Coordinated Activation of the Nuclear Ubiquitin Ligase Cul3-SPOP by the Generation of Phosphatidylinositol 5-Phosphate

Abstract

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 II␤ PIPK isoform (PIPKII␤) and SPOP (speckle-type POZ domain protein), a nuclear speckle-associated protein that recruits substrates to Cul3-based ubiquitin ligases. PIPKII␤ and SPOP interact and co-localize at nuclear speckles in mammalian cells, and SPOP mediates the ubiquitylation of PIPKII␤ 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 PIPKII␤ mutant enhanced ubiquitylation of Cul3-SPOP substrates. The kinase-dead PIPKII␤ 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 PIPKII␤ as a key regulator of this signaling pathway through its association with the Cul3-SPOP complex.Phosphoinositide signaling pathways modulate a diverse array of cellular processes in eukaryotes. Modification of the inositol ring by lipid kinases and phosphatases produces distinct phosphatidylinositol phosphate (PIP) 2 isomers. These phosphatidylinositol phosphate isomers in turn selectively modulate the activities of effector proteins. In the cytosol, phosphoinositides regulate numerous processes, including actin polymerization, focal adhesion dynamics, ion channel activity, growth factor receptor signaling, and vesicle trafficking (1-4).In the nucleus, an autonomous phosphoinositide cycle regulates processes, including differentiation, proliferation, cell cycle progression, and apoptosis (5). Phosphatidylinositol 4,5-bisphosphate (PI-4,5-P 2 ) is a critical phosphoinositide in eukaryotic cells. PI-4,5-P 2 is not only itself a potent signaling molecule but is also a precursor of other second messengers such as phosphatidylinositol 3,4,5-trisphosphate, inositol 1,4,5-trisphosphate, and diacylglycerol. Because of its multipotent signaling capacity, the regulated synthesis of PI-4,5-P 2 is essential for eukaryotes. In mammalian cells, PI-4,5-P 2 is synthesized by two classes of phosphatidylinositol phosphate kinases (PIPKs) (6). Type I PIPKs (PIPKIs) preferentially use phosphatidylinos...

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Prostate cancer-associated mutations in speckle-type POZ protein (SPOP) regulate steroid receptor coactivator 3 protein turnover

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Phosphoinositides: Tiny Lipids With Giant Impact on Cell Regulation

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