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CaM kinase I alpha-induced phosphorylation of Drp1 regulates mitochondrial morphology.

Han XJ, Lu YF, Li SA, Kaitsuka T, Sato Y, Tomizawa K, Nairn AC, Takei K, Matsui H, Matsushita M - J. Cell Biol. (2008)

Bottom Line: VDCC-associated Ca2+ signaling stimulates phosphorylation of dynamin-related protein 1 (Drp1) at serine 600 via activation of Ca2+/calmodulin-dependent protein kinase Ialpha (CaMKIalpha).In neurons and HeLa cells, phosphorylation of Drp1 at serine 600 is associated with an increase in Drp1 translocation to mitochondria, whereas in vitro, phosphorylation of Drp1 results in an increase in its affinity for Fis1.CaMKIalpha is a widely expressed protein kinase, suggesting that Ca2+ is likely to be functionally important in the control of mitochondrial dynamics through regulation of Drp1 phosphorylation in neurons and other cell types.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and 2Department of Neuroscience, Okayama University Graduate School of Medicine and Dentistry, Okayama 700-8558, Japan.

ABSTRACT
Mitochondria are dynamic organelles that frequently move, divide, and fuse with one another to maintain their architecture and functions. However, the signaling mechanisms involved in these processes are still not well characterized. In this study, we analyze mitochondrial dynamics and morphology in neurons. Using time-lapse imaging, we find that Ca2+ influx through voltage-dependent Ca2+ channels (VDCCs) causes a rapid halt in mitochondrial movement and induces mitochondrial fission. VDCC-associated Ca2+ signaling stimulates phosphorylation of dynamin-related protein 1 (Drp1) at serine 600 via activation of Ca2+/calmodulin-dependent protein kinase Ialpha (CaMKIalpha). In neurons and HeLa cells, phosphorylation of Drp1 at serine 600 is associated with an increase in Drp1 translocation to mitochondria, whereas in vitro, phosphorylation of Drp1 results in an increase in its affinity for Fis1. CaMKIalpha is a widely expressed protein kinase, suggesting that Ca2+ is likely to be functionally important in the control of mitochondrial dynamics through regulation of Drp1 phosphorylation in neurons and other cell types.

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Phosphorylation regulates Drp1 and hFis1 binding in vitro. (A) Recombinant Drp1-6His was preincubated in the absence or presence of CaMKIα (KI) and ATP and mixed with GST-hFis1 as indicated. Proteins were recovered using glutathione-Sepharose and analyzed by immunoblotting using an anti-6His antibody to detect Drp1 (top; bottom shows Drp1 input). Quantitation of the results is shown in the bar graph. The results were otained in three independent experiments. Error bars indicate SEM in each group. (B) Drp1-6His was maximally phosphorylated by CaMKI, and the sample was mixed in different ratios with nonphosphorylated Drp1-6His (values are expressed as percent P-Drp1/Drp1; see x axis). GST pull-down assays were performed as in A. The data represent means from three independent experiments. (C) The binding of mutant Drp1-S600A-6His to GST-hFis1 was compared with unphosphorylated and phosphorylated wild-type Drp1-6His. Drp1 was detected by immunoblotting as described above. Similar results were obtained from two independent experiments.
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fig9: Phosphorylation regulates Drp1 and hFis1 binding in vitro. (A) Recombinant Drp1-6His was preincubated in the absence or presence of CaMKIα (KI) and ATP and mixed with GST-hFis1 as indicated. Proteins were recovered using glutathione-Sepharose and analyzed by immunoblotting using an anti-6His antibody to detect Drp1 (top; bottom shows Drp1 input). Quantitation of the results is shown in the bar graph. The results were otained in three independent experiments. Error bars indicate SEM in each group. (B) Drp1-6His was maximally phosphorylated by CaMKI, and the sample was mixed in different ratios with nonphosphorylated Drp1-6His (values are expressed as percent P-Drp1/Drp1; see x axis). GST pull-down assays were performed as in A. The data represent means from three independent experiments. (C) The binding of mutant Drp1-S600A-6His to GST-hFis1 was compared with unphosphorylated and phosphorylated wild-type Drp1-6His. Drp1 was detected by immunoblotting as described above. Similar results were obtained from two independent experiments.

Mentions: In mammalian cells, Drp1 has been shown to regulate mitochondrial fission through an interaction with Fis1 (Yoon et al., 2003). Therefore, we examined in vitro whether CaMKIα-dependent phosphorylation of Drp1 could facilitate the interaction of Drp1 with hFis1. In the absence of prior phosphorylation by CaMKIα, a small amount of Drp1-6His was pulled down by GST-hFis1. Phosphorylation of wild-type Drp1 by CaMKIα resulted in a substantial increase in the amount of Drp1-6His pulled down by GST-hFis1 (Fig. 9 A). The amount of Drp1 bound to GST-Fis1 increased in proportion to the level of phosphorylation of Drp1 (Fig. 9 B). In contrast to wild-type Drp1, there was little measurable binding found between Drp1-S600A and GST-Fis1 (Fig. 9 C). Finally, after down-regulation of Fis1 with siRNA in HeLa cells, the effect of high K+ on the punctate distribution of YFP-Drp1 was reduced (Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200802164/DC1).


CaM kinase I alpha-induced phosphorylation of Drp1 regulates mitochondrial morphology.

Han XJ, Lu YF, Li SA, Kaitsuka T, Sato Y, Tomizawa K, Nairn AC, Takei K, Matsui H, Matsushita M - J. Cell Biol. (2008)

Phosphorylation regulates Drp1 and hFis1 binding in vitro. (A) Recombinant Drp1-6His was preincubated in the absence or presence of CaMKIα (KI) and ATP and mixed with GST-hFis1 as indicated. Proteins were recovered using glutathione-Sepharose and analyzed by immunoblotting using an anti-6His antibody to detect Drp1 (top; bottom shows Drp1 input). Quantitation of the results is shown in the bar graph. The results were otained in three independent experiments. Error bars indicate SEM in each group. (B) Drp1-6His was maximally phosphorylated by CaMKI, and the sample was mixed in different ratios with nonphosphorylated Drp1-6His (values are expressed as percent P-Drp1/Drp1; see x axis). GST pull-down assays were performed as in A. The data represent means from three independent experiments. (C) The binding of mutant Drp1-S600A-6His to GST-hFis1 was compared with unphosphorylated and phosphorylated wild-type Drp1-6His. Drp1 was detected by immunoblotting as described above. Similar results were obtained from two independent experiments.
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Related In: Results  -  Collection

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fig9: Phosphorylation regulates Drp1 and hFis1 binding in vitro. (A) Recombinant Drp1-6His was preincubated in the absence or presence of CaMKIα (KI) and ATP and mixed with GST-hFis1 as indicated. Proteins were recovered using glutathione-Sepharose and analyzed by immunoblotting using an anti-6His antibody to detect Drp1 (top; bottom shows Drp1 input). Quantitation of the results is shown in the bar graph. The results were otained in three independent experiments. Error bars indicate SEM in each group. (B) Drp1-6His was maximally phosphorylated by CaMKI, and the sample was mixed in different ratios with nonphosphorylated Drp1-6His (values are expressed as percent P-Drp1/Drp1; see x axis). GST pull-down assays were performed as in A. The data represent means from three independent experiments. (C) The binding of mutant Drp1-S600A-6His to GST-hFis1 was compared with unphosphorylated and phosphorylated wild-type Drp1-6His. Drp1 was detected by immunoblotting as described above. Similar results were obtained from two independent experiments.
Mentions: In mammalian cells, Drp1 has been shown to regulate mitochondrial fission through an interaction with Fis1 (Yoon et al., 2003). Therefore, we examined in vitro whether CaMKIα-dependent phosphorylation of Drp1 could facilitate the interaction of Drp1 with hFis1. In the absence of prior phosphorylation by CaMKIα, a small amount of Drp1-6His was pulled down by GST-hFis1. Phosphorylation of wild-type Drp1 by CaMKIα resulted in a substantial increase in the amount of Drp1-6His pulled down by GST-hFis1 (Fig. 9 A). The amount of Drp1 bound to GST-Fis1 increased in proportion to the level of phosphorylation of Drp1 (Fig. 9 B). In contrast to wild-type Drp1, there was little measurable binding found between Drp1-S600A and GST-Fis1 (Fig. 9 C). Finally, after down-regulation of Fis1 with siRNA in HeLa cells, the effect of high K+ on the punctate distribution of YFP-Drp1 was reduced (Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200802164/DC1).

Bottom Line: VDCC-associated Ca2+ signaling stimulates phosphorylation of dynamin-related protein 1 (Drp1) at serine 600 via activation of Ca2+/calmodulin-dependent protein kinase Ialpha (CaMKIalpha).In neurons and HeLa cells, phosphorylation of Drp1 at serine 600 is associated with an increase in Drp1 translocation to mitochondria, whereas in vitro, phosphorylation of Drp1 results in an increase in its affinity for Fis1.CaMKIalpha is a widely expressed protein kinase, suggesting that Ca2+ is likely to be functionally important in the control of mitochondrial dynamics through regulation of Drp1 phosphorylation in neurons and other cell types.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and 2Department of Neuroscience, Okayama University Graduate School of Medicine and Dentistry, Okayama 700-8558, Japan.

ABSTRACT
Mitochondria are dynamic organelles that frequently move, divide, and fuse with one another to maintain their architecture and functions. However, the signaling mechanisms involved in these processes are still not well characterized. In this study, we analyze mitochondrial dynamics and morphology in neurons. Using time-lapse imaging, we find that Ca2+ influx through voltage-dependent Ca2+ channels (VDCCs) causes a rapid halt in mitochondrial movement and induces mitochondrial fission. VDCC-associated Ca2+ signaling stimulates phosphorylation of dynamin-related protein 1 (Drp1) at serine 600 via activation of Ca2+/calmodulin-dependent protein kinase Ialpha (CaMKIalpha). In neurons and HeLa cells, phosphorylation of Drp1 at serine 600 is associated with an increase in Drp1 translocation to mitochondria, whereas in vitro, phosphorylation of Drp1 results in an increase in its affinity for Fis1. CaMKIalpha is a widely expressed protein kinase, suggesting that Ca2+ is likely to be functionally important in the control of mitochondrial dynamics through regulation of Drp1 phosphorylation in neurons and other cell types.

Show MeSH
Related in: MedlinePlus