PLK1 (polo like kinase 1) inhibits MTOR complex 1 and promotes autophagy.

Mechanistic target of rapamycin complex 1 (MTORC1) and polo like kinase 1 (PLK1) are major drivers of cancer cell growth and proliferation, and inhibitors of both protein kinases are currently being investigated in clinical studies. To date, MTORC1's and PLK1's functions are mostly studied separately, and reports on their mutual crosstalk are scarce. Here, we identify PLK1 as a physical MTORC1 interactor in human cancer cells. PLK1 inhibition enhances MTORC1 activity under nutrient sufficiency and in starved cells, and PLK1 directly phosphorylates the MTORC1 component RPTOR/RAPTOR in vitro. PLK1 and MTORC1 reside together at lysosomes, the subcellular site where MTORC1 is active. Consistent with an inhibitory role of PLK1 toward MTORC1, PLK1 overexpression inhibits lysosomal association of the PLK1-MTORC1 complex, whereas PLK1 inhibition promotes lysosomal localization of MTOR. PLK1-MTORC1 binding is enhanced by amino acid starvation, a condition known to increase autophagy. MTORC1 inhibition is an important step in autophagy activation. Consistently, PLK1 inhibition mitigates autophagy in cancer cells both under nutrient starvation and sufficiency, and a role of PLK1 in autophagy is also observed in the invertebrate model organism Caenorhabditis elegans. In summary, PLK1 inhibits MTORC1 and thereby positively contributes to autophagy. Since autophagy is increasingly recognized to contribute to tumor cell survival and growth, we propose that cautious monitoring of MTORC1 and autophagy readouts in clinical trials with PLK1 inhibitors is needed to develop strategies for optimized (combinatorial) cancer therapies targeting MTORC1, PLK1, and autophagy.

LC3 is a novel substrate for the mammalian Hippo kinases, STK3/STK4.

The Atg8 family protein LC3 is indispensible for autophagy and plays critical roles in multiple steps of the process. Despite this functional significance, the regulation of LC3 activity at the posttranslational level remains poorly understood. In a recent study, we report that the conserved Ste20 kinases STK3 and STK4, the mammalian orthologs of Hippo kinase, are essential for autophagy in diverse organisms, and both can phosphorylate LC3 on amino acid Thr50. STK3/STK4-mediated phosphorylation is critical for fusion of autophagosomes with lysosomes, as well as the ability of cells to clear intracellular bacteria, an established cargo for autophagy. Our discovery of a novel mode of autophagy regulation involving direct phosphorylation of LC3 by STK3/STK4 significantly enhances our molecular understanding of the autophagy process. Moreover, our findings raise the exciting possibility that STK3/STK4's known roles in immunity are exerted through their ability to regulate autophagy via LC3 phosphorylation.

Phosphorylation of LC3 by the Hippo kinases STK3/STK4 is essential for autophagy.

The protein LC3 is indispensible for the cellular recycling process of autophagy and plays critical roles during cargo recruitment, autophagosome biogenesis, and completion. Here, we report that LC3 is phosphorylated at threonine 50 (Thr(50)) by the mammalian Sterile-20 kinases STK3 and STK4. Loss of phosphorylation at this site blocks autophagy by impairing fusion of autophagosomes with lysosomes, and compromises the ability of cells to clear intracellular bacteria, an established cargo for autophagy. Strikingly, mutation of LC3 mimicking constitutive phosphorylation at Thr(50) reverses the autophagy block in STK3/STK4-deficient cells and restores their capacity to clear bacteria. Loss of STK3/STK4 impairs autophagy in diverse species, indicating that these kinases are conserved autophagy regulators. We conclude that phosphorylation of LC3 by STK3/STK4 is an essential step in the autophagy process. Since several pathological conditions, including bacterial infections, display aberrant autophagy, we propose that pharmacological agents targeting this regulatory circuit hold therapeutic potential.

Analysis of aging in Caenorhabditis elegans.

This chapter is dedicated to the study of aging in Caenorhabditis elegans (C. elegans). The assays are divided into two sections. In the first section, we describe detailed protocols for performing life span analysis in solid and liquid medium. In the second section, we describe various assays for measuring age-related changes. Our laboratory has been involved in several fruitful collaborations with non-C. elegans researchers keen on testing a role for their favorite gene in modulating aging (Carrano et al., 2009; Dong et al., 2007; Raices et al., 2008; Wolff et al., 2006). But even with the guidance of trained worm biologists, this undertaking can be daunting. We hope that this chapter will serve as a worthy compendium for those researchers who may or may not have immediate access to laboratories studying C. elegans.

Fine-tuning of Drp1/Fis1 availability by AKAP121/Siah2 regulates mitochondrial adaptation to hypoxia.

Defining the mechanisms underlying the control of mitochondrial fusion and fission is critical to understanding cellular adaptation to diverse physiological conditions. Here we demonstrate that hypoxia induces fission of mitochondrial membranes, dependent on availability of the mitochondrial scaffolding protein AKAP121. AKAP121 controls mitochondria dynamics through PKA-dependent inhibitory phosphorylation of Drp1 and PKA-independent inhibition of Drp1-Fis1 interaction. Reduced availability of AKAP121 by the ubiquitin ligase Siah2 relieves Drp1 inhibition by PKA and increases its interaction with Fis1, resulting in mitochondrial fission. High AKAP121 levels, seen in cells lacking Siah2, attenuate fission and reduce apoptosis of cardiomyocytes under simulated ischemia. Infarct size and degree of cell death were reduced in Siah2(-/-) mice subjected to myocardial infarction. Inhibition of Siah2 or Drp1 in hatching C. elegans reduces their life span. Through modulating Fis1/Drp1 complex availability, our studies identify Siah2 as a key regulator of hypoxia-induced mitochondrial fission and its physiological significance in ischemic injury and nematode life span.

Chromatin-bound p53 anchors activated Smads and the mSin3A corepressor to confer transforming-growth-factor-beta-mediated transcription repression.

In hepatic cells, Smad and SnoN proteins converge with p53 to repress transcription of AFP, an oncodevelopmental tumor marker aberrantly reactivated in hepatoma cells. Using p53- and SnoN-depleted hepatoma cell clones, we define a mechanism for repression mediated by this novel transcriptional partnership. We find that p53 anchors activated Smads and the corepressor mSin3A to the AFP distal promoter. Sequential chromatin immunoprecipitation analyses and molecular modeling indicate that p53 and Smad proteins simultaneously occupy overlapping p53 and Smad regulatory elements to establish repression of AFP transcription. In addition to its well-known function in antagonizing transforming growth factor beta (TGF-beta) responses, we find that SnoN actively participates in AFP repression by positively regulating mSin3A protein levels. We propose that activation of TGF-beta signaling restores a dynamic interplay between p53 and TGF-beta effectors that cooperate to effectively target mSin3A to tumor marker AFP and reestablish transcription repression.

A direct intersection between p53 and transforming growth factor beta pathways targets chromatin modification and transcription repression of the alpha-fetoprotein gene.

We purified the oncoprotein SnoN and found that it functions as a corepressor of the tumor suppressor p53 in the regulation of the hepatic alpha-fetoprotein (AFP) tumor marker gene. p53 promotes SnoN and histone deacetylase interaction at an overlapping Smad binding, p53 regulatory element (SBE/p53RE) in AFP. Comparison of wild-type and p53-null mouse liver tissue by using chromatin immunoprecipitation (ChIP) reveals that the absence of p53 protein correlates with the disappearance of SnoN at the SBE/p53RE and loss of AFP developmental repression. Treatment of AFP-expressing hepatoma cells with transforming growth factor-beta1 (TGF-beta1) induced SnoN transcription and Smad2 activation, concomitant with AFP repression. ChIP assays show that TGF-beta1 stimulates p53, Smad4, P-Smad2 binding, and histone H3K9 deacetylation and methylation, at the SBE/p53RE. Depletion, by small interfering RNA, of SnoN and/or p53 in hepatoma cells disrupted repression of AFP transcription. These findings support a model of cooperativity between p53 and TGF-beta effectors in chromatin modification and transcription repression of an oncodevelopmental tumor marker gene.