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Rapid calcium-dependent activation of Aurora-A kinase.

Plotnikova OV, Pugacheva EN, Dunbrack RL, Golemis EA - Nat Commun (2010)

Bottom Line: This activation is mediated by direct Ca(2+)-dependent calmodulin (CaM) binding to multiple motifs on AurA.On the basis of structure-function analysis and molecular modelling, we map two primary regions of CaM-AurA interaction to unfolded sequences in the AurA N- and C-termini.This unexpected mechanism for AurA activation provides a new context for evaluating the function of AurA and its inhibitors in normal and cancerous cells.

View Article: PubMed Central - PubMed

Affiliation: Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, Pennsylvania 19111, USA.

ABSTRACT
Oncogenic hyperactivation of the mitotic kinase Aurora-A (AurA) in cancer is associated with genomic instability. Increasing evidence indicates that AurA also regulates critical processes in normal interphase cells, but the source of such activity has been obscure. We report here that multiple stimuli causing release of Ca(2+) from intracellular endoplasmic reticulum stores rapidly and transiently activate AurA, without requirement for second messengers. This activation is mediated by direct Ca(2+)-dependent calmodulin (CaM) binding to multiple motifs on AurA. On the basis of structure-function analysis and molecular modelling, we map two primary regions of CaM-AurA interaction to unfolded sequences in the AurA N- and C-termini. This unexpected mechanism for AurA activation provides a new context for evaluating the function of AurA and its inhibitors in normal and cancerous cells.

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Intracellular calcium release is sufficient for AurA activation.(a) HEK293 cells overexpressing AurA were incubated with 5 μM thapsigargin for the indicated periods of time, and then cell lysates were analysed by western blot. Graph shown below indicates ratio of phAurA to total AurA. (b) AurA immunoprecipitation and in vitro kinase assay, after thapsigargin treatment. HEK293 cells overexpressing AurA were treated with 5 μM thapsigargin or control dimethylsulpfoxide (DMSO) for 5 min, AurA was immunoprecipitated and incubated with histone H3 substrate in an in vitro kinase assay. phHH3, antibody to phosphorylated histone H3. *P<0.05. (c) Left, an experiment similar to that shown in a, except that it is performed in the presence of 50 μM BAPTA-AM or control DMSO. Right, HEK293 cells overexpressing AurA were incubated with 20 μM BAPTA-AM (30 min) plus 7.5 mM EGTA (2 min) before the addition of 5 μM thapsigargin for the indicated periods of time, with analysis as for a. (d) An experiment similar to that shown in a, except that it is performed in the presence of 5 mM EGTA alone for the indicated periods of time (right panel) or in the presence of 5 mM EGTA (2 min) before the addition of 5 μM thapsigargin. (e) HEK293 cells overexpressing AurA were incubated with 5 μM thapsigargin and indicated concentration of ionomycin for the indicated periods of time, and then cell lysates were analysed by western blot using indicated antibodies. (f) HK2 cells were treated with DMSO, 5 μM thapsigargin (Tg) or 0.5 μM ionomycin (Ion), and then T288-phAurA levels were analysed by enzyme-linked immunosorbent assay (ELISA), based on A450 nm. To confirm the specificity of ELISA signal, a parallel group of HK2 cells were pre-treated with the AurA inhibitor PHA680632 (PHA) at 500 nM concentration. *P=0.05. Lysis buffer was used as a negative control. (g) AurA-overexpressing HEK293 cells were transfected with siRNA-depleting NEDD9 (siNEDD9) or scrambled control siRNA (scr), and then were incubated with 5-μm thapsigargin and processed as in a. For this and subsequent SDS–PAGE analyses, each experiment was performed three times independently, with error bars indicating the standard error. *P<0.05.
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f2: Intracellular calcium release is sufficient for AurA activation.(a) HEK293 cells overexpressing AurA were incubated with 5 μM thapsigargin for the indicated periods of time, and then cell lysates were analysed by western blot. Graph shown below indicates ratio of phAurA to total AurA. (b) AurA immunoprecipitation and in vitro kinase assay, after thapsigargin treatment. HEK293 cells overexpressing AurA were treated with 5 μM thapsigargin or control dimethylsulpfoxide (DMSO) for 5 min, AurA was immunoprecipitated and incubated with histone H3 substrate in an in vitro kinase assay. phHH3, antibody to phosphorylated histone H3. *P<0.05. (c) Left, an experiment similar to that shown in a, except that it is performed in the presence of 50 μM BAPTA-AM or control DMSO. Right, HEK293 cells overexpressing AurA were incubated with 20 μM BAPTA-AM (30 min) plus 7.5 mM EGTA (2 min) before the addition of 5 μM thapsigargin for the indicated periods of time, with analysis as for a. (d) An experiment similar to that shown in a, except that it is performed in the presence of 5 mM EGTA alone for the indicated periods of time (right panel) or in the presence of 5 mM EGTA (2 min) before the addition of 5 μM thapsigargin. (e) HEK293 cells overexpressing AurA were incubated with 5 μM thapsigargin and indicated concentration of ionomycin for the indicated periods of time, and then cell lysates were analysed by western blot using indicated antibodies. (f) HK2 cells were treated with DMSO, 5 μM thapsigargin (Tg) or 0.5 μM ionomycin (Ion), and then T288-phAurA levels were analysed by enzyme-linked immunosorbent assay (ELISA), based on A450 nm. To confirm the specificity of ELISA signal, a parallel group of HK2 cells were pre-treated with the AurA inhibitor PHA680632 (PHA) at 500 nM concentration. *P=0.05. Lysis buffer was used as a negative control. (g) AurA-overexpressing HEK293 cells were transfected with siRNA-depleting NEDD9 (siNEDD9) or scrambled control siRNA (scr), and then were incubated with 5-μm thapsigargin and processed as in a. For this and subsequent SDS–PAGE analyses, each experiment was performed three times independently, with error bars indicating the standard error. *P<0.05.

Mentions: The fact that rapid AurA activation occurred after stimulation of cells grown without extracellular Ca2+ implied that the elevated cytoplasmic Ca2+ levels resulting from ER release might regulate AurA activation without intrinsic requirement for second messengers transmitting signals from membrane-localized receptors. To further assess this mechanism, we treated AurA-transfected HEK293 cells with the Ca2+ uptake inhibitor thapsigargin, which blocks the recycling of cytoplasmic Ca2+ into ER stores32, thus increasing cytoplasmic Ca2+ levels without the involvement of upstream activators. Thapsigargin treatment caused a rapid (<1 min) increase in levels of activated T288-AurA, and increased the in vitro kinase activity of immunoprecipitated AurA against defined substrates (Fig. 2a,b).


Rapid calcium-dependent activation of Aurora-A kinase.

Plotnikova OV, Pugacheva EN, Dunbrack RL, Golemis EA - Nat Commun (2010)

Intracellular calcium release is sufficient for AurA activation.(a) HEK293 cells overexpressing AurA were incubated with 5 μM thapsigargin for the indicated periods of time, and then cell lysates were analysed by western blot. Graph shown below indicates ratio of phAurA to total AurA. (b) AurA immunoprecipitation and in vitro kinase assay, after thapsigargin treatment. HEK293 cells overexpressing AurA were treated with 5 μM thapsigargin or control dimethylsulpfoxide (DMSO) for 5 min, AurA was immunoprecipitated and incubated with histone H3 substrate in an in vitro kinase assay. phHH3, antibody to phosphorylated histone H3. *P<0.05. (c) Left, an experiment similar to that shown in a, except that it is performed in the presence of 50 μM BAPTA-AM or control DMSO. Right, HEK293 cells overexpressing AurA were incubated with 20 μM BAPTA-AM (30 min) plus 7.5 mM EGTA (2 min) before the addition of 5 μM thapsigargin for the indicated periods of time, with analysis as for a. (d) An experiment similar to that shown in a, except that it is performed in the presence of 5 mM EGTA alone for the indicated periods of time (right panel) or in the presence of 5 mM EGTA (2 min) before the addition of 5 μM thapsigargin. (e) HEK293 cells overexpressing AurA were incubated with 5 μM thapsigargin and indicated concentration of ionomycin for the indicated periods of time, and then cell lysates were analysed by western blot using indicated antibodies. (f) HK2 cells were treated with DMSO, 5 μM thapsigargin (Tg) or 0.5 μM ionomycin (Ion), and then T288-phAurA levels were analysed by enzyme-linked immunosorbent assay (ELISA), based on A450 nm. To confirm the specificity of ELISA signal, a parallel group of HK2 cells were pre-treated with the AurA inhibitor PHA680632 (PHA) at 500 nM concentration. *P=0.05. Lysis buffer was used as a negative control. (g) AurA-overexpressing HEK293 cells were transfected with siRNA-depleting NEDD9 (siNEDD9) or scrambled control siRNA (scr), and then were incubated with 5-μm thapsigargin and processed as in a. For this and subsequent SDS–PAGE analyses, each experiment was performed three times independently, with error bars indicating the standard error. *P<0.05.
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Related In: Results  -  Collection

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f2: Intracellular calcium release is sufficient for AurA activation.(a) HEK293 cells overexpressing AurA were incubated with 5 μM thapsigargin for the indicated periods of time, and then cell lysates were analysed by western blot. Graph shown below indicates ratio of phAurA to total AurA. (b) AurA immunoprecipitation and in vitro kinase assay, after thapsigargin treatment. HEK293 cells overexpressing AurA were treated with 5 μM thapsigargin or control dimethylsulpfoxide (DMSO) for 5 min, AurA was immunoprecipitated and incubated with histone H3 substrate in an in vitro kinase assay. phHH3, antibody to phosphorylated histone H3. *P<0.05. (c) Left, an experiment similar to that shown in a, except that it is performed in the presence of 50 μM BAPTA-AM or control DMSO. Right, HEK293 cells overexpressing AurA were incubated with 20 μM BAPTA-AM (30 min) plus 7.5 mM EGTA (2 min) before the addition of 5 μM thapsigargin for the indicated periods of time, with analysis as for a. (d) An experiment similar to that shown in a, except that it is performed in the presence of 5 mM EGTA alone for the indicated periods of time (right panel) or in the presence of 5 mM EGTA (2 min) before the addition of 5 μM thapsigargin. (e) HEK293 cells overexpressing AurA were incubated with 5 μM thapsigargin and indicated concentration of ionomycin for the indicated periods of time, and then cell lysates were analysed by western blot using indicated antibodies. (f) HK2 cells were treated with DMSO, 5 μM thapsigargin (Tg) or 0.5 μM ionomycin (Ion), and then T288-phAurA levels were analysed by enzyme-linked immunosorbent assay (ELISA), based on A450 nm. To confirm the specificity of ELISA signal, a parallel group of HK2 cells were pre-treated with the AurA inhibitor PHA680632 (PHA) at 500 nM concentration. *P=0.05. Lysis buffer was used as a negative control. (g) AurA-overexpressing HEK293 cells were transfected with siRNA-depleting NEDD9 (siNEDD9) or scrambled control siRNA (scr), and then were incubated with 5-μm thapsigargin and processed as in a. For this and subsequent SDS–PAGE analyses, each experiment was performed three times independently, with error bars indicating the standard error. *P<0.05.
Mentions: The fact that rapid AurA activation occurred after stimulation of cells grown without extracellular Ca2+ implied that the elevated cytoplasmic Ca2+ levels resulting from ER release might regulate AurA activation without intrinsic requirement for second messengers transmitting signals from membrane-localized receptors. To further assess this mechanism, we treated AurA-transfected HEK293 cells with the Ca2+ uptake inhibitor thapsigargin, which blocks the recycling of cytoplasmic Ca2+ into ER stores32, thus increasing cytoplasmic Ca2+ levels without the involvement of upstream activators. Thapsigargin treatment caused a rapid (<1 min) increase in levels of activated T288-AurA, and increased the in vitro kinase activity of immunoprecipitated AurA against defined substrates (Fig. 2a,b).

Bottom Line: This activation is mediated by direct Ca(2+)-dependent calmodulin (CaM) binding to multiple motifs on AurA.On the basis of structure-function analysis and molecular modelling, we map two primary regions of CaM-AurA interaction to unfolded sequences in the AurA N- and C-termini.This unexpected mechanism for AurA activation provides a new context for evaluating the function of AurA and its inhibitors in normal and cancerous cells.

View Article: PubMed Central - PubMed

Affiliation: Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, Pennsylvania 19111, USA.

ABSTRACT
Oncogenic hyperactivation of the mitotic kinase Aurora-A (AurA) in cancer is associated with genomic instability. Increasing evidence indicates that AurA also regulates critical processes in normal interphase cells, but the source of such activity has been obscure. We report here that multiple stimuli causing release of Ca(2+) from intracellular endoplasmic reticulum stores rapidly and transiently activate AurA, without requirement for second messengers. This activation is mediated by direct Ca(2+)-dependent calmodulin (CaM) binding to multiple motifs on AurA. On the basis of structure-function analysis and molecular modelling, we map two primary regions of CaM-AurA interaction to unfolded sequences in the AurA N- and C-termini. This unexpected mechanism for AurA activation provides a new context for evaluating the function of AurA and its inhibitors in normal and cancerous cells.

Show MeSH
Related in: MedlinePlus