Dysfunctional mechanotransduction through the YAP/TAZ/Hippo pathway as a feature of chronic disease

In order to ascertain their external environment, cells and tissues have the capability to sense and process a variety of stresses, including stretching and compression forces. These mechanical forces, as experienced by cells and tissues, are then converted into biochemical signals within the cell, leading to a number of cellular mechanisms being activated, including proliferation, differentiation and migration. If the conversion of mechanical cues into biochemical signals is perturbed in any way, then this can be potentially implicated in chronic disease development and processes such as neurological disorders, cancer and obesity. This review will focus on how the interplay between mechanotransduction, cellular structure, metabolism and signalling cascades led by the Hippo-YAP/TAZ axis can lead to a number of chronic diseases and suggest how we can target various pathways in order to design therapeutic targets for these debilitating diseases and conditions

A distinctive requirement for p53 in the genome protective Topoisomerase 2a-dependent G2 arrest in hTERT positive cancer cells

Topoisomerase 2a (Topo2a)-dependent G2 arrest engenders faithful segregation of sister chromatids, yet in certain tumor cell lines where this arrest is dysfunctional, a PKCε-dependent failsafe pathway can be triggered. Here we elaborate on recent advances in understanding the underlying mechanisms associated with this G2 arrest by determining that p53-p21 signaling is essential for efficient arrest in cell lines, in patient-derived cells, and in colorectal cancer organoids. Regulation of this p53 axis required the SMC5/6 complex, which is distinct from the p53 pathways observed in the DNA damage response. Topo2a inhibition specifically during S phase did not trigger G2 arrest despite affecting completion of DNA replication. Moreover, in cancer cells reliant upon the alternative lengthening of telomeres (ALT) mechanism, a distinct form of Topo2a-dependent, p53-independent G2 arrest was found to be mediated by BLM and Chk1. Importantly, the previously described PKCε-dependent mitotic failsafe was engaged in hTERT-positive cells when Topo2a-dependent G2 arrest was dysfunctional and where p53 was absent, but not in cells dependent on the ALT mechanism. In PKCε knockout mice, p53 deletion elicited tumors were less aggressive than in PKCε-replete animals and exhibited a distinct pattern of chromosomal rearrangements. This evidence suggests the potential of exploiting synthetic lethality in arrest-defective hTERT-positive tumors through PKCε-directed therapeutic intervention.SignificanceThe identification of a requirement for p53 in stringent Topo2a-dependent G2 arrest and engagement of PKCε failsafe pathways in arrest-defective hTERT-positive cells provides a therapeutic opportunity to induce selective synthetic lethality

Nieuwe inzichten in de farmacologische inhibitie en isovormspecifieke regulatie van Proteine Kinase D2

Protein kinases are essential to life. They catalyze the phosphorylation of their substrates, which in turn alters their function. Expectedly, kinase dysregulation has been associated with multiple disorders, including cancer, and they are often targets for small-molecule inhibition. In this manuscript we studied the Protein Kinase D (PKD) family. This family belongs to the Calmodulin kinase (CAMK) group and consists of three isoenzymes in humans. While the isoenzymes are structurally highly similar, they are not always functionally redundant, and sometimes the isoenzymes even have opposing roles. The molecular basis for these isoenzyme-specific functions is not well understood. PKDs are classically activated via signaling pathways that generate diacylglycerol downstream of G-protein coupled receptors or receptor tyrosine kinases, but they are also activated in oxidative stress conditions. Remarkably, we uncovered an isoenzyme-specific regulation of PKD2 in oxidative stress through the Abl-mediated phosphorylation of a highly conserved Tyr residue in the P+1 loop (i.e. the substrate binding loop) of the activation segment. Phosphorylation specifically occurs in PKD2 due to an Abl recognition motif that is divergent between PKD1/2/3. Phosphorylation of this Tyr residue results in higher substrate turnover by PKD2. We also observed isoform-specific signaling to NF-κB in oxidative stress conditions, but this was independent of P+1 loop Tyr phosphorylation. Interestingly, while P+1 loop Tyr phosphorylation of PKD2 is dependent of upstream kinases in cells, PKDs (all isoforms) can autophosphorylate this residue in vitro. This activity is only seen in in vitro assays. This is due to the fact that PKDs associate with an autophosphorylation-inhibiting factor in cells which prohibits Tyr autophosphorylation. Interestingly, we show that the molecular determinants for Tyr autophosphorylation are different from those for Ser-autophosphorylation and trans-phosphorylation of peptide. Additionally, we show that Tyr autophosphorylation activity is dependent on the presence of an unusual Cys residue in the HxD motif of the catalytic loop. This residue is an Arg in most kinases, but is often substituted in dual-specificity kinases. Because of the role of PKD isoforms in cancer, we were also interested in the development of PKD inhibitors. In this regard, bulky PP1 analogs (e.g. 1-NM-PP1), which normally do not bind WT kinases due to clashes with the ‘gatekeeper’ amino-acid were an interesting starting point for the rational design of a potent PKD inhibitor, since they unexpectedly inhibit WT PKDs. Our structure-activity relationships (SAR) studies of 1-NM-PP-1 derivates uncovered an unusual binding mode of these type of inhibitors to PKD and resulted in the development of the most active compound of this class as of yet. These data thus provide new insights for the potential development of future PKD-specific compounds.status: publishe

Function and Regulation of Protein Kinase D in Oxidative Stress: A Tale of Isoforms

Oxidative stress is a condition that arises when cells are faced with levels of reactive oxygen species (ROS) that destabilize the homeostatic redox balance. High levels of ROS can cause damage to macromolecules including DNA, lipids, and proteins, eventually resulting in cell death. Moderate levels of ROS however serve as signaling molecules that can drive and potentiate several cellular phenotypes. Increased levels of ROS are associated with a number of diseases including neurological disorders and cancer. In cancer, increased ROS levels can contribute to cancer cell survival and proliferation via the activation of several signaling pathways. One of the downstream effectors of increased ROS is the protein kinase D (PKD) family of kinases. In this review, we will discuss the regulation and function of this family of ROS-activated kinases and describe their unique isoform-specific features, in terms of both kinase regulation and signaling output

Protein kinase D2: a versatile player in cancer biology

Protein kinase D2 (PKD2) is a serine/threonine kinase that belongs to the PKD family of calcium-calmodulin kinases, which comprises three isoforms: PKD1, PKD2, and PKD3. PKD2 is activated by many stimuli including growth factors, phorbol esters, and G-protein-coupled receptor agonists. PKD2 participation to uncontrolled growth, survival, neovascularization, metastasis, and invasion has been documented in various tumor types including pancreatic, colorectal, gastric, hepatic, lung, prostate, and breast cancer, as well as glioma multiforme and leukemia. This review discusses the versatile functions of PKD2 from the perspective of cancer hallmarks as described by Hanahan and Weinberg. The PKD2 status, signaling pathways affected in different tumor types and the molecular mechanisms that lead to tumorigenesis and tumor progression are presented. The latest developments of small-molecule inhibitors selective for PKD/PKD2, as well as the need for further chemotherapies that prevent, slow down, or eliminate tumors are also discussed in this review.status: publishe

Protein kinase D displays intrinsic Tyr autophosphorylation activity: insights into mechanism and regulation

The protein kinase D (PKD) family is regulated through multi-site phosphorylation, including autophosphorylation. For example, PKD displays in vivo autophosphorylation on Ser-742 (and Ser-738 in vitro) in the activation loop and Ser-910 in the C-tail (hPKD1 numbering). In this paper, we describe the surprising observation that PKD also displays in vitro autocatalytic activity towards a Tyr residue in the P + 1 loop of the activation segment. We define the molecular determinants for this unusual activity and identify a Cys residue (C705 in PKD1) in the catalytic loop as of utmost importance. In cells, PKD Tyr autophosphorylation is suppressed through the association of an inhibitory factor. Our findings provide important novel insights into PKD (auto)regulation.status: publishe

Loss of ADAMTS5 enhances brown adipose tissue mass and promotes browning of white adipose tissue via CREB signaling

A potential strategy to treat obesity - and the associated metabolic consequences - is to increase energy expenditure. This could be achieved by stimulating thermogenesis through activation of brown adipose tissue (BAT) and/or the induction of browning of white adipose tissue (WAT). Over the last years, it has become clear that several metalloproteinases play an important role in adipocyte biology. Here, we investigated the potential role of ADAMTS5.status: publishe

Discovery of a potent protein kinase D inhibitor: insights in the binding mode of pyrazolo[3,4-d] pyrimidine analogues

In this study, we set out to rationally optimize PKD inhibitors based on the pyrazolo[3,4-d]pyrimidine scaffold. The lead compound for this study was 1-NM-PP1, which was previously found by us and others to inhibit PKD. In our screening we identified one compound (3-IN-PP1) displaying a 10-fold increase in potency over 1-NM-PP1, opening new possibilities for specific protein kinase inhibitors for kinases that show sensitivity towards pyrazolo[3,4-d]pyrimidine derived compounds. Interestingly the observed SAR was not in complete agreement with the commonly observed binding mode where the pyrazolo[3,4-d]pyrimidine compounds are bound in a similar fashion as PKD's natural ligand ATP. Therefore we suggest an alternate binding mode where the compounds are flipped 180 degrees. This possible alternate binding mode for pyrazolo[3,4-d]pyrimidine based compounds could pave the way for a new class of specific protein kinase inhibitors for kinases sensitive towards pyrazolo[3,4-d]pyrmidines.crosscheck: This document is CrossCheck deposited related_data: Supplementary Information identifier: Klaas Verschueren (ORCID) identifier: Joachim Demaerel (ORCID) identifier: Arnout R. D. Voet (ResearcherID) identifier: Wim M. De Borggraeve (ORCID) identifier: Wim M. De Borggraeve (ResearcherID) copyright_licence: The Royal Society of Chemistry has an exclusive publication licence for this journal copyright_licence: This article is freely available. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence (CC BY 3.0) history: Received 2 December 2016; Accepted 31 January 2017; Accepted Manuscript published 9 February 2017; Advance Article published 15 February 2017; Version of Record published 23 March 2017status: publishe