Regulation of Centrosomes by Cyclin-Dependent Kinases - The Centrosome - page 186

Fig. 11.3 The centrosome and DNA replication cycles. Both are initiated by the activity of CDK2-
cyclin E at G1/S and maintained by CDK2-cyclin A in S/G2 phases. CDK1-cyclin B coordinates
centrosome maturation with nuclear envelope breakdown and chromosome condensation at G2/M.
Known centrosomal CDK2 substrates NPM, Mps1 and CP110 are shown (pink)
11.2.2 CDK2 Centrosomal Substrates
Once the importance of CDK2-cyclin E in centrosome replication was established,
efforts turned to identifying its centrosomal targets. At least three centrosome
proteins have been found to be directly phosphorylated by CDK2 in complex with
cyclin E and/or cyclin A. These are Nucleophosmin (NPM) (Okuda et al. 2000 ),
Monopolar spindle 1 (Mps1) (Fisk and Winey 2001 ), and Centrosome Protein of
110 kDa (CP110) (Chen et al. 2002 ) (Fig. 11.3 ).
11.2.2.1 NPM
To identify centrosomal CDK2-cyclin E targets, the Fukasawa laboratory performed
an in vitro kinase reaction using centrosomes isolated from quiescent 3T3 mouse
fibroblast cells, as substrate (Okuda et al. 2000 ). A single protein in the centrosome
prep was found to be phosphorylated by CDK2-cyclin E and was identified as NPM
(Okuda et al. 2000 ), also known as B23, a previously identified component of
nucleolar granules (Yung et al. 1985 ). NPM was found to be recruited to the cen-
trosomes during mitosis and remained at the unreplicated centrosome in early G1
phase, but was then lost following centrosome replication (Okuda et al. 2000 ).
Microinjection of antibodies to NPM or overexpression of either a deletion mutant
(NPMD186-239) or a non-phosphorylable mutant (NPM-T199A) of NPM blocked
centrosome replication (Okuda et al. 2000 ; Tokuyama et al. 2001 ). The NPM-T199A
mutant also remained associated with the centrosomes throughout the cell cycle and
resulted in the formation of monopolar spindles in mitosis (Tokuyama et al. 2001 ).