Phosphoinositides are a family of lipid signalling molecules that regulate many cellular functions in eukaryotes. Phosphatidylinositol-4,5-bisphosphate (PtdIns4,5P2), the central component in the phosphoinositide signalling circuitry, is generated primarily by type I phosphatidylinositol 4-phosphate 5-kinases (PIPKIalpha, PIPKIbeta and PIPKIgamma). In addition to functions in the cytosol, phosphoinositides are present in the nucleus, where they modulate several functions; however, the mechanism by which they directly regulate nuclear functions remains unknown. PIPKIs regulate cellular functions through interactions with protein partners, often PtdIns4,5P2 effectors, that target PIPKIs to discrete subcellular compartments, resulting in the spatial and temporal generation of PtdIns4,5P2 required for the regulation of specific signalling pathways. Therefore, to determine roles for nuclear PtdIns4,5P2 we set out to identify proteins that interacted with the nuclear PIPK, PIPKIalpha. Here we show that PIPKIalpha co-localizes at nuclear speckles and interacts with a newly identified non-canonical poly(A) polymerase, which we have termed Star-PAP (nuclear speckle targeted PIPKIalpha regulated-poly(A) polymerase) and that the activity of Star-PAP can be specifically regulated by PtdIns4,5P2. Star-PAP and PIPKIalpha function together in a complex to control the expression of select mRNAs, including the transcript encoding the key cytoprotective enzyme haem oxygenase-1 (refs 8, 9) and other oxidative stress response genes by regulating the 3'-end formation of their mRNAs. Taken together, the data demonstrate a model by which phosphoinositide signalling works in tandem with complement pathways to regulate the activity of Star-PAP and the subsequent biosynthesis of its target mRNA. The results reveal a mechanism for the integration of nuclear phosphoinositide signals and a method for regulating gene expression.

We have recently identified Star-PAP, a nuclear poly(A) polymerase that associates with phosphatidylinositol-4-phosphate 5-kinase I␣ (PIPKI␣) and is required for the expression of a specific subset of mRNAs. Star-PAP activity is directly modulated by the PIPKI␣ product phosphatidylinositol 4,5-bisphosphate (PI-4,5-P 2 ), linking nuclear phosphoinositide signaling to gene expression. Here, we show that PI-4,5-P 2 -dependent protein kinase activity is also a part of the Star-PAP protein complex. We identify the PI-4,5-P 2 -sensitive casein kinase I␣ (CKI␣) as a protein kinase responsible for this activity and further show that CKI␣ is capable of directly phosphorylating Star-PAP. Both CKI␣ and PIPKI␣ are required for the synthesis of some but not all Star-PAP target mRNA, and like Star-PAP, CKI␣ is associated with these messages in vivo. Taken together, these data indicate that CKI␣, PIPKI␣, and Star-PAP function together to modulate the production of specific Star-PAP messages. The Star-PAP complex therefore represents a location where multiple signaling pathways converge to regulate the expression of specific mRNAs.Polyadenylation of most mRNAs is required for their efficient transcription and export as well as regulating their stability and translational efficiency (1). Polyadenylation of mRNA is achieved through the activity of poly(A) polymerases (PAPs). 2There are multiple PAPs in mammalian cells, including PAP␣, which is thought to be primarily responsible for the polyadenylation of newly transcribed mRNAs in the nucleus (2, 3). Additionally, there are "non-canonical" PAPs, including Gld2 and Trf4, which regulate stability and degradation of specific RNAs through polyadenylation (4, 5).Star-PAP is a non-canonical poly(A) polymerase that is distinct from all other currently characterized PAP enzymes (6). Like the canonical PAP␣, Star-PAP has an RNA recognition motif and is a nuclear enzyme. However, similar to non-canonical PAPs, the Star-PAP protein complex and architecture differ significantly from PAP␣, and consequently, Star-PAP specifically targets a select subset of mRNAs. This suggests that Star-PAP is a hybrid PAP that is required for the 3Ј-end formation of newly transcribed pre-mRNAs but functions in a regulatory role to control mRNA expression levels. Star-PAP has a unique domain structure relative to all other known PAPs (6). One unique feature is a 205-amino acid proline-rich region (PRR) inserted into the catalytic PAP core. The PRR splits the catalytic PAP domain, and this region represents a potential site for regulation of Star-PAP function.Poly(A) polymerases operate as large multiprotein complexes responsible for the 3Ј-processing of RNAs (7, 8). The Star-PAP polyadenylation complex is similar to that of poly(A) polymerases that utilizes mRNA as a substrate and includes cleavage and polyadenylation specificity factor (CPSF) subunits, cleavage stimulatory factor (CstF) subunits, symplekin, and RNA polymerase II (6, 9). However, unlike other poly(A) polymerase complexes, the Star-PAP complex...

The phosphatidylinositol phosphate (PIP) kinases are a unique family of enzymes that generate an assortment of lipid messengers, including the pivotal second messenger phosphatidylinositol 4,5-bisphosphate (PI4,5P2). While members of the PIP kinase family function by catalyzing a similar phosphorylation reaction, the specificity loop of each PIP kinase subfamily determines substrate preference and partially influences distinct subcellular targeting. Specific protein-protein interactions that are unique to particular isoforms or splice variants play a key role in targeting PIP kinases to appropriate subcellular compartments to facilitate the localized generation of PI4,5P2 proximal to effectors, a mechanism key for the function of PI4,5P2 as a second messenger. This review documents the discovery of the PIP kinases and their signaling products, and summarizes our current understanding of the mechanisms underlying the localized generation of PI4,5P2 by PIP kinases for the regulation of cellular events including actin cytoskeleton dynamics, vesicular trafficking, cell migration, and an assortment of nuclear events.

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