Coordinated activation of the nuclear ubiquitin ligase Cul3-SPOP by the generation of phosphatidylinositol 5-phosphate.
Phosphoinositide signaling pathways regulate numerous processes in eukaryotic cells, including migration, proliferation, and survival. The regulatory lipid phosphatidylinositol 4,5-bisphosphate is synthesized by two distinct classes of phosphatidylinositol phosphate kinases (PIPKs), the type I and II PIPKs. Although numerous physiological functions have been identified for type I PIPKs, little is known about the functions and regulation of type II PIPK. Using a yeast two-hybrid screen, we identified an interaction between the type IIbeta PIPK isoform (PIPKIIbeta) and SPOP (speckle-type POZ domain protein), a nuclear speckle-associated protein that recruits substrates to Cul3-based ubiquitin ligases. PIPKIIbeta and SPOP interact and co-localize at nuclear speckles in mammalian cells, and SPOP mediates the ubiquitylation of PIPKIIbeta by Cul3-based ubiquitin ligases. Additionally, stimulation of the p38 MAPK pathway enhances the ubiquitin ligase activity of Cul3-SPOP toward multiple substrate proteins. Finally, a kinase-dead PIPKIIbeta mutant enhanced ubiquitylation of Cul3-SPOP substrates. The kinase-dead PIPKIIbeta mutant increases the cellular content of its substrate lipid phosphatidylinositol 5-phosphate (PI5P), suggesting that PI5P may stimulate Cul3-SPOP activity through a p38-dependent signaling pathway. Expression of phosphatidylinositol-4,5-bisphosphate 4-phosphatases that generate PI5P dramatically stimulated Cul3-SPOP activity and was blocked by the p38 inhibitor SB203580. Taken together, these data define a novel mechanism whereby the phosphoinositide PI5P leads to stimulation of Cul3-SPOP ubiquitin ligase activity and also implicate PIPKIIbeta as a key regulator of this signaling pathway through its association with the Cul3-SPOP complex.
Bunce
Matthew W
MW
Department of Pharmacology, University of Wisconsin, Madison, WI 53706, USA.
Boronenkov
Igor V
IV
Anderson
Richard A
RA
D021581:Active Transport, Cell Nucleus; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D044842:Cullin Proteins / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D048669:MAP Kinase Kinase 6 / Q000378:metabolism; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism; D011485:Protein Binding; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011725:Pyridines / Q000494:pharmacology; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D012680:Sensitivity and Specificity; D015398:Signal Transduction / Q000187:drug effects; D013379:Substrate Specificity; D054875:Ubiquitination; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
C503645:CUL3 protein, human; D018797:Cell Cycle Proteins; D044842:Cullin Proteins; D007093:Imidazoles; D009687:Nuclear Proteins; D018129:Phosphatidylinositol Phosphates; D047428:Protein Kinase Inhibitors; D011725:Pyridines; D012097:Repressor Proteins; C509761:SPOP protein, human; C113567:phosphatidylinositol 5-phosphate; D017853:Phosphotransferases (Alcohol Group Acceptor); C020077:1-phosphatidylinositol-4-phosphate 5-kinase; D048051:p38 Mitogen-Activated Protein Kinases; D048669:MAP Kinase Kinase 6; C093642:SB 203580