by Plk1 regulates its binding to microtubules and is crucial for chromosome alignment [105]. CLIP-170 appears to promote kinetochore-microtubule attachments and chromosome congression by counteracting Dynein/Dynactin [106].…”

CLIP-170 is required to recruit PLK1 to kinetochores during early mitosis for chromosome alignment

“…CLIP-170 is thought to facilitate KT-MT attachment by binding to microtubules through its N-terminus, which contains cytoskeleton-associated protein-glycine-rich (CAP-Gly) domains (Tanenbaum et al, 2006). Recently, it was reported that PLK1 also phosphorylates CLIP-170 at S312, which regulates the binding of CLIP-170 to microtubules and is pivotal for chromosome alignment (Kakeno et al, 2014). Therefore, PLK1 recruitment by CLIP-170 is expected to promote KT-MT attachment directly through CLIP-170 by regulating the kinetochore localization and microtubule-binding activity of the latter.…”