Mechanisms of protein kinase D activation in response to P2Y(2) and P2X7 receptors in primary astrocytes.
Protein kinase D (PKD) is a family of serine/threonine kinases that can be activated by many stimuli via protein kinase C in a variety of cells. This is the first report where PKD activation and localization is studied in glial cells. Herein, we demonstrate that P2Y(2) and P2X7 receptor stimulation of primary rat cerebellar astrocytes rapidly increases PKD1/2 phosphorylation and activity. P2Y(2) receptor response evokes a PKD1/2 activation that is dependent on a pertussis toxin-insensitive G protein, phospholipase C (PLC)-mediated generation of diacylglycerol, and protein kinase C. This mechanism is similar to the one described for other G-protein coupled receptors. In contrast, the way the ionotropic P2X7 receptor activates PKD1/2 is significantly different. Importantly, this response is not dependent on calcium entry, but depends on the activity of several phospholipases, including phosphoinositide-phospholipase C (PI-PLC), phosphatidylcholine-phospholipase C (PC-PLC) and also phospholipase D (PLD). Immunoblot and confocal microscopy analysis show that PKD1/2 activation by nucleotides is transient. The active kinase first moves to and concentrates in certain plasma membrane domains. Then, phosphorylated-PKD1/2 translocates to intracellular vesicles, where it remains active. All together, our results open the perspective of PKD1/2 being involved in many physiological functions where nucleotides play important roles not only in astrocytes but in other cell types bearing these receptors.
Carrasquero
Luz María G
LM
Departamento de Bioquímica, Facultad de Veterinaria, Universidad Complutense de Madrid, Madrid 28040, Spain.
Delicado
Esmerilda G
EG
Sánchez-Ruiloba
Lucía
L
Iglesias
Teresa
T
Miras-Portugal
Maria Teresa
MT
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D001253:Astrocytes / Q000187:drug effects / Q000378:metabolism*; D002531:Cerebellum / Q000166:cytology; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects / Q000502:physiology*; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009711:Nucleotides / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism*; D011732:Pyridoxal Phosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D018048:Receptors, Purinergic P2 / Q000172:deficiency / Q000502:physiology*; D058486:Receptors, Purinergic P2X7; D058473:Receptors, Purinergic P2Y2; D012694:Serine / Q000378:metabolism; D014162:Transfection / Q000379:methods
D004791:Enzyme Inhibitors; D009711:Nucleotides; C546468:P2RX7 protein, human; C546385:P2RY2 protein, human; C546469:P2rx7 protein, mouse; C546470:P2rx7 protein, rat; C546386:P2ry2 protein, mouse; C546387:P2ry2 protein, rat; D010975:Platelet Aggregation Inhibitors; D018048:Receptors, Purinergic P2; D058486:Receptors, Purinergic P2X7; D058473:Receptors, Purinergic P2Y2; D049452:Green Fluorescent Proteins; C077792:pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid; D012694:Serine; C033901:3'-O-(4-benzoyl)benzoyladenosine 5'-triphosphate; D011732:Pyridoxal Phosphate; D000255:Adenosine Triphosphate; C089212:protein kinase D; D011493:Protein Kinase C