Control of centrin stability by Aurora A.

Aurora A is an oncogenic serine/threonine kinase which can cause cell transformation and centrosome amplification when over-expressed. Human breast tumors show excess Aurora A and phospho-centrin in amplified centrosomes. Here, we show that Aurora A mediates the phosphorylation of and localizes with centrin at the centrosome, with both proteins reaching maximum abundance from prophase through metaphase, followed by their precipitous loss in late stages of mitosis. Over-expression of Aurora A results in excess phospho-centrin and centrosome amplification. In contrast, centrosome amplification is not seen in cells over-expressing Aurora A in the presence of a recombinant centrin mutant lacking the serine phosphorylation site at residue 170. Expression of a kinase dead Aurora A results in a decrease in mitotic index and abrogation of centrin phosphorylation. Finally, a recombinant centrin mutation that mimics centrin phosphorylation increases centrin's stability against APC/C-mediated proteasomal degradation. Taken together, these results suggest that the stability of centrin is regulated in part by Aurora A, and that excess phosphorylated centrin may promote centrosome amplification in cancer.

Aurora A, centrosome structure, and the centrosome cycle.

The centrosome, also known as the microtubule organizing center of the cell, is a membrane-less organelle composed of a pair of barrel-shaped centrioles surrounded by electron-dense pericentriolar material. The centrosome progresses through the centrosome cycle in step with the cell cycle such that centrosomes are duplicated in time to serve as the spindle poles during mitosis and that each resultant daughter cell contains a single centrosome. Regulation of the centrosome cycle with relation to the cell cycle is an essential process to maintain the ratio of one centrosome per new daughter cell. Numerous mitosis-specific kinases have been implicated in this regulation, and phosphorlyation plays an important role in coordinating the centrosome and cell cycles. Centrosome amplification can occur when the cycles are uncoupled, and this amplification is associated with cancer and with an increase in the levels of chromosomal instability. The aurora kinases A, B, and C are serine/threonine kinases that are active during mitosis. Aurora A is associated with centrosomes, being localized at the centrosome just prior to the onset of mitosis and for the duration of mitosis. Overexpression of aurora A leads to centrosome amplification and cellular transformation. The activity of aurora A is regulated by phosphorlyation and proteasomal degradation.

Cisplatin abrogates the geldanamycin-induced heat shock response.

Benzoquinone ansamycin antibiotics such as geldanamycin (GA) bind to the NH(2)-terminal ATP-binding domain of heat shock protein (Hsp) 90 and inhibit its chaperone functions. Despite in vitro and in vivo studies indicating promising antitumor activity, derivatives of GA, including 17-allylaminogeldanamycin (17-AAG), have shown little clinical efficacy as single agents. Thus, combination studies of 17-AAG and several cancer chemotherapeutics, including cisplatin (CDDP), have begun. In colony-forming assays, the combination of CDDP and GA or 17-AAG was synergistic and caused increased apoptosis compared with each agent alone. One measurable response that results from treatment with Hsp90-targeted agents is the induction of a heat shock factor-1 (HSF-1) heat shock response. Treatment with GA + CDDP revealed that CDDP suppresses up-regulation of HSF-1 transcription, causing decreased levels of stress-inducible proteins such as Hsp27 and Hsp70. However, CDDP treatment did not prevent trimerization and nuclear localization of HSF-1 but inhibited DNA binding of HSF-1 as shown by chromatin immunoprecipitation. Melphalan, but not camptothecin, caused similar inhibition of GA-induced HSF-1-mediated Hsp70 up-regulation. 3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt cell survival assays revealed that deletion of Hsp70 caused increased sensitivity to GA (Hsp70(+/+) IC(50) = 63.7 +/- 14.9 nmol/L and Hsp70(-/-) IC(50) = 4.3 +/- 2.9 nmol/L), which confirmed that a stress response plays a critical role in decreasing GA sensitivity. Our results suggest that the synergy of GA + CDDP is due, in part, to CDDP-mediated abrogation of the heat shock response through inhibition of HSF-1 activity. Clinical modulation of the HSF-1-mediated heat shock response may enhance the efficacy of Hsp90-directed therapy.