ABSTRACT
The hepatitis C virus encodes a single polyprotein that is processed by host and viral proteases to yield at least 10 mature viral proteins. The nonstructural (NS) protein 5A is a phosphoprotein, and experimental data indicate that the phosphorylation state of NS5A is important for the outcome of viral RNA replication. We were able to identify kinase inhibitors that specifically inhibit the formation of the hyperphosphorylated form of NS5A (p58) in cells. These kinase inhibitors were used for inhibitor affinity chromatography in order to identify the cellular targets of these compounds. The kinases casein kinase I (CKI), p38 MAPK, CIT (Citron Rho-interacting kinase), GAK, JNK2, PKA, RSK1/2, and RIPK2 were identified in the high affinity binding fractions of two NS5A hyperphosphorylation inhibitors (NS5A-p58-i). Even though these kinases are targets of the NS5A-p58-i, the only kinase showing an effect on NS5A hyperphosphorylation was confirmed to be CKI-alpha. Although this finding does not exclude the possibility that other kinase(s) might be involved in basal or regulatory phosphorylation of NS5A, we show here that NS5A is a direct substrate of CKI-alpha. Moreover, in vitro phosphorylation of NS5A by CKI-alpha resulted for the first time in the production of basal and hyperphosphorylated forms resembling those produced in cells. In vitro kinase reactions performed with NS5A peptides show that Ser-2204 is a preferred substrate residue for CKI-alpha after pre-phosphorylation of Ser-2201.
Subject(s)
Casein Kinase Ialpha/metabolism , Hepacivirus/metabolism , Protein Processing, Post-Translational/physiology , Viral Nonstructural Proteins/metabolism , Virus Replication/physiology , Animals , Casein Kinase Ialpha/chemistry , Casein Kinase Ialpha/isolation & purification , Cell Line , Chromatography, Affinity , Hepacivirus/chemistry , Humans , Phosphorylation/drug effects , Protein Kinase Inhibitors/pharmacology , Protein Processing, Post-Translational/drug effects , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/isolation & purification , Virus Replication/drug effectsABSTRACT
FADD is essential for death receptor (DR)-induced apoptosis. However, it is also critical for cell cycle progression and proliferation, activities that are regulated by phosphorylation of its C-terminal Ser194, which has also been implicated in sensitizing cancer cells to chemotherapeutic drugs and in regulating FADD's intracellular localization. We now demonstrate that casein kinase Ialpha (CKIalpha) phosphorylates FADD at Ser194 both in vitro and in vivo. FADD-CKIalpha association regulates the subcellular localization of FADD, and phosphorylated FADD was found to colocalize with CKIalpha on the spindle poles in metaphase. Inhibition of CKIalpha diminished FADD phosphorylation, prevented the ability of Taxol to arrest cells in mitosis, and blocked mitogen-induced proliferation of mouse splenocytes. In contrast, a low level of cycling splenocytes from mice expressing FADD with a mutated phosphorylation site was insensitive to CKI inhibition. These data suggest that phosphorylation of FADD by CKI is a crucial event during mitosis.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Casein Kinase Ialpha/metabolism , Serine/metabolism , Adaptor Proteins, Signal Transducing/genetics , Amino Acid Sequence , Animals , Apoptosis , Binding Sites/genetics , Casein Kinase Ialpha/genetics , Casein Kinase Ialpha/isolation & purification , Cell Cycle/drug effects , Cell Cycle/physiology , Cell Line , Cell Nucleus/metabolism , Concanavalin A/pharmacology , Cytosol/metabolism , Enzyme Inhibitors/pharmacology , Fas-Associated Death Domain Protein , HeLa Cells , Humans , Isoquinolines/pharmacology , Jurkat Cells , Lymphocyte Activation , Mice , Mice, Inbred C57BL , Mice, Mutant Strains , Mitosis/drug effects , Mitosis/physiology , Molecular Sequence Data , Mutation/genetics , Paclitaxel/pharmacology , Phosphorylation , Protein Binding , Protein Transport/genetics , RNA, Small Interfering/genetics , Sequence Homology, Amino Acid , Spindle Apparatus/metabolism , T-Lymphocytes/metabolism , Tetradecanoylphorbol Acetate/pharmacology , TransfectionABSTRACT
MDMX is a homolog of MDM2 that is critical for regulating p53 function during mouse development. MDMX degradation is regulated by MDM2-mediated ubiquitination. Whether there are other mechanisms of MDMX regulation is largely unknown. We found that MDMX binds to the casein kinase 1 alpha isoform (CK1alpha) and is phosphorylated by CK1alpha. Expression of CK1alpha stimulates the ability of MDMX to bind to p53 and inhibit p53 transcriptional function. Regulation of MDMX-p53 interaction requires CK1alpha binding to the central region of MDMX and phosphorylation of MDMX on serine 289. Inhibition of CK1alpha expression by isoform-specific small interfering RNA (siRNA) activates p53 and further enhances p53 activity after ionizing irradiation. CK1alpha siRNA also cooperates with DNA damage to induce apoptosis. These results suggest that CK1alpha is a functionally relevant MDMX-binding protein and plays an important role in regulating p53 activity in the absence or presence of stress.
