p62 (SQSTM1) and cyclic AMP phosphodiesterase-4A4 (PDE4A4) locate to a novel, reversible protein aggregate with links to autophagy and proteasome degradation pathways.
Chronic challenge of cyclic AMP phosphodiesterase-4A4 (PDE4A4) with certain PDE4 selective inhibitors causes it to reversibly form intracellular aggregates that are not membrane-encapsulated. These aggregates are neither stress granules (SGs) nor processing bodies (PBs) as they contain neither PABP-1 nor Dcp1a, respectively. However, the PDE4 inhibitor rolipram decreases arsenite-induced SGs and increases the amount of PBs, while arsenite challenge ablates rolipram-induced PDE4A4 aggregates. PDE4A4 aggregates are neither autophagic vesicles (autophagosomes) nor aggresomes, although microtubule disruptors ablate PDE4A4 aggregate formation. PDE4A4 constitutively co-immunoprecipitates with p62 protein (sequestosome1, SQSTM1), which locates to both PDE4A4 aggregates and autophagosomes in cells constitutively challenged with rolipram. The mTor inhibitor, rapamycin, activates autophagy, prevents PDE4A4 from forming intracellular aggregates and triggers the loss of bound p62 from PDE4A4. siRNA-mediated knockdown of p62 attenuates PDE4A4 aggregate formation. The p62-binding protein, light chain 3 (LC3), is not found in PDE4A4 aggregates. Blockade of proteasome activity and activation of autophagy with MG132 both increases the level of ubiquitinated proteins found associated with PDE4A4 and inhibits PDE4A4 aggregate formation. Activation of autophagy with either thapsigargin or ionomycin inhibits PDE4A4 aggregate formation. Inhibition of autophagy with either wortmannin or LY294002 activates PDE4A4 aggregate formation. The protein kinase C inhibitors, RO 320432 and GO 6983, and the ERK inhibitors UO 126 and PD 98059 all activated PDE4A4 aggregate formation, whilst roscovitine, thalidomide and the tyrosine kinase inhibitors, genistein and AG17, all inhibited this process. We suggest that the fate of p62-containing protein aggregates need not necessarily be terminal, through delivery to autophagic vesicles and aggresomes. Instead, we propose a novel regulatory mechanism where a sub-population of p62-containing protein aggregates would form in a rapid, reversible manner so as to sequester specific cargo away from their normal, functionally important site(s) within the cell. Thus an appropriate conformational change in the target protein would confer reversible recruitment into a sub-population of p62-containing protein aggregates and so provide a regulatory function by removing these cargo proteins from their functionally important site(s) in a cell.
Christian
Frank
F
Neuroscience and Molecular Pharmacology, Wolfson and Davidson Buildings, Faculty of Biomedical & Life Sciences, University of Glasgow, Glasgow, G12 8QQ, Scotland, United Kingdom.
Anthony
Diana F
DF
Vadrevu
Surakiran
S
Riddell
Tracy
T
Day
Jonathan P
JP
McLeod
Ruth
R
Adams
David R
DR
Baillie
George S
GS
Houslay
Miles D
MD
D048868:Adaptor Proteins, Signal Transducing / Q000032:analysis*; D000818:Animals; D001343:Autophagy; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4 / Q000032:analysis*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D003594:Cytoplasmic Granules / Q000201:enzymology; D022162:Cytoplasmic Vesicles / Q000201:enzymology; D006367:HeLa Cells; D006801:Humans; D007976:Leupeptins / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D061988:Proteasome Inhibitors; D020889:Rolipram / Q000037:antagonists & inhibitors / Q000494:pharmacology; D000071456:Sequestosome-1 Protein; D054875:Ubiquitination
D048868:Adaptor Proteins, Signal Transducing; D015853:Cysteine Proteinase Inhibitors; D007976:Leupeptins; D061988:Proteasome Inhibitors; C099718:SQSTM1 protein, human; D000071456:Sequestosome-1 Protein; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; D046988:Proteasome Endopeptidase Complex; D020889:Rolipram; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde