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Plk1 phosphorylates CLIP-170 and regulates its binding to microtubules for chromosome alignment

Mai Kakeno¹, Kenji Matsuzawa, Toshinori Matsui

¹Department of Cell Pharmacology, Nagoya University Graduate School of Medicine.

Cell Structure and Function

|January 24, 2014

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.

Insights

Phosphorylation of cytoplasmic linker protein (CLIP)-170 by Polo-like kinase 1 (Plk1) during mitosis regulates microtubule interactions. This phosphorylation is crucial for proper chromosome alignment in dividing cells.

Area of Science:

Cell Biology
Molecular and Cellular Biology
Cytoskeleton Dynamics

Background:

Microtubule (MT) organization is vital for cell functions, regulated by MT-associated proteins.
Plus-end-tracking proteins (+TIPs) like CLIP-170 are key regulators of MT dynamics and interactions.
The precise control mechanisms for CLIP-170 activity during cell division remain largely unelucidated.

Purpose of the Study:

To investigate the spatial and temporal regulation of CLIP-170 activity during mitosis.
To identify the kinase responsible for CLIP-170 modification during cell division.
To determine the functional consequences of CLIP-170 phosphorylation on MT interactions and chromosome alignment.

Main Methods:

Analysis of CLIP-170 phosphorylation at Ser312 during mitosis.
In vitro binding assays using purified CLIP-170 fragments and Polo-like kinase 1 (Plk1).
Assessment of kinetochore-MT attachment and chromosome alignment in mitotic cells.

Main Results:

CLIP-170 phosphorylation at Ser312 increases during mitosis, mediated by Plk1.
Plk1-mediated phosphorylation reduces CLIP-170 binding to MT ends and lattice, but not EB3.
Proper chromosome alignment and stable kinetochore-MT attachment require CLIP-170 and its phosphorylation/dephosphorylation cycle.

Conclusions:

CLIP-170 phosphorylation by Plk1 modulates its interaction with MTs, regulating chromosome alignment in mitotic cells.
The phosphorylation state of CLIP-170 is a critical determinant of its activity and function.
Cellular context dictates CLIP-170 activity through stringent control of its phosphorylation status.

Read full analysis

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