PMC:4349302 / 1428-8212 JSONTXT

{"project":"2_test","denotations":[{"id":"25889812-15086783-14899089","span":{"begin":404,"end":405},"obj":"15086783"},{"id":"25889812-14685229-14899090","span":{"begin":592,"end":593},"obj":"14685229"},{"id":"25889812-16824007-14899091","span":{"begin":594,"end":595},"obj":"16824007"},{"id":"25889812-21421913-14899092","span":{"begin":1006,"end":1007},"obj":"21421913"},{"id":"25889812-22226883-14899093","span":{"begin":1127,"end":1128},"obj":"22226883"},{"id":"25889812-20431265-14899094","span":{"begin":1129,"end":1130},"obj":"20431265"},{"id":"25889812-16100512-14899095","span":{"begin":1337,"end":1338},"obj":"16100512"},{"id":"25889812-17003043-14899096","span":{"begin":1568,"end":1569},"obj":"17003043"},{"id":"25889812-18525025-14899097","span":{"begin":1570,"end":1571},"obj":"18525025"},{"id":"25889812-16601923-14899098","span":{"begin":3298,"end":3300},"obj":"16601923"},{"id":"25889812-17314061-14899098","span":{"begin":3298,"end":3300},"obj":"17314061"},{"id":"25889812-19416656-14899098","span":{"begin":3298,"end":3300},"obj":"19416656"},{"id":"25889812-15907394-14899099","span":{"begin":3492,"end":3494},"obj":"15907394"},{"id":"25889812-19416656-14899100","span":{"begin":3624,"end":3626},"obj":"19416656"},{"id":"25889812-14685263-14899101","span":{"begin":3784,"end":3786},"obj":"14685263"},{"id":"25889812-16905542-14899101","span":{"begin":3784,"end":3786},"obj":"16905542"},{"id":"25889812-17449912-14899101","span":{"begin":3784,"end":3786},"obj":"17449912"},{"id":"25889812-16824007-14899102","span":{"begin":4038,"end":4039},"obj":"16824007"},{"id":"25889812-11689438-14899103","span":{"begin":4040,"end":4042},"obj":"11689438"},{"id":"25889812-11729268-14899103","span":{"begin":4040,"end":4042},"obj":"11729268"},{"id":"25889812-15824133-14899103","span":{"begin":4040,"end":4042},"obj":"15824133"},{"id":"25889812-17591919-14899104","span":{"begin":4103,"end":4105},"obj":"17591919"},{"id":"25889812-19005213-14899105","span":{"begin":4210,"end":4212},"obj":"19005213"},{"id":"25889812-17442665-14899106","span":{"begin":4363,"end":4365},"obj":"17442665"},{"id":"25889812-18165232-14899107","span":{"begin":4415,"end":4417},"obj":"18165232"},{"id":"25889812-17442665-14899108","span":{"begin":4498,"end":4500},"obj":"17442665"},{"id":"25889812-17591919-14899109","span":{"begin":4616,"end":4618},"obj":"17591919"},{"id":"25889812-9852146-14899110","span":{"begin":5072,"end":5074},"obj":"9852146"},{"id":"25889812-12530516-14899110","span":{"begin":5072,"end":5074},"obj":"12530516"},{"id":"25889812-14645071-14899110","span":{"begin":5072,"end":5074},"obj":"14645071"},{"id":"25889812-24001971-14899110","span":{"begin":5072,"end":5074},"obj":"24001971"},{"id":"25889812-19428735-14899111","span":{"begin":5726,"end":5728},"obj":"19428735"},{"id":"25889812-23385484-14899112","span":{"begin":6400,"end":6402},"obj":"23385484"}],"text":"Background\nAll transmembrane proteins and soluble proteins that are secreted from a cell into the extracellular space generally pass through the endoplasmic reticulum (ER) and Golgi complex (GC). In mammalian cells the GC constitutes a highly ordered organelle composed of cisternal stacks, the main function of which is the modification of proteins and sorting to their distinct membrane localizations [1]. Cargo is received from the ER at the cis-Golgi complex, traverses the medial Golgi stacks, then reaches the trans-Golgi side where vesicles bud off from the trans-Golgi network (TGN) [2,3]. The formation of these vesicles is driven by a complex interaction of proteins and lipids and is still only partly understood. One of the key players of this network is protein kinase D, which comprises a family of three closely related serine-threonine kinases (PKD1, PKD2 and PKD3). PKD has an established role in the regulation of constitutive but also regulated vesicular transport processes at the TGN [4].\nIn the following, we shortly describe the molecular interaction network of PKD at the TGN (Figure 1 and reviewed in [5,6]). The first TGN-localized PKD substrate identified was PI4KIII β whose phosphorylation on serine 294 increased the activity of the lipid kinase, resulting in the enhanced production of its substrate PI4P [7]. This phosphorylated lipid is especially important in defining Golgi membrane identity and providing a local signaling platform to which several PI4P-binding proteins are recruited, including the ceramide transfer protein CERT [8,9].\nFigure 1 Model of molecular interactions at the TGN, comprising the key proteins PKD, PI4KIII β and CERT. Chemical conversions and changes of locations are represented by solid edges with number labels. Catalytic reactions and effective regulatory influences are represented by dashed edges. PKD and PI4KIII β are present as inactive or active TGN bound forms (variables PKD, PKDpDAG, PI4KIII β and PI4KIII βp, respectively). Active TGN bound PKD activates PI4KIII β (R1) and detaches CERT from the TGN membrane (R2) via phosphorylation (R3). Active PI4KIII β attracts CERT to the TGN (R5) by producing PI4P (R4). Two alternatives for CERT dependent ceramide transport are depicted. (A) In model A, ceramide transfer is ensured by a circular reaction scheme of CERT: Unphosphorylated CERT that is bound to both membranes (CERTaTGN) can deliver ceramide to the TGN (R10). Extraction of new ceramide requires detachment from the TGN (R3) via phosphorylation (CERTpER) and subsequent dephosphorylation (R9) at the ER (CERTaER). Thereby, PKD induced CERT phosphorylation (R2) and ceramide transfer (R10) dependent PKD activation (R8) constitute a positive feedback mechanism (R6, R7, R8, R2, R10), indicated by blue arrows. (B) In model B, unphosphorylated CERT (CERTa) is only transport-active in an ER-TGN double bound state (CERTaERTGN), constantly transferring ceramide (R10). Here, CERT phosphorylation (CERTp) (R3) causes an interruption of the transfer process and forms a negative feedback between PKD and CERT (R6, R7, R8, R2, R10), indicated by blue arrows.\nCeramide is produced at the ER and then transported to the TGN in a non-vesicular manner by CERT, which is recruited by the ER-resident membrane protein VAP [10-12]. The mechanistic understanding is that CERT takes up a ceramide molecule into its START domain, which forms a hydrophobic pocket, followed by the release of ceramide at the TGN membrane [13]. This transport is thought to occur at so-called membrane contact sites (MCS) where the ER and the TGN come into close contact [12]. At the TGN, sphingomyelin synthases (SMS) catalyze the conversion of ceramide and phosphatidylcholine (PC) to sphingomyelin (SM) and diacylglycerol (DAG) [14-16]. DAG fulfills several central functions at the TGN by activating novel PKC isoforms which phosphorylate and activate PKD, by recruiting and activating PKD, and by directly impacting membrane curvature involved in the process of vesicle formation [3,17-19]. CERT, in turn, is phosphorylated by PKD at serine 132 [20], which serves as a priming site for multiple phosphorylations by CKI γ2 in the serine rich (SR) motif [21]. This hyperphosphorylation of CERT negatively affects its affinity for PI4P and induces a conformational change that inhibits START domain function [22]. At the ER, CERT is dephosphorylated by PP2C ε [23], enhancing the interaction of CERT with the Golgi membranes and also with VAP [22]. Thereby, the proteins PKD, PI4KIII β and CERT are involved in a complex scenario of interrelated feedback loops [20]. Despite the knowledge on the qualitative effects of phosphorylation on CERT membrane binding and function, it is currently still unclear how this complex molecular interaction network impacts the efficacy of ceramide transport at ER-Golgi MCS.\nOnly few studies have attempted to address aspects of TGN function by mathematical modeling. The existing models describe mechanisms for vesicle kinetics, membrane physics, and feedback in the SMS reaction [24-27]. Thus far, no quantitative model exists that describes the kinetics of key protein interactions at the TGN important for secretory transport. Such a quantitative dynamic model would contribute to an increased understanding of molecule interactions and organelle function and is therefore valuable to basic research. In addition, such a model has broader relevance, as the vesicular transport from the TGN to the plasma membrane drives the polarized migration of cancer cells. A further application are biotechnological interventions in the secretory pathway targeting the optimization of the production of therapeutic proteins in mammalian cells [28].\nHere, we use mathematical modeling and perturbation experiments to address how the feedback loops within the PKD-CERT network interact to coordinate ceramide transport. We present a quantitative dynamical model for the molecular interactions at the TGN that is based on chemical reaction kinetics. Biochemical time series experiments after perturbation of the system and absolute protein quantification measurements are used for model calibration. As the information in the data is not sufficient to identify all model parameters uniquely, we use statistical Bayesian approaches, which are computationally demanding but particularly tailored to deal with this problem [29-31].\nOur calibrated model is able to capture dynamic interactions between PKD, PI4KIII β and CERT on an average cellular level. Furthermore, validation experiments confirm that we are able to reliably predict different perturbation scenarios. Our model-based analysis thus provides insight into the regulatory network of key components underlying ceramide transfer at ER-Golgi MCS."}