Phosphorylation of PKC activation loop plays an important role in receptor-mediated translocation of PKC.
Protein kinase C (PKC) is translocated to various cellular regions in a subtype and stimulation-dependent manner. Thereafter, the activated PKC phosphorylates its substrate and causes subsequent cellular responses (PKC targeting). The 3-phosphoinositide-dependent protein kinase-1 (PDK1) has an essential role in the maturation of PKC by phosphorylating a threonine residue in the PKC activation loop. To elucidate the role of PDK1 in PKC targeting, we expressed mutant gamma- or delta-PKC fused with GFP (gamma- or delta-PKC-ALM (activation loop mutant)-GFP), whose threonine residue in the activation loop was replaced with alanine, and compared their P2Y receptor-mediated translocation with wild-type PKC-GFP in CHO cells. ATP (1 mm) induced the transient translocation of wild-type gamma- or delta-PKC-GFP from cytoplasm to plasma membrane and following retranslocation from membrane to the cytoplasm. gamma- or delta-PKC-ALM-GFP was also translocated to plasma membrane, which was, however, retained at the membrane for a longer period than wild type. Similar results were observed in kinase-negative PKC mutants, indicating that the phosphorylation by PDK1 affects the retranslocation step of PKC by regulating the kinase activity. The simultaneous monitoring of [Ca2+]i and diacylglycerol (DG) levels with the translocation of PKC demonstrated that PKC-ALM induced the prolonged accumulation of DG, resulting in the prolonged retention of PKC-ALM at the plasma membrane. It is possible that PKC-ALM with decreased kinase activity could delay the conversion of DG at the plasma membrane. Our present study suggests that the activation loop phosphorylation plays an important role in receptor-mediated PKC targeting.
Seki
Takahiro
T
Department of Molecular and Pharmacological Neuroscience, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima 734-8551, Japan.
Matsubayashi
Hiroaki
H
Amano
Taku
T
Shirai
Yasuhito
Y
Saito
Naoaki
N
Sakai
Norio
N
D000818:Animals; D016466:CHO Cells; D002462:Cell Membrane / Q000201:enzymology / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D019852:Diacylglycerol Kinase / Q000235:genetics / Q000378:metabolism; D017930:Genes, Reporter; D015151:Immunoblotting; D009154:Mutation; D010766:Phosphorylation; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D019852:Diacylglycerol Kinase; D011493:Protein Kinase C