Cdc2-mediated Phosphorylation of CLIP-170 Is Essential for Its Inhibition of Centrosome Reduplication

Abstract

CLIP-170, the founding member of microtubule "plus ends tracking" proteins, is involved in many critical microtubulerelated functions, including recruitment of dynactin to the microtubule plus ends and formation of kinetochore-microtubule attachments during metaphase. Although it has been reported that CLIP-170 is a phosphoprotein, neither have individual phosphorylation sites been identified nor have the associated kinases been extensively studied. Herein, we identify Cdc2 as a kinase that phosphorylates CLIP-170. We show that Cdc2 interacts with CLIP-170 mediating its phosphorylation on Thr 287 in vivo. Significantly, expression of CLIP-170 with a threonine 287 to alanine substitution (T287A) results in its mislocalization, accumulation of Plk1 and cyclin B, and block of the G2/M transition. Finally, we found that depletion of CLIP-170 leads to centrosome reduplication and that Cdc2 phosphorylation of CLIP-170 is required for the process. These results demonstrate that Cdc2-mediated phosphorylation of CLIP-170 is essential for the normal function of this protein during cell cycle progression.Microtubule dynamics consist of alternating phases of growth and shortening, a pattern of behavior known as dynamic instability (1). This process is tightly regulated by a group of proteins that bind specifically to the plus ends of the growing microtubules (2). Cytoplasmic linker protein (CLIP) 3 -170, the founding member of the microtubule plus end family (3), is composed of three separate regions: N terminus, central coiled-coil region, and C terminus. In addition to two conserved cytoskeleton-associated protein glycine-rich (CAP-Gly) domains, the N terminus has three serine-rich regions. The N-terminal domain plays an essential role in microtubule targeting (4), the long central coiled-coil domain is responsible for dimerization of the protein, and the C-terminal region, which contains two zinc-finger domains interferes with microtubule binding by interacting with the N terminus (5). Experiments in a variety of organisms have demonstrated that CLIP-170 plays an important role in microtubule dynamics (6, 7). In addition to its positive role in regulating microtubule growth in both yeast and humans (8, 9), CLIP-170 is involved in recruitment of dynactin to the microtubule plus ends and in linking microtubules to the cortex through Cdc42 and IQGAP (10, 11). The role of CLIP-170 during mitosis was recently examined by loss-offunction approaches. It was shown that CLIP-170 localizes to unattached kinetochores in prometaphase and that such localization is essential for the formation of kinetochore-microtubule attachments (12, 13).It was previously reported that CLIP-170 is a phosphoprotein and that overall phosphorylation of CLIP-170 affects its microtubule binding ability (14). More recently, metabolic labeling experiments indicated that CLIP-170 is phosphorylated at multiple sites (15). However, individual phosphorylation sites have not been identified. Moreover, the FKBP12-rapamycin-associated protein (FRAP) is the only...

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

“…The mutation of Ser312 to alanine markedly reduced CLIP-170 phosphorylation by Plk1 but not by Cdk1 ( Fig. 2A), indicating that Cdk1 phosphorylates other residue(s), including Thr287 as previously reported (Yang et al, 2009). Immunoblot analysis with the anti-phosphorylated CLIP-170 antibody further demonstrated that Plk1 indeed phosphorylated Ser312 (Fig.…”

“…Recent studies have demonstrated that CLIP-170 is indeed phosphorylated and successfully identified the responsible kinases (Yang et al, 2009;Lee et al, 2010;Li et al, 2010;Nakano et al, 2010). We previously found that AMPK .…”

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

“…Both phosphorylation events are required for the dynactin-dependent kinetochore localization of CLIP-170 and the formation of KT-MT attachments (Li et al, 2010). Moreover, CLIP-170 is phosphorylated on T287 by cyclin-dependent kinase 1 (CDK1) -an event that is essential for cell cycle progression (Yang et al, 2009). However, the mechanism of the role of CLIP-170 in chromosome alignment remains unknown.…”

Phosphorylation of CLIP-170 by LRRK1 regulates EGFR trafficking by promoting recruitment of p150Glued to MT plus-ends

“…CLIP-170 can be phosphorylated on multiple residues (Rickard and Kreis, 1991;Choi et al, 2002;Yang et al, 2009). Previous studies have demonstrated that CLIP-170 is phosphorylated in the third serine-rich region adjacent to the second CAP-Gly microtubule-binding domain (Lee et al, 2010;Nakano et al, 2010).…”