Interaction of CDK5RAP2 with EB1 to Track Growing Microtubule Tips and to Regulate Microtubule Dynamics

Department of Biochemistry, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China.
Molecular biology of the cell (Impact Factor: 4.47). 07/2009; 20(16):3660-70. DOI: 10.1091/mbc.E09-01-0009
Source: PubMed


Mutations in cdk5rap2 are linked to autosomal recessive primary microcephaly, and attention has been paid to its function at centrosomes. In this report, we demonstrate that CDK5RAP2 localizes to microtubules and concentrates at the distal tips in addition to centrosomal localization. CDK5RAP2 interacts directly with EB1, a prototypic member of microtubule plus-end tracking proteins, and contains the basic and Ser-rich motif responsible for EB1 binding. The EB1-binding motif is conserved in the CDK5RAP2 sequences of chimpanzee, bovine, and dog but not in those of rat and mouse, suggesting a function gained during the evolution of mammals. The mutation of the Ile/Leu-Pro dipeptide within the motif abolishes EB1 interaction and plus-end attachment. In agreement with the mutational analysis, suppression of EB1 expression inhibits microtubule tip-tracking of CDK5RAP2. We have also found that the CDK5RAP2-EB1 complex regulates microtubule dynamics and stability. CDK5RAP2 depletion by RNA interference impacts the dynamic behaviors of microtubules. The CDK5RAP2-EB1 complex induces microtubule bundling and acetylation when expressed in cell cultures and stimulates microtubule assembly and bundle formation in vitro. Collectively, these results show that CDK5RAP2 targets growing microtubule tips in association with EB1 to regulate microtubule dynamics.

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Available from: ka wing Fong, May 04, 2014
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    • "As mentioned above, AKAP450 interacts with p150Glued that directly binds to MTs and EB1. CDK5Rap2 was shown to bind to growing MT tips by associating with EB1, suggesting that it could, in this way, regulate the plus-end dynamics of MTs [71]. Moreover, two myomegalin isoforms differing at their N-terminus have been identified in RPE-1 cells [29]. "
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    ABSTRACT: A shared feature among all microtubule (MT)-dependent processes is the requirement for MTs to be organized in arrays of defined geometry. At a fundamental level, this is achieved by precisely controlling the timing and localization of the nucleation events that give rise to new MTs. To this end, MT nucleation is restricted to specific subcellular sites called MT-organizing centres. The primary MT-organizing centre in proliferating animal cells is the centrosome. However, the discovery of MT nucleation capacity of the Golgi apparatus (GA) has substantially changed our understanding of MT network organization in interphase cells. Interestingly, MT nucleation at the Golgi apparently relies on multiprotein complexes, similar to those present at the centrosome, that assemble at the cis-face of the organelle. In this process, AKAP450 plays a central role, acting as a scaffold to recruit other centrosomal proteins important for MT generation. MT arrays derived from either the centrosome or the GA differ in their geometry, probably reflecting their different, yet complementary, functions. Here, I review our current understanding of the molecular mechanisms involved in MT nucleation at the GA and how Golgi- and centrosome-based MT arrays work in concert to ensure the formation of a pericentrosomal polarized continuous Golgi ribbon structure, a critical feature for cell polarity in mammalian cells. In addition, I comment on the important role of the Golgi-nucleated MTs in organizing specialized MT arrays that serve specific functions in terminally differentiated cells.
    Full-text · Article · Sep 2014 · Philosophical Transactions of The Royal Society B Biological Sciences
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    • "The generation and maintenance of a U2OS subline stably expressing low levels of EB1- GFP were reported previously (Fong et al., 2009). Cells seeded on 35-mm glassbottomed culture dishes were used for acquiring time-lapse image sequences on a spinning-disk microscope (Carl Zeiss Axio- Observer Z1; Yokogawa CSU-X1 spinning disk; Yokogawa, Japan) equipped with an Andor Neo sCMOS camera (Andor, Belfast, UK). "
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    ABSTRACT: As the primary microtubule nucleator in animal cells, the γ-tubulin ring complex (γTuRC) plays a crucial role in microtubule organization, but little is known about how the activity of the γTuRC is regulated. Recently, isolated γTuRC was found to contain NME7, a poorly characterized member of the NME family. Here, we report that NME7 is a γTuRC component that regulates the microtubule-nucleating activity of the γTuRC. NME7 contains 2 putative kinase domains (A and B) and shows autophosphorylating activity. Whereas domain A is involved in the autophosphorylation, domain B is inactive. NME7 interacts with the γTuRC through both A and B domains, with Arg322 in domain B being crucial to the binding. In association with the γTuRC, NME7 localizes to centrosomes throughout the cell cycle and to mitotic spindles during mitosis. Suppression of NME7 expression does not affect γTuRC assembly or localization to centrosomes, but impairs centrosome-based microtubule nucleation. Importantly, wild-type NME7 promotes γTuRC-dependent nucleation of microtubules, but kinase-deficient NME7 does so only poorly. These results suggest that NME7 functions in the γTuRC in a kinase-dependent manner to facilitate microtubule nucleation.
    Full-text · Article · May 2014 · Molecular biology of the cell
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    • "CDK5RAP2 exists as two isoforms resembling the CM-MMG isoform and also binds EB1 through an SxLP motif (Fong et al., 2009). The role of this interaction is still unclear because CDK5RAP2 inhibition only slightly affects MT dynamics and the SxLP motif in CDK5RAP2 is not conserved in all vertebrate species. "
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    ABSTRACT: The generation of cellular microtubules is initiated at specific sites such as the centrosome and the Golgi apparatus that contain nucleation complexes rich in γ-tubulin. The microtubule growing plus-ends are stabilized by plus-end tracking proteins (+TIPs), mainly EB1 and associated proteins. Myomegalin was identified as a centrosome/Golgi protein associated with cyclic nucleotide phosphodiesterase. We show here that Myomegalin exists as several isoforms. We characterize two of them. One isoform, CM-MMG, harbors a conserved domain (CM1), recently described as a nucleation activator, and is related to a family of γ-tubulin binding proteins, which includes Drosophila centrosomin. It localizes at the centrosome and at the cis-Golgi in an AKAP450-dependent manner. It recruits γ-tubulin nucleating complexes and promotes microtubule nucleation. The second isoform, EB-MMG, is devoid of CM1 domain and has a unique N-terminus with potential EB1-binding sites. It localizes at the cis-Golgi and can localize to microtubule plus-ends. EB-MMG binds EB1 and affects its loading on microtubules and microtubule growth. Depletion of Myomegalin by small interfering RNA delays microtubule growth from the centrosome and Golgi apparatus, and decreases directional migration of RPE1 cells. In conclusion, the Myomegalin gene encodes different isoforms that regulate microtubules. At least two of these have different roles, demonstrating a previously unknown mechanism to control microtubules in vertebrate cells.
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