CDK5RAP2 Is a Pericentriolar Protein That Functions in Centrosomal Attachment of the γ-Tubulin Ring Complex

Abstract

Microtubule nucleation and organization by the centrosome require ␥-tubulin, a protein that exists in a macromolecular complex called the ␥-tubulin ring complex (␥TuRC). We report characterization of CDK5RAP2, a novel centrosomal protein whose mutations have been linked to autosomal recessive primary microcephaly. In somatic cells, CDK5RAP2 localizes throughout the pericentriolar material in all stages of the cell cycle. When overexpressed, CDK5RAP2 assembled a subset of centrosomal proteins including ␥-tubulin onto the centrosomes or under the microtubule-disrupting conditions into microtubule-nucleating clusters in the cytoplasm. CDK5RAP2 associates with the ␥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 ␥TuRC attachment to the centrosome but not for ␥TuRC assembly. Perturbing CDK5RAP2 function delocalized ␥-tubulin from the centrosomes and inhibited centrosomal microtubule nucleation, thus leading to disorganization of interphase microtubule arrays and formation of anastral mitotic spindles. Together, CDK5RAP2 is a pericentriolar structural component that functions in ␥TuRC attachment and therefore in the microtubule organizing function of the centrosome. Our findings suggest that centrosome malfunction due to the CDK5RAP2 mutations may underlie autosomal recessive primary microcephaly. INTRODUCTIONIn animal cells, the centrosome is the primary microtubule (MT) organizing center (MTOC), which plays a key role in the control of the temporal and spatial distribution of MT networks (Ou and Rattner, 2004;Doxsey et al., 2005;Luders and Stearns, 2007). Typically, centrosomes are positioned at the focus of a radial array of MTs during interphase and are incorporated into spindle poles during mitosis. Interphase centrosomes are composed of a pair of centrioles embedded in a cloud of electron-dense pericentriolar material (PCM). The centriole has a well-defined structure with MT triplets arranged into a cylinder, whereas the organization of the PCM is less apparent. Early studies have shown the existence of a salt (2 M KI)-insoluble scaffold/matrix underlying the PCM (Moritz et al., 1998;Schnackenberg et al., 1998). This matrix, formed with a high content of large coiled-coil proteins, provides binding sites for the tubulin family member ␥-tubulin and other proteins associated with centrosomal functions, such as MT nucleation. Through the cell cycle, the PCM varies in volume and the MT-nucleating activity, which are smallest in G1-phase and biggest during mitosis. In addition, proteins have a precise and cell cycle-specific placement within the PCM that has been observed as a tubular structure located along the surface of the centriole and around its proximal end but not around its distal end (Ou and Rattner, 2000;Ou et al., 2003). The outer surface of the PCM is dynamic and PCM proteins found in the cytoplasm transit to the PCM either by diffusion or via MTs.Distributed throughout the PCM, ␥-tubulin ...

Intact Cell MALDI-TOF MS on Sperm: A Molecular Test For Male Fertility Diagnosis

“…Several references can be found in the literature that relates the differential abundance of these proteins with fertility (36 -40). Along with these, we also observed a fragment of the CDK5RAP2, a protein that displays several functions in the control of centrosome and microtubule organization (41,42). The role of this regulatory protein in spermatozoa has not been investigated, but it is known that it might play a role in keeping correct function of the centrosome (41).…”

“…Along with these, we also observed a fragment of the CDK5RAP2, a protein that displays several functions in the control of centrosome and microtubule organization (41,42). The role of this regulatory protein in spermatozoa has not been investigated, but it is known that it might play a role in keeping correct function of the centrosome (41). An additional member of the models mass lists is voltage-dependent anion channel 2.…”

Centrosomal targeting of tyrosine kinase activity does not enhance oncogenicity in chronic myeloproliferative disorders

“…To the contrary, CDK5RAP2-PDGFRaFan inframe fusion between the N-terminal 494 out of 1205 amino acids of CDK5RAP2 and truncated exon 12 of PDGFRa, cloned from a patient with chronic eosinophilic leukemia 13 -displayed a homogenous cytoplasmic localization without enrichment at centrosomes, although centrosomal localization of full-length CDK5RAP2 itself was confirmed in our study. 37,38 Similarly, NDE1-PDGFRb 18 also localized to the cytoplasm without centrosomal enrichment. In previous studies, FOP-FGFR1 was shown to localize to the centrosome, whereas the centrosomal localization of CEP110-FGFR1 was rather equivocal.…”

The Centrosome and Its Duplication Cycle

“…CDKRAP2 and Cep192, human counterparts of CNN and Spd-2, also increase on centrosome maturation as does pericentrin, and the proteins show interdependency for the recruitment of g-tubulin (Haren et al 2009;Lee and Rhee 2011). In part, this reflects the physical association of g-tubulin with the amino-terminal part, and pericentrin with the carboxy-terminal part of CDKRAP2 (Fong et al 2008;Choi et al 2010;Wang et al 2010).…”

“…The nuclear receptor, Spc110 is a member of the pericentrin family of microtubule-nucleating proteins in which microtubule-nucleating motifs are separated from anchors by extended coiled-coil spacers (Kilmartin et al 1993;Kilmartin and Goh 1996;Sundberg and Davis 1997). These g-tubulin docking motifs are highly conserved from human pericentrin and kendrin through Drosophila centrosomin (CNN) to fission yeast Mto1 and Pcp1 (Flory et al 2002;Zhang and Megraw 2007;Fong et al 2008;Samejima et al 2008;Lin et al 2014). Spc29 links Spc110 to the hexagonal crystalline lattice of Spc42 that comprises the central plaque in a coupling that relies on association of Spc110 with calmodulin (Geiser et al 1993;Stirling et al 1994;Donaldson and Kilmartin 1996;Spang et al 1996;Bullit et al 1997;Sundberg and Davis 1997;Elliott et al 1999).…”