Stabilization of the metaphase spindle by Cdc14 is required for recombinational DNA repair.

Cells are constantly threatened by multiple sources of genotoxic stress that cause DNA damage. To maintain genome integrity, cells have developed a coordinated signalling network called DNA damage response (DDR). While multiple kinases have been thoroughly studied during DDR activation, the role of protein dephosphorylation in the damage response remains elusive. Here, we show that the phosphatase Cdc14 is essential to fulfil recombinational DNA repair in budding yeast. After DNA double-strand break (DSB) generation, Cdc14 is transiently released from the nucleolus and activated. In this state, Cdc14 targets the spindle pole body (SPB) component Spc110 to counterbalance its phosphorylation by cyclin-dependent kinase (Cdk). Alterations in the Cdk/Cdc14-dependent phosphorylation status of Spc110, or its inactivation during the induction of a DNA lesion, generate abnormal oscillatory SPB movements that disrupt DSB-SPB interactions. Remarkably, these defects impair DNA repair by homologous recombination indicating that SPB integrity is essential during the repair process. Together, these results show that Cdc14 promotes spindle stability and DSB-SPB tethering during DNA repair, and imply that metaphase spindle maintenance is a critical feature of the repair process.

Empirical inference of circuitry and plasticity in a kinase signaling network.

Our understanding of physiology and disease is hampered by the difficulty of measuring the circuitry and plasticity of signaling networks that regulate cell biology, and how these relate to phenotypes. Here, using mass spectrometry-based phosphoproteomics, we systematically characterized the topology of a network comprising the PI3K/Akt/mTOR and MEK/ERK signaling axes and confirmed its biological relevance by assessing its dynamics upon EGF and IGF1 stimulation. Measuring the activity of this network in models of acquired drug resistance revealed that cells chronically treated with PI3K or mTORC1/2 inhibitors differed in the way their networks were remodeled. Unexpectedly, we also observed a degree of heterogeneity in the network state between cells resistant to the same inhibitor, indicating that even identical and carefully controlled experimental conditions can give rise to the evolution of distinct kinase network statuses. These data suggest that the initial conditions of the system do not necessarily determine the mechanism by which cancer cells become resistant to PI3K/mTOR targeted therapies. The patterns of signaling network activity observed in the resistant cells mirrored the patterns of response to several drug combination treatments, suggesting that the activity of the defined signaling network truly reflected the evolved phenotypic diversity.

Calpain interacts with class IA phosphoinositide 3-kinases regulating their stability and signaling activity.

Class IA phosphoinositide 3-kinases (PI3Ks) are signaling enzymes with key roles in the regulation of essential cellular functions and disease, including cancer. Accordingly, their activity is tightly controlled in cells to maintain homeostasis. The formation of multiprotein complexes is a ubiquitous mechanism to regulate enzyme activity but the contribution of protein-protein interactions to the regulation of PI3K signaling is not fully understood. We designed an affinity purification quantitative mass spectrometry strategy to identify proteins interacting dynamically with PI3K in response to pathway activation, with the view that such binding partners may have a functional role in pathway regulation. Our study reveals that calpain small subunit 1 interacts with PI3K and that the association between these proteins is lower in cells stimulated with serum compared to starved cells. Calpain and PI3K activity assays confirmed these results, thus demonstrating that active calpain heterodimers associate dynamically with PI3K. In addition, calpains were found to cleave PI3K proteins in vitro (resulting in a reduction of PI3K lipid kinase activity) and to regulate endogenous PI3K protein levels in vivo. Further investigations revealed that calpains have a role in the negative regulation of PI3K/Akt pathway activity (as measured by Akt and ribosomal S6 phosphorylation) and that their inhibition promotes cell survival during serum starvation. These results indicate that the interaction between calpain and PI3K is a novel mechanism for the regulation of class IA PI3K stability and activity.

Withanolide Metabolites Inhibit PI3K/AKT and MAPK Pro-Survival Pathways and Induce Apoptosis in Acute Myeloid Leukemia Cells.

Acute myeloid leukemia (AML) is an aggressive disease and, despite advances, its treatment remains challenging. Therefore, it remains important to identify new agents for the management of this disease. Withanolides, a group of steroidal lactones found in Solanaceae plants are of potential interest due to their reported anticancer activities in different settings. In this study we investigated the anti-proliferative effects and mode of action of Solanaceae-derived withanolides in AML cell models; these metabolites include withametelin (WTH) and Coagulansin A (CoA) isolated from Datura innoxia and Withania coagluanse, respectively. Both withanolides inhibited the proliferation of AML cells and induced cell death, with WTH being more potent than CoA in the AML models tested. Quantitative label-free proteomics and phosphoproteomics were employed to define the mechanism of action of the studied withanolides. We identified and quantified 5269 proteins and 17,482 phosphosites in cells treated with WTH, CoA or vehicle control. Withanolides modulated the expression of proteins involved in regulating key cellular processes including cell cycle, metabolism, signaling, protein degradation and gene expression. Enrichment analysis of the phosphoproteomics data against kinase substrates, kinase-kinase relationships and canonical pathways showed that the withanolides decreased the activity of kinases such as phosphoinositide 3-kinase (PI3K), protein kinase B (PKB; also known as RAC-alpha serine/threonine-protein kinase or AKT), mammalian target of rapamycin (mTOR), extracellular signal-regulated protein kinase 1 and 2 (ERK1/2) and the serine/threonine-protein kinase A-Raf (ARAF), while increasing the activation of DNA repair kinases. These results indicate that withanolide metabolites have pleiotropic effects in the modulation of oncogenic pro-survival and pro-apoptotic signaling pathways that regulate the induction of apoptosis. Withanolide mediated apoptosis was confirmed by immunoblotting showing increased expression of cleaved PARP and Caspases 3, 8 and 9 as a result of treatment. Overall, our results suggest that WTH and CoA have therapeutic potential against AML with WTH exhibiting more potent effects and should be explored further.