Subject(s)
Cell Nucleus/metabolism , Gene Expression Regulation , Isoenzymes/metabolism , Phosphatidylinositols/metabolism , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Polynucleotide Adenylyltransferase/metabolism , Casein Kinase Ialpha/genetics , Casein Kinase Ialpha/metabolism , Humans , Isoenzymes/genetics , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Nucleotidyltransferases , Phosphatidylinositol 4,5-Diphosphate/metabolism , Phosphotransferases (Alcohol Group Acceptor)/genetics , Polynucleotide Adenylyltransferase/genetics , Signal Transduction/physiologyABSTRACT
We have recently identified Star-PAP, a nuclear poly(A) polymerase that associates with phosphatidylinositol-4-phosphate 5-kinase Ialpha (PIPKIalpha) and is required for the expression of a specific subset of mRNAs. Star-PAP activity is directly modulated by the PIPKIalpha 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 Ialpha (CKIalpha) as a protein kinase responsible for this activity and further show that CKIalpha is capable of directly phosphorylating Star-PAP. Both CKIalpha and PIPKIalpha are required for the synthesis of some but not all Star-PAP target mRNA, and like Star-PAP, CKIalpha is associated with these messages in vivo. Taken together, these data indicate that CKIalpha, PIPKIalpha, 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.
Subject(s)
Casein Kinase Ialpha/metabolism , Gene Expression Regulation , Polynucleotide Adenylyltransferase/metabolism , Amino Acid Sequence , Animals , Cell Line , Humans , Models, Biological , Molecular Sequence Data , Nucleotidyltransferases , Phosphatidylinositol 4,5-Diphosphate/metabolism , Phosphorylation , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Polynucleotide Adenylyltransferase/chemistry , Proline , Protein Binding , Protein Kinases/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Sequence DeletionABSTRACT
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.
Subject(s)
Cell Nucleus/metabolism , Phosphatidylinositol Phosphates/metabolism , Polynucleotide Adenylyltransferase/metabolism , RNA 3' End Processing , Animals , Cell Line , Cell Nucleus/enzymology , Cell Nucleus/genetics , Heme Oxygenase-1/genetics , Humans , Mice , Multiprotein Complexes/metabolism , Nucleotidyltransferases , Oxidative Stress/genetics , Phosphatidylinositol 4,5-Diphosphate , Phosphotransferases (Alcohol Group Acceptor)/deficiency , Phosphotransferases (Alcohol Group Acceptor)/genetics , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Polynucleotide Adenylyltransferase/chemistry , Polynucleotide Adenylyltransferase/deficiency , Polynucleotide Adenylyltransferase/genetics , Protein Binding , RNA, Messenger/genetics , RNA, Messenger/metabolism , Substrate Specificity , Transcription, GeneticABSTRACT
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.
