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.