cAMP/PKA signalling reinforces the LATS–YAP pathway to fully suppress YAP in response to actin cytoskeletal changes

Actin cytoskeletal damage induces inactivation of the oncoprotein YAP (Yes‐associated protein). It is known that the serine/threonine kinase LATS (large tumour suppressor) inactivates YAP by phosphorylating its Ser127 and Ser381 residues. However, the events downstream of actin cytoskeletal changes that are involved in the regulation of the LATS–YAP pathway and the mechanism by which LATS differentially phosphorylates YAP on Ser127 and Ser381 in vivo have remained elusive. Here, we show that cyclic AMP (cAMP)‐dependent protein kinase (PKA) phosphorylates LATS and thereby enhances its activity sufficiently to phosphorylate YAP on Ser381. We also found that PKA activity is involved in all contexts previously reported to trigger the LATS–YAP pathway, including actin cytoskeletal damage, G‐protein‐coupled receptor activation, and engagement of the Hippo pathway. Inhibition of PKA and overexpression of YAP cooperate to transform normal cells and amplify neural progenitor pools in developing chick embryos. We also implicate neurofibromin 2 as an AKAP (A‐kinase‐anchoring protein) scaffold protein that facilitates the function of the cAMP/PKA–LATS–YAP pathway. Our study thus incorporates PKA as novel component of the Hippo pathway.     

14‐3‐3γ mediates Cdc25A proteolysis to block premature mitotic entry after DNA damage

14‐3‐3 proteins control various cellular processes, including cell cycle progression and DNA damage checkpoint. At the DNA damage checkpoint, some subtypes of 14‐3‐3 (β and ζ isoforms in mammalian cells and Rad24 in fission yeast) bind to Ser345‐phosphorylated Chk1 and promote its nuclear retention. Here, we report that 14‐3‐3γ forms a complex with Chk1 phosphorylated at Ser296, but not at ATR sites (Ser317 and Ser345). Ser296 phosphorylation is catalysed by Chk1 itself after Chk1 phosphorylation by ATR, and then ATR sites are rapidly dephosphorylated on Ser296‐phosphorylated Chk1. Although Ser345 phosphorylation is observed at nuclear DNA damage foci, it occurs more diffusely in the nucleus. The replacement of endogenous Chk1 with Chk1 mutated at Ser296 to Ala induces premature mitotic entry after ultraviolet irradiation, suggesting the importance of Ser296 phosphorylation in the DNA damage response. Although Ser296 phosphorylation induces the only marginal change in Chk1 catalytic activity, 14‐3‐3γ mediates the interaction between Chk1 and Cdc25A. This ternary complex formation has an essential function in Cdc25A phosphorylation and degradation to block premature mitotic entry after DNA damage.     

YAP‐TEAD signaling promotes basal cell carcinoma development via a c‐JUN/AP1 axis

The mammalian Hippo signaling pathway, through its effectors YAP and TAZ, coerces epithelial progenitor cell expansion for appropriate tissue development or regeneration upon damage. Its ability to drive rapid tissue growth explains why many oncogenic events frequently exploit this pathway to promote cancer phenotypes. Indeed, several tumor types including basal cell carcinoma (BCC) show genetic aberrations in the Hippo (or YAP/TAZ) regulators. Here, we uncover that while YAP is dispensable for homeostatic epidermal regeneration, it is required for BCC development. Our clonal analyses further demonstrate that the few emerging Yap‐null dysplasia have lower fitness and thus are diminished as they progress to invasive BCC. Mechanistically, YAP depletion in BCC tumors leads to effective impairment of the JNK‐JUN signaling, a well‐established tumor‐driving cascade. Importantly, in this context, YAP does not influence canonical Wnt or Hedgehog signaling. Overall, we reveal Hippo signaling as an independent promoter of BCC pathogenesis and thereby a viable target for drug‐resistant BCC.     

NDR1/STK38 potentiates NF‐κB activation by its kinase activity

Human NDR1/STK38 belongs to the nuclear‐Dbf2‐related (NDR) family of Ser/Thr kinases. It has been implicated to function in centrosome duplication, control of cell cycle and apoptosis. However, the mechanism of NDR1 signaling pathway remains largely elusive. Here, we report a novel role of NDR1 in NF‐κB activation. By overexpression, NDR1 potentiates NF‐κB activation induced by TNFα, whereas knockdown of NDR1 expression inhibits NF‐κB activation induced by TNFα. Coimmunoprecipitation shows that NDR1 interacts with multiple signal components except p65 in NF‐κB signaling pathway. Furthermore, both phosphorylation and kinase dead mutants of NDR1 lose their synergistic effects on TNFα‐induced NF‐κB activation. siRNA oligo against NDR1 and kinase dead mutant as well mainly block the NF‐κB activation induced by TRAF2 but not RIP1. Furthermore, kinase dead mutant of NDR1 fails to interact with TRAF2. Taken together, our findings suggest an unknown function of NDR1, which may regulate NF‐κB activation by its kinase activity. Copyright © 2012 John Wiley & Sons, Ltd.     

The truncated NLR protein TIR‐NBS13 is a MOS6/IMPORTIN‐α3 interaction partner required for plant immunity

Importin‐α proteins mediate the translocation of nuclear localization signal (NLS)‐containing proteins from the cytoplasm into the nucleus through nuclear pore complexes (NPCs). Genetically, Arabidopsis IMPORTIN‐α3/MOS6 (MODIFIER OF SNC1, 6) is required for basal plant immunity and constitutive disease resistance activated in the autoimmune mutant snc1 (suppressor of npr1‐1, constitutive 1), suggesting that MOS6 plays a role in the nuclear import of proteins involved in plant defense signaling. Here, we sought to identify and characterize defense‐regulatory cargo proteins and interaction partners of MOS6. We conducted both in silico database analyses and affinity purification of functional epitope‐tagged MOS6 from pathogen‐challenged stable transgenic plants coupled with mass spectrometry. We show that among the 13 candidate MOS6 interactors we selected for further functional characterization, the TIR‐NBS‐type protein TN13 is required for resistance against Pseudomonas syringae pv. tomato (Pst) DC3000 lacking the type‐III effector proteins AvrPto and AvrPtoB. When expressed transiently in N. benthamiana leaves, TN13 co‐immunoprecipitates with MOS6, but not with its closest homolog IMPORTIN‐α6, and localizes to the endoplasmic reticulum (ER), consistent with a predicted N‐terminal transmembrane domain in TN13. Our work uncovered the truncated NLR protein TN13 as a component of plant innate immunity that selectively binds to MOS6/IMPORTIN‐α3 in planta. We speculate that the release of TN13 from the ER membrane in response to pathogen stimulus, and its subsequent nuclear translocation, is important for plant defense signal transduction.     

Light modulated activity of root alkaline/neutral invertase involves the interaction with 14‐3‐3 proteins

Alkaline/neutral invertases (A/N‐Invs) are now recognized as essential proteins in plant life. They catalyze the irreversible breakdown of sucrose into glucose and fructose and thus supply the cells with energy as well as signaling molecules. In this study we report on a mechanism that affects the activity of the cytosolic invertase AtCINV1 (At‐A/N‐InvG or AT1G35580). We demonstrate that Ser547 at the extreme C‐terminus of the AtCINV1 protein is a substrate of calcium‐dependent kinases (CPK3 and 21) and that phosphorylation creates a high‐affinity binding site for 14‐3‐3 proteins. The invertase as such has basal activity, but we provide evidence that interaction with 14‐3‐3 proteins enhances its activity. The analysis of three quadruple 14‐3‐3 mutants generated from six T‐DNA insertion mutants of the non‐epsilon family shows both specificity as well as redundancy for this function of 14‐3‐3 proteins. The strong reduction in hexose levels in the roots of one 14‐3‐3 quadruple mutant plant is in line with the activating function of 14‐3‐3 proteins. The physiological relevance of this mechanism that affects A/N‐invertase activity is underscored by the light‐induced activation and is another example of the central role of 14‐3‐3 proteins in mediating dark/light signaling. The nature of the light‐induced signal that travels from the shoot to root and the question whether this signal is transmitted via cytosolic Ca⁺⁺ changes that activate calcium‐dependent kinases, await further study.     

A 14‐3‐3 Mode‐1 Binding Motif Initiates Gap Junction Internalization During Acute Cardiac Ischemia

Altered phosphorylation and trafficking of connexin 43 (Cx43) during acute ischemia contributes to arrhythmogenic gap junction remodeling, yet the critical sequence and accessory proteins necessary for Cx43 internalization remain unresolved. 14‐3‐3 proteins can regulate protein trafficking, and a 14‐3‐3 mode‐1 binding motif is activated upon phosphorylation of Ser373 of the Cx43 C‐terminus. We hypothesized that Cx43^Ser373 phosphorylation is important to pathological gap junction remodeling. Immunofluorescence in human heart reveals the enrichment of 14‐3‐3 proteins at intercalated discs, suggesting interaction with gap junctions. Knockdown of 14‐3‐3τ in cell lines increases gap junction plaque size at cell–cell borders. Cx43^S373A mutation prevents Cx43/14‐3‐3 complexing and stabilizes Cx43 at the cell surface, indicating avoidance of degradation. Using Langendorff‐perfused mouse hearts, we detect phosphorylation of newly internalized Cx43 at Ser373 and Ser368 within 30 min of no‐flow ischemia. Phosphorylation of Cx43 at Ser368 by protein kinase C and Ser255 by mitogen‐activated protein kinase has previously been implicated in Cx43 internalization. The Cx43^S373A mutant is resistant to phosphorylation at both these residues and does not undergo ubiquitination, revealing Ser373 phosphorylation as an upstream gatekeeper of a posttranslational modification cascade necessary for Cx43 internalization. Cx43^Ser373 phosphorylation is a potent target for therapeutic interventions to preserve gap junction coupling in the stressed myocardium.      

Theaflavin‐3,3´‐digallate increases the antibacterial activity of β‐lactam antibiotics by inhibiting metallo‐β‐lactamase activity

Metallo‐β‐lactamases (MBLs) are some of the best known β‐lactamases produced by common Gram‐positive and Gram‐negative pathogens and are crucial factors in the rise of bacterial resistance against β‐lactam antibiotics. Although many types of β‐lactamase inhibitors have been successfully developed and used in clinical settings, no MBL inhibitors have been identified to date. Nitrocefin, checkerboard and time‐kill assays were used to examine the enzyme behaviour in vitro. Molecular docking calculation, molecular dynamics simulation, calculation of the binding free energy and ligand‐residue interaction decomposition were used for mechanistic research. The behaviour of the enzymes in vivo was investigated by a mouse infection experiment. We showed that theaflavin‐3,3´‐digallate (TFDG), a natural compound lacking antibacterial activities, can inhibit the hydrolysis of MBLs. In the checkerboard and time‐kill assays, we observed a synergistic effect of TFDG with β‐lactam antibiotics against methicillin‐resistant Staphylococcus aureus BAA1717. Molecular dynamics simulations were used to identify the mechanism of the inhibition of MBLs by TFDG, and we observed that the hydrolysis activity of the MBLs was restricted by the binding of TFDG to Gln242 and Ser369. Furthermore, the combination of TFDG with β‐lactam antibiotics showed effective protection in a mouse Staphylococcus aureus pneumonia model. These findings suggest that TFDG can effectively inhibit the hydrolysis activity of MBLs and enhance the antibacterial activity of β‐lactam antibiotics against pathogens in vitro and in vivo.     

β‐Guanidinopropionic acid extends the lifespan of Drosophila melanogaster via an AMP‐activated protein kinase‐dependent increase in autophagy

Previous studies have demonstrated that AMP‐activated protein kinase (AMPK) controls autophagy through the mammalian target of rapamycin (mTOR) and Unc‐51 like kinase 1 (ULK1/Atg1) signaling, which augments the quality of cellular housekeeping, and that β‐guanidinopropionic acid (β‐GPA), a creatine analog, leads to a chronic activation of AMPK. However, the relationship between β‐GPA and aging remains elusive. In this study, we hypothesized that feeding β‐GPA to adult Drosophila produces the lifespan extension via activation of AMPK‐dependent autophagy. It was found that dietary administration of β‐GPA at a concentration higher than 900 mm induced a significant extension of the lifespan of Drosophila melanogaster in repeated experiments. Furthermore, we found that Atg8 protein, the homolog of microtubule‐associated protein 1A/1B‐light chain 3 (LC3) and a biomarker of autophagy in Drosophila, was significantly upregulated by β‐GPA treatment, indicating that autophagic activity plays a role in the effect of β‐GPA. On the other hand, when the expression of Atg5 protein, an essential protein for autophagy, was reduced by RNA interference (RNAi), the effect of β‐GPA on lifespan extension was abolished. Moreover, we found that AMPK was also involved in this process. β‐GPA treatment significantly elevated the expression of phospho‐T172‐AMPK levels, while inhibition of AMPK by either AMPK‐RNAi or compound C significantly attenuated the expression of autophagy‐related proteins and lifespan extension in Drosophila. Taken together, our results suggest that β‐GPA can induce an extension of the lifespan of Drosophila via AMPK‐Atg1‐autophagy signaling pathway.