The centrosome/centriole duplication cycle is coupled with the DNA replication cycle, which is one of the important mechanisms that ensure centrosomes to duplicate only once in a single cell cycle. The coupling of these two events is at least in part achieved by the late G1 phase-specific activation of CDK2-cyclin E. CDK2-cyclin E targets a number of proteins, which leads to initiation of both centrosome duplication and DNA replication. We have previously shown that NPM/B23 is one of the targets of CDK2-cyclin E to initiate centrosome duplication (Okuda et al., 2000
; Tokuyama et al., 2001
). NPM/B23 phosphorylated by CDK2-cyclin E binds to and super-activates ROCK II, which is a critical event for the timely initiation of centrosome duplication (Ma et al., 2006
). Activation of ROCK II also requires the binding of Rho-GTPase to the Rho binding domain at C-terminus, which releases the kinase domain from the autoinhibitory domain. Although all Rho isoforms (RhoA, RhoB, and RhoC) are capable of binding to and activating ROCK II, we found that RhoA and RhoC, but not RhoB, participate in the regulation of centrosome duplication. For instance, knock-down of either RhoA or RhoC resulted in suppression of centrosome duplication, while knock-down of RhoB had no effect on centrosome duplication. The activity of Rho is regulated by three classes of proteins: guanine nucleotide exchange factors (GEFs), which facilitate the exchange of GDP to GTP, GTPase-activating proteins (GAPs), which increase the rate of GTP hydrolysis to GDP, and GDP dissociating inhibitors (GDIs), which inhibit spontaneous GDP-GTP exchange of Rho (reviewed in Kjoller and Hall, 1999
). The failure of RhoB to promote centrosome duplication is not due to the failure to be activated by these regulatory proteins, since expression of constitutively active form of RhoB, unlike that of RhoA and RhoC, fails to efficiently promote centrosome duplication. Instead, we found that constitutively active RhoA and RhoC localize to centrosomes, while RhoB failed to do so. Because ROCK II localizes to centrosomes throughout the cell cycle, and drives centrosome duplication at centrosomes (Ma et al., 2006
), the ability of Rho to be recruited to centrosomes is expected to be required for controlling centrosome duplication. Thus, the failure of RhoB to control centrosome duplication is likely because of its inability to localize to centrosomes. Although our present studies show that RhoB is not involved in centrosome duplication during normal cell cycle or centrosome re-duplication in late G1 and S phases, our studies do not exclude the involvement of RhoB in centrosome amplification that occurs in late G2 phase. For instance, there is one study implicating RhoB in radiation-induced centrosome amplification, which is known to occur in late G2 phase of the cell cycle (Milia et al., 2005
In respect to the centrosome localization activity of Rho, we further found that GDP-bound inactive forms of RhoA and RhoC fail to localize to centrosomes or do so very inefficiently. However, this observation does not answer whether RhoA/C is recruited to centrosomes as a GDP-bound form or GTP-bound form. It is possible that RhoA/C may be recruited to centrosomes as a GDP-bound form, and GDP-GTP exchange may occur at centrosomes. In support of this possibility, some RhoGEFs such as Ect2 (Wolf et al., 2006
) and ARHGEF10 (Aoki et al., 2009
) have been shown to reside at centrosomes, indicating that GDP-GTP exchanges of Rho can occur at centrosomes.
The observation that depletion of RhoA alone and that of RhoC alone both resulted in suppression of centrosome duplication raises a question of whether there is any functional difference between RhoA and RhoC to control centrosome duplication. Our present studies indicate that ROCK II is a primary target of both RhoA and RhoC to control centrosome duplication. For instance, the ROCK II-binding/activation defective mutant RhoA and RhoC both failed to promote centrosome duplication. Based on the finding that introduction of constitutively active RhoC in the RhoA RNAi cells as well as introduction of constitutively active RhoA in the RhoC RNAi cells restores the centrosome duplication potential of the cells, it is possible that RhoA and RhoC may comprise the intracellular concentration of the total Rho proteins required for initiation of centrosome duplication. However, alternatively, although the primary target of both RhoA and RhoC is ROCK II, RhoA and RhoC may possess their own unique functions toward initiation of centrosome duplication besides ROCK II activation, and such unique function(s) of one isoform may be readily compensated by the overexpression/overactivation of the other isoform.
Our present findings put forward to several significant clinical implications. Overactivation of RhoA as well as RhoC is commonly found in human cancers (reviewed in Gómez del Pulgar et al., 2005
; Ellenbroek and Collard, 2007
; Vega and Ridley, 2008
). We found that overexpression/overactivation of RhoA and RhoC promotes centrosome duplication via ROCK II. Moreover, stable expression of constitutively active forms of RhoA and RhoC results in a high frequency of centrosome amplification and chromosome instability (data not shown). Because the presence of amplified centrosomes destabilizes chromosomes, centrosome amplification is likely an important factor contributing to carcinogenesis associated with overexpression/overactivation of RhoA and RhoC. The other implication is in association with aberrant activation of receptor tyrosine kinases (RTKs) and carcinogenesis. The uncontrolled activation of RTKs is one of the most common features of cancers. It has been recognized that oncogenic activation of many receptor tyrosine kinases leads to destabilization of chromosomes. However, this phenomenon has been belittled as an indirect consequence of the continuous firing of the cell cycle signaling. Because Rho is one of the immediate effectors of a wide variety of RTKs (reviewed in Kjoller and Hall, 1999
), our present finding, in which over-activation of Rho leads to centrosome amplification via ROCK II, suggest that oncogenic activation of RTKs may be more directly involved in destabilization of chromosomes through generation of amplified centrosomes by continual activation of the Rho-ROCK II pathway. Consistently to this possibility, it has been shown that continual (oncogenic) activation of EGF receptor has been shown to promote centrosome duplication (Balczon et al.
, 1996). Similarly, stable expression and continual activation of the Met hepatocyte growth factor (HGF) receptor in fibroblasts (i.e.
, M114 cell line supplemented with excess HGF, ref. Shinomiya et al., 2004
) leads to centrosome amplification in a ROCK II-dependent manner (Supplemental Information: Fig. 4