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Transmembrane transport and protein phosphorylation in the regulation of cell function

Research project P5/05 (Research action P5)


Persons :


Description :

The eight partners in this network collaborate on three major interrelated processes that control virtually every function of eukaryotic cells:

(1) transmembrane transport of ions, water and solutes;
(2) the dynamics of Ca2+ as a second messenger; and
(3) protein phosphorylation in the final execution of the message.

1. Transport through the plasma membrane - The network has two targets for joint investigation:

1.1. The plant H+-ATPases undergo reversible phosphorylation which induces activation by binding of regulatory 14-3-3 proteins. The unconventional phosphorylation site prompts investigation of the protein kinases and phosphatases involved. Other studies will address whether expression and phosphorylation of H+-ATPases relate to plant development and growth in adverse environmental conditions.

1.2. Aquaporins are water channels, but homologous proteins in plant and animal cells appear to transport other molecules, which shall be identified in a collaborative approach. Some aquaporins are phosphorylated. The phosphorylation dynamics and its functional effects need to be investigated.

2. Ca2+ signals depend on influx, release, and re-uptake of Ca2+, which are all being investigated:

2.1. Ca2+-entry by TRP-channels will be a major study object for multiple partners: modulation of channel activity by phosphorylation as well as by sphingolipids, PUFA, and derivatives thereof; identification of TRP partner-proteins; interaction of TRP with the cytoskeleton in the assembly of signalling complexes.

2.2. Ca2+ controls Ca2+ release through the IP3-receptor (IP3R). Ca2+ and/or CaM-dependent kinases or phosphatases may hereby play an important role. Their exact role, their precise interaction mode with the IP3R, and the phosphorylation site(s) of the IP3R, all need to be established. Also, the prospect that the metabolism of IP3 itself is targeted by protein phosphorylation will be directly addressed.

2.3. The two Belgian inositol-phosphate teams will expand their ongoing studies, using overexpression of additional IP3-metabolizing enzymes, to study the intracellular Ca2+-signal dynamics. They will also apply this technique to investigate the propagation of Ca2+ waves between cells (in cell types using distinct communication modes), and also to study Ca2+-signal dynamics in subcellular compartments.

2.4. The role of the Golgi apparatus in setting up intracellular Ca2+ signals will be analysed by multiple approaches: the importance of PIP2 will be investigated by overexpression of a specific 5-phosphatase; further, a recently discovered ATP-driven Ca2+ pump will be the target of functional and molecular studies; also, the role of sphingolipids in the release of Ca2+ by non-IP3-mechanisms will be explored.

2.5. The specific roles of the sarco(endo)plasmic reticulum Ca2+ ATPases SERCA2b and SERCA2a (the latter with its phosphorylatable regulator, phospholamban) will be tackled, taking advantage of a trans-genic mouse that expresses only SERCA2b in the sarcoplasmic reticulum. Also the reason underlying the high phosphorylation level of phospholamban in the early postnatal period will be investigated.

3. The control of cell functions by reversible protein phosphorylation:

3.1. Modulation of insulin signalling to metabolic pathways: several partners will collaborate to purify, clone and characterize the newly discovered protein kinase WISK, to delineate the role of PKB and to study the interference by SHIP2. Another effort will focus on the metabolism of methylglyoxal, which may be involved in the generation of AGEs that contribute to the pathogenesis of diabetic complications.

3.2. Identification of physiological substrates of protein phosphatases: for this purpose cells will be stably transfected with expression vectors for inhibitory polypeptide domains that tightly bind to either PP1 or PP2A, as a fusion with a targeting sequence for various subcellular locations.

3.3. Phospholipids and the control of the organization of the cytoskeleton: an ongoing collaboration will aim at characterizing the phosphatase(s) catalyzing lysophosphatidate-stimulated dephosphorylation of AFK-phosphorylated actin; a second multi-partner effort will focus on the emerging role of lysophosphatidate as a general cellular mediator, using e.g. a newly synthesized, fluorescent derivative.


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