The PtdIns(4,5)P2 ligand itself influences the localization of PKCalpha in the plasma membrane of intact living cells.
Rapamycin-triggered heterodimerization strategy is becoming an excellent tool for rapidly modifying phosphatidylinositol(4,5)-bisphosphate [PtdIns(4,5)P2] levels at the plasma membrane and for studying their influence in different processes. In this work, we studied the effect of modulation of the PtdIns(4,5)P2 concentration on protein kinase C (PKC) alpha membrane localization in intact living cells. We showed that an increase in the PtdIns(4,5)P2 concentration enlarges the permanence of PKCalpha in the plasma membrane when PC12 cells are stimulated with ATP, independently of the diacylglycerol generated. The depletion of this phosphoinositide decreases both the percentage of protein able to translocate to the plasma membrane and its permanence there. Our results demonstrate that the polybasic cluster located in the C2 domain of PKCalpha is responsible for this phosphoinositide-protein interaction. Furthermore, the C2 domain acts as a dominant interfering module in the neural differentiation process of PC12 cells, a fact that was also supported by the inhibitory effect obtained by knocking down PKCalpha with small interfering RNA duplexes. Taken together, these data demonstrate that PtdIns(4,5)P2 itself targets PKCalpha to the plasma membrane through the polybasic cluster located in the C2 domain, with this interaction being critical in the signaling network involved in neural differentiation.
Marín-Vicente
Consuelo
C
Dpto. de Bioquímica y Biología Molecular (A), Facultad de Veterinaria, Universidad de Murcia, Apdo. 4021, E-30100 Murcia, Spain.
Nicolás
Francisco E
FE
Gómez-Fernández
Juan C
JC
Corbalán-García
Senena
S
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002454:Cell Differentiation / Q000235:genetics; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D004075:Diglycerides / Q000378:metabolism; D008024:Ligands; D008954:Models, Biological; D020932:Nerve Growth Factor / Q000494:pharmacology; D016501:Neurites / Q000187:drug effects; D016716:PC12 Cells; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism / Q000494:pharmacology*; D011485:Protein Binding; D051571:Protein Kinase C-alpha / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary / Q000235:genetics / Q000502:physiology; D021381:Protein Transport / Q000187:drug effects; D034741:RNA, Small Interfering / Q000494:pharmacology; D051381:Rats; D020123:Sirolimus / Q000494:pharmacology
D004075:Diglycerides; D008024:Ligands; D019269:Phosphatidylinositol 4,5-Diphosphate; D034741:RNA, Small Interfering; D000255:Adenosine Triphosphate; D020932:Nerve Growth Factor; D051571:Protein Kinase C-alpha; D020123:Sirolimus