Several candidates have been proposed as mammalian anchoring proteins for γ-TuRC at the centrosome, such as Ctr100, pericentrin, and kendrin (Tassin et al., 1997
; Dictenberg et al., 1998
; Flory et al., 2000
). However, none of them have been demonstrated to interact with components of γ-TuRC or to mediate microtubule nucleation, although pericentrin was found in the complex containing γ-tubulin (Dictenberg et al., 1998
). In this study, we have shown that the amino-terminal regions of CG-NAP and kendrin indirectly associated with γ-tubulin through interaction with other components of γ-TuRC, GCP2/GCP3 and GCP2, respectively. Interaction among endogenous proteins was also detected by immunoprecipitation study (Figure ), although it remains to be established that CG-NAP and kendrin constitute primary anchoring sites for γ-TuRC in vivo. Furthermore, pretreatment of isolated centrosomes by antibodies to CG-NAP and/or kendrin suppressed the initiation of microtubule nucleation (Figure C), suggesting the involvement of CG-NAP and kendrin in microtubule nucleation from the centrosome. The antigens used to generate these antibodies are located in the amino-terminal region that interacted with γ-TuRC; thus, the epitopes might be close to or overlap with the binding sites for γ-TuRC. It was revealed that the amount of γ-tubulin was decreased in the centrosomes treated with antibodies to CG-NAP and/or kendrin by immunoblotting analysis (our unpublished results). Therefore, these antibodies suppressed microtubule nucleation, probably by displacement of γ-TuRC from CG-NAP and/or kendrin in the centrosome. The majority of microtubule nucleation appears to be limited to the centrosome despite the presence of substantial amounts of cytoplasmic γ-TuRC. Therefore, on recruitment to CG-NAP and/or kendrin in the centrosome, γ-TuRC might represent the active form, or activation of γ-TuRC might be prerequisite for the recruitment. Additional studies will be necessary to address each of these possibilities.
CG-NAP and kendrin were localized to the centrosome via their carboxyl-terminal regions, which were found to associate with calmodulin. The role of calmodulin in the targeting of CG-NAP and kendrin to the centrosome remains unclear. It was reported that green fluorescent protein–tagged calmodulin is localized to the centrosome only at mitotic phase in HeLa cells (Li et al., 1999
). We could also detect only a small amount of calmodulin in the centrosomal fractions and almost no specific staining of endogenous calmodulin at the centrosomes (our unpublished results). Centrosomal targeting of CG-NAP and kendrin might be mediated by calmodulin at mitotic phase and by some undefined protein(s) at interphase. Another possibility is that calmodulin may serve to chaperone the carboxyl-terminal region of CG-NAP and kendrin, similar to the role of Cmd1p in the proper assembly of Spc110p at SPB (Sundberg et al., 1996
), and be released after centrosomal targeting, as discussed by Gillingham and Munro (2000)
. It is also unclear whether calmodulin binding to these proteins is regulated by Ca2+
: it was Ca2+
-dependent in vitro (Figure B) but Ca2+
-independent in the immunoprecipitation study (Figure C). The interaction between these proteins might be modified by the presence of some other protein(s) under cellular conditions. In addition to the calmodulin-binding region, the centrosomal-localization regions contain several conserved sequences between CG-NAP and kendrin (Figure B), which may provide interaction sites for other proteins. Our screening using CG-NAP3510–3828
as bait thus far has yielded only calmodulin clones, which might be attributed to the abundance of calmodulin mRNA in mammalian cells. Further screening by using different bait constructs may be one approach to assess these possibilities.
CG-NAP and kendrin have coiled-coil regions (Figure ) that may form a filamentous complex. Indeed, endogenously and exogenously expressed CG-NAP and kendrin formed complexes (Figure , A and B). Moreover, certain combinations of CG-NAP and kendrin deletions were coimmunoprecipitated (our unpublished results), suggesting that the interaction between CG-NAP and kendrin is direct rather than indirect as components of a large protein complex. Heterodimers (or oligomers) of CG-NAP and kendrin may serve as components of PCM and provide anchoring sites for γ-TuRC. CG-NAP also forms a homodimer (or oligomer) (Takahashi et al., 1999
); thus, CG-NAP homodimers (or oligomers) may also provide the anchoring sites. Do CG-NAP and kendrin play distinct roles in the centrosome? In yeast, γ-TuRC anchoring proteins Spc110p and Spc72p play independent roles by their different subcellular localization, inner plaque and outer plaque, respectively. Although CG-NAP and kendrin represent differences in terms of the additional localization of CG-NAP in the Golgi apparatus, they are localized to the centrosome in a very similar manner (Figure C). Some difference was observed in the efficiency of binding with γ-TuRC. CG-NAP16–1229
associated with GCP2 and γ-tubulin more efficiently than kendrin1–1189
(compare Figures B and B), which might be attributed to the relatively low homology between these regions (Figure ). We may not be able to conclude that CG-NAP has a higher affinity to γ-TuRC, because these results were obtained with deletion constructs of CG-NAP and kendrin and from cells at random stages of the cell cycle. Cell cycle–dependent phosphorylation and regulation of yeast SPB proteins, such as Spc110p (Friedman et al., 2001
) and Spc98p (Pereira et al., 1998
), have been demonstrated. Similarly, centrosomal proteins may be regulated by phosphorylation, for instance, at mitotic phase. From interphase to metaphase, the amount of γ-tubulin at the centrosome increases at least threefold and decreases rapidly by late anaphase (Khodjakov and Rieder, 1999
). The recruitment of γ-TuRC to the centrosome may contribute to the increase in microtubule number associated with mitotic versus interphase centrosomes (Kuriyama and Borisy, 1981
). It is attractive to postulate that mitotic phosphorylation of CG-NAP or kendrin or GCP2/GCP3 alters affinity among these proteins or to the component(s) of PCM, which may result in increased recruitment of γ-TuRC to the centrosomes.
We have found that CG-NAP interacts with Rho-activated protein kinase PKN (Amano et al., 1996
; Shibata et al., 1996
; Watanabe et al., 1996
), PKA, PKCε, and protein phosphatases PP1 and PP2A, and thus, CG-NAP may target them to the proximity of specific substrates at the centrosome and the Golgi apparatus (Takahashi et al., 1999
). It is possible that the complex containing CG-NAP, kendrin, and γ-TuRC serves as a substrate for these enzymes and is regulated downstream of various signals such as Rho and cAMP. Kendrin also anchors PKA (Diviani et al., 2000
). CG-NAP and kendrin may form matrix as integral components of PCM to provide anchoring sites for γ-TuRC as well as serving as targeting machinery for various signaling enzymes to the centrosome. Further studies will be necessary to elucidate the role of CG-NAP and kendrin in the regulation of recruitment and activity of γ-TuRC at the centrosome.