Conserved Motif of CDK5RAP2 Mediates Its Localization to Centrosomes and the Golgi Complex

Abstract

As the primary microtubule-organizing centers, centrosomes require ␥-tubulin for microtubule nucleation and organization. Located in close vicinity to centrosomes, the Golgi complex is another microtubule-organizing organelle in interphase cells. CDK5RAP2 is a ␥-tubulin complex-binding protein and functions in ␥-tubulin attachment to centrosomes. In this study, we find that CDK5RAP2 localizes to the Golgi complex in an ATPand centrosome-dependent manner and associates with Golgi membranes independently of microtubules. CDK5RAP2 contains a centrosome-targeting domain with its core region highly homologous to the Motif 2 (CM2) of centrosomin, a functionally related protein in Drosophila. This sequence, referred to as the CM2-like motif, is also conserved in related proteins in chicken and zebrafish. Therefore, CDK5RAP2 may undertake a conserved mechanism for centrosomal localization. Using a mutational approach, we demonstrate that the CM2-like motif plays a crucial role in the centrosomal and Golgi localization of CDK5RAP2. Furthermore, the CM2-like motif is essential for the association of the centrosome-targeting domain to pericentrin and AKAP450. The binding with pericentrin is required for the centrosomal and Golgi localization of CDK5RAP2, whereas the binding with AKAP450 is required for the Golgi localization. Although the CM2-like motif possesses the activity of Ca 2؉ -independent calmodulin binding, binding of calmodulin to this sequence is dispensable for centrosomal and Golgi association. Altogether, CDK5RAP2 may represent a novel mechanism for centrosomal and Golgi localization.

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).…”

Centrosomin represses dendrite branching by orienting microtubule nucleation

“…We therefore investigated if Cnn is re--utilized in post--mitotic neurons in order to localize microtubule nucleation by an analogous anchoring process. The mammalian Cnn paralog CDK5RAP2 localizes to somatic Golgi during interphase in non--neuronal cells 33 . Moreover, in addition to their function in dendrite secretory trafficking 34,35 , Golgi outposts were recently identified as a site of acentrosomal microtubule nucleation in dendrites 20 .…”

Probing the Boundaries of Orthology: The Unanticipated Rapid Evolution ofDrosophila centrosomin

“…The function of the carboxy terminus is mostly unknown, although we have shown that loss of the SPD motif and carboxy terminus results in very low levels of truncated Cnn at the centrosome and in high levels at the metaphase plate (Eisman et al 2009). A recent study reported that a motif in the Cnn carboxy terminus is conserved in mouse CDK5RAP2 (Wang et al 2010), but a PsiBLAST with the D. melanogaster motif found minimal homology, with an E-value of 7.3 between fly and mouse. Clearly, future studies of Cnn are required to define the molecular basis of Cnn function.…”

“…C. Eisman and T. C. Kaufman carboxy terminus is conserved in mouse CDK5RAP2 (Wang et al 2010), but a PsiBLAST with the D. melanogaster motif found minimal homology, with an E-value of 7.3 between fly and mouse. Clearly, future studies of Cnn are required to define the molecular basis of Cnn function.…”