Minimal Plus-end Tracking Unit of the Cytoplasmic Linker Protein CLIP-170

Abstract

Cytoplasmic linker protein 170 (CLIP-170) is the prototype microtubule (MT) plus-end tracking protein (؉TIP) and is involved in regulating MT dynamics. A comprehensive understanding of the process by which CLIP-170 tracks MT plus ends would provide insight into its function. However, the precise molecular mechanism of CLIP-170 ؉TIP behavior is unknown, and many potential models have been presented. Here, by separating the two CLIP-170 CAP-Gly domains and their adjacent serine-rich regions into fragments of varied size, we have characterized the minimal plus-end tracking unit of CLIP-170 in vivo. Each CLIP-170 fragment was also characterized for its tubulin polymerization activity in vitro. We found that the two CAP-Gly domains have different activities, whereas CAP-Gly-1 appears incompetent to mediate either ؉TIP behavior or MT nucleation, a CLIP-170 fragment consisting of the second CAPGly domain and its adjacent serine-rich region can both track MT plus ends in vivo and induce tubulin polymerization in vitro. These observations complement recent work on CLIP-170 fragments, demonstrate that CAP-Gly motifs do not require dimerization for ؉TIP and polymerization-promoting activities, and provide insight into CLIP-170 function and mechanism.

Cytoplasmic Linker Proteins Regulate Neuronal Polarization through Microtubule and Growth Cone Dynamics

“…As previous work suggested that CLIPs mediate bundling of microtubules (Gupta et al, 2009), we analyzed bundling of microtubules at the tip of the neurons in DN-CLIP and MBD-CLIP transfected neurons. In control neurons, microtubules of minor neurites and axons of stage 2 and stage 3 neurons were bundled and reached into the tips of the processes (Fig.…”

Microtubule +TIPs at a glance

“…Prominent examples are the CLIP proteins and the large subunit of the dynactin complex p150 glued . A single CAP-Gly domain of CLIP-170, together with the adjacent serine-rich region, can track growing microtubule ends (Gupta et al, 2009).…”

“…These elements mediate the interaction with each other and microtubules, and typically display affinities in the low micromolar range (Gupta et al, 2009;Mishima et al, 2007;Weisbrich et al, 2007).…”

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

“…The C-terminal region of CLIP-170 autonomously and intramolecularly inhibits these interactions, thereby diminishing end-tracking (Lansbergen et al, 2004). Accumulating evidence has shown that the third serine-rich region, which is adjacent to the CAP-Gly domain, also contributes to the end-tracking of CLIP-170 (Gupta et al, 2009(Gupta et al, , 2010. Using the N-terminal fragment of CLIP-170, we demonstrated that phosphorylation at Ser312 in the third serine-rich region reduced binding to MTs without affecting binding to EB3 or to its own C-terminal region (Fig.…”

“…The C-terminal zinc-finger motifs of CLIP-170 mediate its localization at kinetochores for chromosome alignment in prometaphase (Dujardin et al, 1998;Coquelle et al, 2002;Tai et al, 2002;Tanenbaum et al, 2006). The serine-rich regions encompassing the CAP-Gly domains of CLIP-170 also contribute to MT binding (Gupta et al, 2009(Gupta et al, , 2010. Recent studies have shown that phosphorylation(s) in the N-terminal serine-rich regions of CLIP-170 strengthens the aforementioned inhibitory intramolecular association (Lee et al, 2010) and is involved in its localization at kinetochores during mitosis (Li et al, 2010).…”