Mathias Cobbaut

Differential regulation of PKD isoforms in oxidative stress conditions through phosphorylation of a conserved Tyr in the P+1 loop

Protein kinases are essential molecules in life and their crucial function requires tight regulation. Many kinases are regulated via phosphorylation within their activation loop. This loop is embedded in the activation segment, which additionally contains the Mg2+ binding loop and a P + 1 loop that is important in substrate binding. In this report, we identify Abl-mediated phosphorylation of a highly conserved Tyr residue in the P + 1 loop of protein kinase D2 (PKD2) during oxidative stress. Remarkably, we observed that the three human PKD isoforms display very different degrees of P + 1 loop Tyr phosphorylation and we identify one of the molecular determinants for this divergence. This is paralleled by a different activation mechanism of PKD1 and PKD2 during oxidative stress. Tyr phosphorylation in the P + 1 loop of PKD2 increases turnover for Syntide-2, while substrate specificity and the role of PKD2 in NF-κB signaling remain unaffected. Importantly, Tyr to Phe substitution renders the kinase inactive, jeopardizing its use as a non-phosphorylatable mutant. Since large-scale proteomics studies identified P + 1 loop Tyr phosphorylation in more than 70 Ser/Thr kinases in multiple conditions, our results do not only demonstrate differential regulation/function of PKD isoforms under oxidative stress, but also have implications for kinase regulation in general.

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

Objective 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. Methods Mice deficient in ADAMTS5 (Adamts5−/−) and wild-type (Adamts5+/+) littermates were kept on a standard of Western-type diet for 15 weeks. Energy expenditure and heat production was followed by indirect calorimetry. To activate thermogenesis, mice were treated with the β3-adrenergic receptor (β3-AR) agonist CL-316,243 or alternatively, exposed to cold for 2 weeks. Results Compared to Adamts5+/+ mice, Adamts5−/− mice have significantly more interscapular BAT and marked browning of their subcutaneous (SC) WAT. Thermogenic pathway analysis indicated, in the absence of ADAMTS5, enhanced β3-AR signaling via activation of the cAMP response element-binding protein (CREB). Additional β3-AR stimulation with CL-316,243 promoted browning of WAT in Adamts5+/+ mice but had no additive effect in Adamts5−/− mice. However, cold exposure induced more pronounced browning of WAT in Adamts5−/− mice. Conclusions These data indicate that ADAMTS5 plays a functional role in development of BAT and browning of WAT. Hence, selective targeting of ADAMTS5 could provide a novel therapeutic strategy for treatment/prevention of obesity and metabolic diseases.

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.

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.

Abstract B08: Differential regulation of Protein Kinase D isoforms in oxidative stress conditions via tyrosine phosphorylation in the activation segment

The protein kinase D (PKD) family of kinases belongs to the CAMK group and consists of three highly homologous enzymes. They play a versatile role in cancer cell signaling, primarily downstream of PKC isoforms. Classically PKDs are activated upon co-localization with PKC at DAG containing membranes, where PKC phosphorylates the PKD activation loop, generating active PKD. Besides this, PKDs are also activated in oxidative stress conditions, where PKD1 protects against cellular damage via signaling to NF-kB. While the three isoforms are highly homologous, they are not redundant in their biological function and oncogenic potential. A picture has emerged which associates PKD1 expression and activity with decreased migration and inhibition of epithelial mesenchymal transition (EMT). PKD2 and PKD3 on the other hand are upregulated in various cancers, and show oncogenic potential e.g. via enhanced ECM breakdown and potentiation of angiogenesis. While non-redundant biological roles continue to surface, the biochemical mechanisms behind these discrepant functions remain mostly elusive. Here, we identify an isoform specific regulation of PKD2 in oxidative stress conditions via the phosphorylation of a key Tyrosine residue in the substrate binding loop of the activation segment which is highly conserved in most Ser/Thr kinases. Phosphorylation of this residue selectively occurs in PKD2 (and not in PKD1). While wild-type PKD1 signals to NF-kB in oxidative stress, we could show that wild-type PKD2 signaling to NF-kB is impeded in these conditions. Substitution of the activation segment Tyr-717 in PKD2 with a non-phosphorylatable Phe restores signaling to NF-kB, indicating that Tyr-717 phosphorylation is a key determinant for the signaling outcome of PKDs in oxidative stress. Note: This abstract was not presented at the conference. Citation Format: Mathias Cobbaut, Rita Derua, Etienne Waelkens, Peter Storz, Veerle Janssens, Johan Van Lint. Differential regulation of Protein Kinase D isoforms in oxidative stress conditions via tyrosine phosphorylation in the activation segment [abstract]. In: Proceedings of the AACR International Conference: New Frontiers in Cancer Research; 2017 Jan 18-22; Cape Town, South Africa. Philadelphia (PA): AACR; Cancer Res 2017;77(22 Suppl):Abstract nr B08.