Polo kinase at the mitochondria

Sci. Signal.  26 Apr 2016:
Vol. 9, Issue 425, pp. ec98
DOI: 10.1126/scisignal.aaf9390

Mitochondria require the proper balance of Ca2+ for metabolic function and also help maintain cellular Ca2+ homeostasis and limit Ca2+ cytotoxicity. As Shanmughapriya et al. showed in cultured cells, there is a complex interaction between cellular Ca2+ signals, expression of the gene encoding the mitochondrial Ca2+ uniporter complex MCU, and mitochondrial metabolism. This week, Lee et al. identify the Polo-like kinase (PLK) as a key regulator of the mitochondrial guanosine triphosphatase (GTPase) Miro in controlling mitochondrial Ca2+ handling and consequently function in Drosophila neural stem cells. Both a loss-of-function (LOF) mutation in dMiro and gain of function (GOF) by overexpression reduced the number of neural stem cells in the Drosophila brain in a cell-intrinsic manner. Although Miro is best known for linking mitochondria to microtubules and thus controlling mitochondrial distribution and motility, the phenotypes associated with LOF or GOF of dMiro were not recapitulated by altering the function of the Miro partner that is involved in mitochondrial motility, suggesting a different role for Miro in neural stem cells. Instead, experiments with fly brains with cells expressing genetically encoded mitochondrial calcium reporters showed that dMiro GOF increased both the basal mitochondrial Ca2+ concentration and the mitochondrial Ca2+ concentration resulting from uptake after histamine-stimulated release from the endoplasmic reticulum (ER), whereas LOF reduced the mitochondrial Ca2+ concentration. Consistent with these differences in mitochondrial Ca2+ concentration, the GOF clones in the brain had increased mitochondrial reactive oxygen species (ROS) and increased markers of apoptosis, indicating Ca2+ toxicity due to overload. In contrast, brains of the LOF mutant had reduced ATP, and LOF clones in the brain had increased abundance of the phosphorylated and inhibited form of pyruvate dehydrogenase, indicating reduced mitochondrial metabolic activity due to insufficient Ca2+. Bioinformatic analysis revealed putative PLK phosphorylation sites in the N-terminal GTPase domain of Miro that are conserved from fly to human. In vitro kinase assays and mutational analysis showed that Ser66 was the site that was predominantly phosphorylated by PLK1 and that PLK-mediated phosphorylation increased the GTPase activity of Miro. Expression of the S66A mutant failed to rescue the loss of neural stem cell phenotype observed with the LOF mutant, and overexpression of the S66A mutant did not cause increased mitochondrial ROS, indicating that this mutant did not increase mitochondrial Ca2+ uptake. Expression of constitutively active Polo (the fly PLK homolog) reduced neural stem cell numbers, which was rescued by either knockdown of dMiro, expression of the S66A mutant, or knockdown of genes encoding proteins involved in ER-mitochondrial Ca2+ transfer. Whereas total Miro was uniformly distributed along mitochondria, Miro phosphorylated at Ser66 exhibited a punctate pattern that overlapped with components of the ER-mitochondrial contact site (ERMCS). Consistent with a regulatory role for phosphorylation of Miro in the stability of the ERMCS, expression of Miro-S66A in flies reduced the coimmunoprecipitation of an ER component and a mitochondrial component of the ERMCS, whereas expression of a phosphomimetic mutant of Miro enhanced this interaction. Thus, PLK-mediated phosphorylation of Miro contributes to the stabilization of the ERMCS, which enhances ER-mitochondrial Ca2+ transfer.

S. Shanmughapriya, S. Rajan, N. E. Hoffman, X. Zhang, S. Guo, J. E. Kolesar, K. J. Hines, J. Ragheb, N. R. Jog, R. Caricchio, Y. Baba, Y. Zhou, B. A. Kaufman, J. Y. Cheung, T. Kurosaki, D. L. Gill, M. Madesh, Ca2+ signals regulate mitochondrial metabolism by stimulating CREB-mediated expression of the mitochondrial Ca2+ uniporter gene MCU. Sci. Signal. 8, ra23 (2015). [Abstract]

S. Lee, K.-S. Lee, S. Huh, S. Liu, D.-Y. Lee, S. H. Hong, K. Yu, B. Lu, Polo kinase phosphorylates Miro to control ER-mitochondria contact sites and mitochondrial Ca2+ homeostasis in neural stem cell development. Dev. Cell 37, 174–189 (2016). [PubMed]