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Functional characterization of a novel protein, CDK5RAP2, in microtubule organization and regulation

Authors Fong, Ka Wing
Issue Date 2008
Summary In animal cells, centrosome is the primary microtubule organizing center (MTOC) which plays a key role in the control of the temporal and spatial distribution of microtubule (MT) networks. Typically, centrosomes are positioned at the focus of a radial array of MTs during interphase and are incorporated into spindle poles during mitosis. MT nucleation and organization by the centrosome require γ-tubulin, a protein that exists in a macromolecular complex called the γ-tubulin ring complex (γ-TuRC). There is a large cytoplasmic pool of the γ-TuRC, which is recruited to the centrosome and incorporated into the pericentriolar matrix (PCM). Several proteins, including Pericentrin/Kendrin, AKAP450, Ninein, Ninein-like protein (N1p), and NEDD1/GCP-WD have been implicated in the integration of the γ-TuRC with the centrosome. Their collaborative efforts are required to proper γ-TuRC function at the centrosome. CDK5RAP2 was discovered in a yeast two-hybrid screen for proteins that interact with the neuronal p35 activator of Cyclin dependent kinase 5 (Cdk5). Although CDK5RAP2 is highly expressed in the brain, its RNA transcript has been detected in all human tissues examined. Genetic mutations of CDK5RAP2 cause autosomal recessive primary microcephaly (MCPH), a cell cycle disorder of neural progenitors during neurogenesis. Moreover, CDK5RAP2 was found in human centrosome. In my study, we found that CDK5RAP2 localizes throughout the PCM in all stages of the cell cycle. When over-expressed, CDK5RAP2 assembled a subset of centrosomal proteins including γ-tubulin onto the centrosomes or under the MT-disrupting conditions into MT-nucleating clusters in the cytoplasm. CDK5RAP2 associates with the γ-TuRC via a short conserved sequence present in several related proteins found in a range of organisms from fungi to mammals. The binding of CDK5RAP2 is required for γ-TuRC attachment to the centrosome but not for γ-TuRC assembly. Perturbing CDK5RAP2 function delocalized γ-tubulin from the centrosome and inhibited centrosomal MT nucleation, thus leading to disorganization of interphase MT arrays and formation of anastral mitotic spindles. Taken together, we identified CDK5RAP2 as one of the key γ-TuRC anchoring proteins which is required for regulating γ-TuRC function at the centrosome. To further explore the function of CDK5RAP2, we conducted the yeast two-hybrid screen by using human adult brain cDNA library to identify novel CDK5RAP2 binding proteins. We fished out End Binding protein 3 (EB3) as a novel CDK5RAP2 binding protein. EB is the prototype of MT plus-end tracking proteins (+TIPs) which plays essential role to control MT plus-end dynamics. CDK5RAP2 was also shown to bind to EB1, which is ubiquitously expressed protein whereas EB3 is preferentially expressed in brain and muscle. The EB1 binding site in CDK5RAP2 was defined as the conserved Leu938-Pro939 dipeptide. We demonstrated for the first time the MT plus-end tracking behavior of CDK5RAP2 by employing the time-lapse microscopy of live cells stably expressing GFP-CDK5RAP2. Either mutation of EB1 binding sites in CDK5RAP2 or depletion of eb1 by RNA interference abolished CDK5RAP2 plus-end tracking ability. Besides, CDK5RAP2 acted with EB1 to promote MT polymerization in vitro and facilitated the formation of EB1 induced MT bundles in vivo. To sum up, we identified CDK5RAP2 as a novel +TIP and characterized CDK5RAP2 function to cooperate with EB1 to regulate MT dynamics in the MT plus-end. To conclude, CDK5RAP2 exists in PCM of the centrosome that functions in γ-TuRC attachment and therefore the MT organizing function of the centrosome. In addition, CDK5RAP2 is able to bind to EB1 and cooperates with EB1 to regulate MT plus-end dynamics.
Note Thesis (Ph.D.)--Hong Kong University of Science and Technology, 2008
Language English
Format Thesis
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