Suppression of Synaptotagmin II restrains phorbolester-induced downregulation of protein kinase Calpha by diverting the kinase from a degradative pathway to the recycling endocytic compartment.
Downregulation of protein kinase Calpha (PKCalpha) following long-term exposure to phorbol esters such as TPA is traffic dependent and involves delivery of the active, membrane-associated PKCalpha to endosomes. In this study, we show that synaptotagmin II (Syt II), a member of the Syt family of proteins, is required for TPA-induced degradation of PKCalpha. Thus, whereas the kinase half-life in TPA-treated cultured mast cells (the mast cell line rat basophilic leukemia RBL-2H3) is 2 hours, it is doubled in RBL-Syt II(-) cells, in which the cellular level of Syt II is reduced by >95% by transfection with Syt II antisense cDNA. We demonstrate that in TPA-treated RBL cells, PKCalpha travels from the cytosol to the plasma membrane, where it is delivered to early endosomes on its route to degradation. By contrast, in TPA-treated RBL-Syt II(-) cells, PKCalpha is diverted to recycling endosomes and remains distributed between the plasma membrane and the perinuclear recycling endocytic compartment. Notably, in both RBL and RBL-Syt II(-) cells, a fraction of PKCalpha is delivered and maintained in the secretory granules (SG). These results implicate Syt II as a critical factor for the delivery of internalized cargo for degradation. As shown here, one consequence of Syt II suppression is a delay in PKCalpha downregulation, resulting in its prolonged signaling.
Department of Cell Biology and Histology, Sackler School of Medicine, Tel Aviv University, Tel Aviv, 69978, Israel.
Ze
Z
Peng
Elena
E
Grimberg
Ronit
R
Sagi-Eisenberg
D000818:Animals; D016375:Antisense Elements (Genetics) / Q000494:pharmacology; D002451:Cell Compartmentation / Q000187:drug effects / Q000235:genetics; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology*; D003546:Cysteine Endopeptidases / Q000378:metabolism; D003600:Cytosol / Q000187:drug effects / Q000201:enzymology; D015536:Down-Regulation / Q000187:drug effects / Q000502:physiology*; D004705:Endocytosis / Q000187:drug effects / Q000235:genetics; D011992:Endosomes / Q000187:drug effects / Q000201:enzymology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D008407:Mast Cells / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000037:antagonists & inhibitors / Q000172:deficiency* / Q000235:genetics; D010703:Phorbol Esters / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex; D011493:Protein Kinase C / Q000187:drug effects / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D021381:Protein Transport / Q000187:drug effects / Q000235:genetics*; D051381:Rats; D022142:Secretory Vesicles / Q000187:drug effects / Q000201:enzymology; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics; D050861:Synaptotagmin II; D014407:Tumor Cells, Cultured
D016375:Antisense Elements (Genetics); D004791:Enzyme Inhibitors; D009097:Multienzyme Complexes; D009419:Nerve Tissue Proteins; D010703:Phorbol Esters; D050861:Synaptotagmin II; C494264:Syt2 protein, rat; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex