Phosphorylation of serine 212 confers novel activity to human estrogen receptor α

Estrogen receptor α (ERα) can be phosphorylated at various residues, one of which is serine 212 in the DNA binding domain. The majority of human nuclear receptors conserves, as a motif, this serine residue within their DNA binding domain. Among these nuclear receptors, phosphorylation of the corresponding threonine 38 in the nuclear receptor CAR is essential for determining its activity [9]. Here, we have investigated the role of phosphorylated serine 212 in the regulation of ERα activity by comparing it with serine 236, another potential phosphorylation site within the DNA binding domain, and demonstrated that phosphorylation of serine 212 confers upon ERα a distinct activity regulating gene expression in Huh-7 cells. In Western blot analysis, wild type ERα and mutants ERα S212A, ERα S212D, ERα S236A and ERα S236D were equally expressed in the nucleus, thus indicating that phosphorylation does not determine nuclear localization of ERα. ERα S212D, but not ERα S236D, retained its capability of activating an ERE-reporter gene in luciferase assays. Similar results were also obtained for human ERβ; the ERβ S176D mutant retained its trans-activation activity, but the ERβ S200D mutant did not. cDNA microarray and Ingenuity Pathway Analysis, employed on Huh-7 cells ectopically expressing either ERα S212A or ERα S212D, revealed that phosphorylation of serine 212 enabled ERα to regulate a unique set of genes and cellular functions.     

Phosphorylation of the Core Protein of Hepatitis B Virus by a 46-Kilodalton Serine Kinase

Core protein is the major component of the core particle (nucleocapsid) of human hepatitis B virus. Core particles and core proteins are involved in a number of important functions in the replication cycle of the virus, including RNA packaging, DNA synthesis, and recognition of viral envelope proteins. Core protein is a phosphoprotein with most, if not all, of the phosphorylation on C-terminal serine residues. In this study, we identified a serine kinase activity from the ribosome-associated protein fraction of cytoplasm that could specifically bind and phosphorylate the C-terminal portion of recombinant core protein. This kinase is referred to as core-associated kinase (CAK). CAK could be inhibited by the kinase inhibitors heparin and manganese ions but not by spermidine, DRB, H89, or H7, indicating that CAK is distinct from protein kinase A and protein kinase C. CAK could be partially purified by heparin-Sepharose CL-6B and phosphocellulose P11 columns. By using a far-Western assay, three specific proteins, of 46, 35, and 13 kDa, were shown to interact with the C-terminal part of the core protein. These three proteins were present only in the eluted fractions that contains the CAK activity. An in-gel kinase assay showed that a 46-kDa kinase in the same fraction could bind and phosphorylate the C-terminal part of the recombinant core protein. These results indicate that this 46-kDa kinase is most probably CAK. A similar 46-kDa kinase, which exhibits the same profile of sensitivity to kinase inhibitors as that of CAK, is present in both purified intracellular core particles and extracellular 42-nm virions, suggesting that CAK is a candidate for the core particle-associated kinase.     

The arginine-rich domain of hepatitis B virus precore and core proteins contains a signal for nuclear transport.

Precore and core proteins are two related co-carboxy-terminal proteins of hepatitis B virus. Precore protein contains the entire sequence of core protein plus an amino-terminal extension of 29 amino acid residues. Both proteins can display a common antigenic determinant known as core antigen (HBcAg). Clinically, HBcAg is detected in the nucleus, cytoplasm, or both of hepatitis B virus-infected hepatocytes. In order to understand the mechanism that regulates nuclear transport of HBcAg, various portions of precore and core proteins were linked to a reporter protein, human alpha-globin, and expressed in mammalian cells. Our results indicate that the precore protein-specific sequence, although important for nuclear transport, does not contain a nuclear localization signal. Instead, a signal for nuclear transport is located near the carboxy termini of precore and core proteins in the arginine-rich domain. This signal is made up of a set of two direct PRRRRSQS repeats and is highly conserved among mammalian hepadnaviruses.     

A CRM1-dependent nuclear export pathway is involved in the regulation of IRF-5 subcellular localization

Interferon regulatory factors (IRFs) are involved in gene regulation in many biological processes including the antiviral, growth regulatory, and immune modulatory functions of the interferon system. Several studies have demonstrated that IRF-3, IRF-5, and IRF-7 specifically contribute to the innate antiviral response to virus infection. It has been reported that virus-specific phosphorylation leads to IRF-5 nuclear localization and up-regulation of interferon, cytokine, and chemokine gene expression. Two nuclear localization signals have been identified in IRF-5, both of which are sufficient for nuclear translocation and retention in virus-infected cells. In the present study, we demonstrate that a CRM1-dependent nuclear export pathway is involved in the regulation of IRF-5 subcellular localization. IRF-5 possesses a functional nuclear export signal (NES) that controls dynamic shuttling between the cytoplasm and the nucleus. The NES element is dominant in unstimulated cells and results in the predominant cytoplasmic localization of IRF-5. Mutation of two leucine residues in the NES motif to alanine, or three adjacent Ser/Thr residues to the phosphomimetic Asp, results in constitutively nuclear IRF-5 and suggests that phosphorylation of adjacent Ser/Thr residues may contribute to IRF-5 nuclear accumulation in virus-induced cells. IKK-related kinases TBK1 and IKKepsilon have been shown to phosphorylate and activate IRF-3 and IRF-7, leading to the production of type 1 interferons and the development of a cellular antiviral state. We examined the phosphorylation and activation of IRF-5 by TBK1 and IKKepsilon kinases. Although IRF-5 is phosphorylated by IKKepsilon and TBK1 in co-transfected cells, the phosphorylation of IRF-5 did not lead to IRF-5 nuclear localization or activation.     

Nucleocytoplasmic distribution of rabies virus P-protein is regulated by phosphorylation adjacent to C-terminal nuclear import and export signals

Nucleocytoplasmic distribution of the rabies virus phosphoprotein is implicated in the evasion of cellular antiviral mechanisms by rabies virus and has been reported to depend on an N-terminal nuclear export sequence and a C-terminal nuclear localization sequence. This paper identifies a second nuclear export sequence that is located between key residues of the nuclear localization sequence in the phosphoprotein C-terminal domain. The C-terminal domain confers predominantly nuclear localization in unstimulated transfected cells, indicating that the nuclear localization sequence is the dominant signal at steady state. However, protein kinase-C activation or mutagenesis to mimic protein kinase-C phosphorylation at a site proximal to the C-terminal nuclear localization/export sequences shifts the targeting activity of the C-terminal domain toward nuclear exclusion, indicating that the nuclear export sequence becomes the dominant signal in activated cells. Mapping of these sequences within the three-dimensional structure of the C-terminal domain indicates that their activities may be coregulated by phosphorylation and/or conformational changes in the domain. The data are consistent with a model in which intimate positioning of the nuclear localization sequence, export sequence, and phosphorylation site within a single domain provides a switch mechanism to rapidly and efficiently balance the reciprocal import and export signals in response to cellular stimuli.     

Analysis of the phosphorylation sites of herpes simplex virus type 1 regulatory protein ICP27

The herpes simplex virus type 1 (HSV-1) regulatory protein ICP27 is a 63-kDa phosphoprotein required for viral replication. ICP27 has been shown to contain both stable phosphate groups and phosphate groups that cycle on and off during infection (K. W. Wilcox, A. Kohn, E. Sklyanskaya, and B. Roizman, J. Virol. 33:167-182, 1980). Despite extensive genetic analysis of the ICP27 gene, there is no information available about the sites of the ICP27 molecule that are phosphorylated during viral infection. In this study, we mapped several of the phosphorylation sites of ICP27 following in vivo radiolabeling. Phosphoamino acid analysis showed that serine is the only amino acid that is phosphorylated during infection. Two-dimensional phosphopeptide mapping showed a complex tryptic phosphopeptide pattern with at least four major peptides and several minor peptides. In addition, ICP27 purified from transfected cells yielded a similar phosphopeptide pattern, suggesting that cellular kinases phosphorylate ICP27 during viral infection. In vitro labeling showed that protein kinase A (PKA), PKC, and casein kinase II (CKII) were able to differentially phosphorylate ICP27, resulting in distinct phosphopeptide patterns. The major phosphorylation sites of ICP27 appeared to cluster in the N-terminal portion of the protein, such that a frameshift mutant that encodes amino acids 1 to 163 yielded a phosphopeptide pattern very similar to that seen with the wild-type protein. Further, using small deletion and point mutations in kinase consensus sites, we have elucidated individual serine residues that are phosphorylated in vivo. Specifically, the serine at residue 114 was highly phosphorylated by PKA and the serine residues at positions 16 and 18 serve as targets for CKII phosphorylation in vivo. These kinase consensus site mutants were still capable of complementing the growth of an ICP27-null mutant virus. Interestingly, phosphorylation of the serine at residue 114, which lies within the major nuclear localization signal, appeared to modulate the efficiency of nuclear import of ICP27.     

Phosphorylation of Cdc6 at serine 74, but not at serine 106, drives translocation of Cdc6 to the cytoplasm

Phosphorylation‐dependent cytoplasmic translocation of human Cdc6 during S phase is sufficient to control its activity after origin firing. Export from the nucleus also serves as a mechanism for preventing re‐replication in mammalian cells. Phosphorylation of the CDK consensus serine residues 54, 74, and 106 has been suggested to be involved in the cytoplasmic translocation of Cdc6. To determine the relative importance of the three phosphorylation sites, we have generated Cdc6 variants by substituting one or more of the three serine residues with alanine or aspartic acid and have assessed their cytoplasmic translocation behavior. Phosphorylation of serine 74 mainly contributes to the cytoplasmic translocation of Cdc6, while serine 54 phosphorylation provides a minor contribution. In contrast, phosphorylation at serine 106 does not affect the nuclear export of Cdc6. Comparative results were found in cells coexpressing the phosphorylation defective mutants of Cdc6 and cyclin A as well as in non‐transfected cells synchronized by their release from a double thymidine block. We conclude that Cdk‐mediated phosphorylation of Cdc6 at serine 74 is required for the cytoplasmic translocalization of Cdc6 during the cell cycle. Phosphorylation of Cdc6 at serine 54 plays a minor role and phosphorylation of serine 106 plays no role in the cytoplasmic localization of Cdc6. The phosphorylation of S74 in Cdc6 could be important for binding to the nuclear export protein for translocalization. J. Cell. Physiol. 228: 1221–1228, 2013. © 2012 Wiley Periodicals, Inc.     

Identification of major ERK-related phosphorylation sites in Gab1

Gab1 (Grb2-associated binder1) belongs to a family of multifunctional docking proteins that play a central role in the integration of receptor tyrosine kinase (RTK) signaling, i.e., mediating cellular growth response, transformation, and apoptosis. In addition to RTK-specific tyrosine phosphorylation, these docking proteins also can be phosphorylated on serine/threonine residues affecting signal transduction. Since serine and threonine phosphorylation are capable of modulating the initial signal one major task to elucidate signal transduction via Gab1 is to determine the exact localization of distinct phosphorylation sites. To address this question in this report we examined extracellular signal-regulated kinases 1/2 (ERK) specific serine/threonine phosphorylation of the entire Gab1 engaged in insulin signaling in more detail in vitro. To elucidate the ERK1/2-specific phosphorylation pattern of Gab1, we used phosphopeptide mapping by two-dimensional HPLC analysis. Subsequently, phosphorylated serine/threonine residues were identified by sequencing the separated phosphopeptides using matrix assisted laser desorption ionization mass spectrometry (MALDI-MS) and Edman degradation. Our results demonstrate that ERK1/2 phosphorylate Gab1 at six serine/threonine residues (T312, S381, S454, T476, S581, S597) in consensus motifs for MAP kinase phosphorylation. Serine residues S454, S581, S597, and threonine residue T476 represent nearly 80% of overall incorporated phosphate. These sites are located adjacent to src homology region-2 (SH2) binding motifs (YVPM-motif: Y447, Y472, Y619) specific for the phosphatidylinositol 3kinase (PI3K). The biological role of identified phosphorylation sites was proven by PI3K and Akt activity in intact cells. These data demonstrate that ERK1/2 modulate insulin action via Gab1 by targeting serine and threonine residues beside YXXM motifs. Accordingly, insulin signaling is blocked at the level of PI3K.     

Dephosphorylation of West Nile virus capsid protein enhances the processes of nucleocapsid assembly

West Nile virus (WNV) capsid (C) protein is one of the three viral structural proteins and it encapsidates the viral RNA to form the nucleocapsid. It is known to be a multifunctional protein involved in assembly and apoptosis. WNV C protein was previously found to be phosphorylated in infected cells and bioinformatic analysis revealed 5 putative phosphorylation sites at serine 26, 36, 83, 99 and threonine 100. Phosphorylation was abolished through mutagenesis of these putative phosphorylation sites to investigate how phosphorylation could affect the processes of nucleocapsid assembly like RNA binding, oligomerization and cellular localization. It was found that phosphorylation attenuated its RNA binding activity. Although oligomerization was not inhibited by mutagenesis of the putative phosphorylation sites, the rate of dimerization and oligomerization was affected. Hypophosphorylation of C protein reduced its nuclear localization efficiency and hence enhanced cytoplasmic localization. This study also revealed that although WNV C is phosphorylated in infected cells, the relative level of phosphorylation is reduced over the course of an infection to promote RNA binding and nucleocapsid formation in the cytoplasm. This is the first report to describe how dynamic phosphorylation of WNV C protein modulates the processes involved in nucleocapsid assembly.     

Analysis of a nuclear localization signal of simian virus 40 major capsid protein Vp1.

The nuclear localization signal of the major structural protein, Vp1, of simian virus 40 was further defined by mutagenesis. The targeting activity was examined in cells microinjected with SV-Vp1 variant viral DNAs bearing either an initiation codon mutation of the agnoprotein or mutations in the Vp1 coding sequence or microinjected with pSG5-Vp1 and pSG5-Vp1 mutant DNAs in which Vp1 or mutant Vp1 is expressed from simian virus 40 early promoter. The Vp1 nuclear localization signal functioned autonomously without agno-protein once the Vp1 protein was synthesized in the cytoplasm. The targeting activity was localized to the amino-terminal 19 residues. While replacement of cysteine 10 with glycine, alanine, or serine did not affect the activity, replacement of arginine 6 with glycine caused the cytoplasmic phenotype. When multiple mutations were introduced among residue 5, 6, 7, 16, 17, or 19, the targeting activity was found to reside in two clusters of basic residues, a cluster of lysine 5, arginine 6, and lysine 7 and a cluster of lysine 16, lysine 17, and lysine 19. The clusters are independently important for nuclear localization activity.     

Hepatitis B virus core antigen has two nuclear localization sequences in the arginine-rich carboxyl terminus.

Expression of the hepatitis B virus core antigen (HBcAg) in mouse NIH 3T3 fibroblasts has been shown previously (A. McLachlan et al., J. Virol. 61:683-692, 1987) to result in the nuclear localization of this polypeptide. Since the carboxyl terminus of HBcAg contains four clusters of arginine residues which resemble nuclear localization sequences identified in other nuclear proteins, a series of carboxyl-terminus-truncated HBcAg polypeptides were expressed in mouse fibroblasts to examine the role of these sequences in the cellular localization of HBcAg. By immunofluorescence and cell fractionation analysis, it was demonstrated that regions of the HBcAg polypeptide including the most carboxyl-terminal (cluster 1) and amino-terminal (cluster 4) clusters of arginine residues represent distinct and independent nuclear localization sequences for this polypeptide. Substitution of a threonine residue for the second arginine residue in cluster 4 inactivates the nuclear localization signal in this region of the HBcAg polypeptide, demonstrating the importance of this residue to this signal sequence. However, HBcAg fails to accumulate in the nucleus only when both nuclear localization signal sequences are simultaneously deleted or disrupted by mutation. The possible significance of the nuclear localization sequences identified in the HBcAg polypeptide is discussed in the context of the role of the nucleocapsid in the hepatitis B virus life cycle.     

Characterization of the phosphorylation status of the hepatitis B virus X-associated protein 2

The cytosolic Ah receptor (AhR) heterocomplex consists of one molecule of the AhR, a 90-kDa heat shock protein (Hsp90) dimer, and one molecule of the hepatitis B virus X-associated protein 2 (XAP2). Serine residues 43,53,131-2, and 329 on XAP2-FLAG were identified as putative phosphorylation sites using site-directed mutagenesis followed by two-dimensional phosphopeptide mapping analysis. Protein kinase CK2 (CK2) was identified as the 45-kDa kinase from COS 1 cell or liver extracts that was responsible for phosphorylation of serine 43 in the XAP2 peptide 39-57. Loss of phosphorylation at any or all of the serine residues did not significantly affect the ability of XAP2-FLAG to bind to the murine AhR in rabbit reticulocyte lysate or Hsp90 in COS-1 cells. Furthermore, all of these serine mutants were able to sequester murine AhR-YFP into the cytoplasm as well as wild-type XAP2. YFP-XAP2 S53A was unable to enter the nucleus, indicating a potential role of phosphorylation in nuclear translocation of XAP2.     

Nuclear localization of the cell cycle regulator CDH1 and its regulation by phosphorylation

The anaphase-promoting complex activated by CDC20 and CDH1 is a major ubiquitination system that controls the destruction of cell cycle regulators. Exactly how ubiquitination is regulated in time and space is incompletely understood. Here we report on the cell cycle-dependent localization of CDH1 and its regulation by phosphorylation. CDH1 localizes dynamically to the nucleus during interphase and to the centrosome during metaphase and anaphase. The nuclear accumulation of CDH1 correlates with a reduction in the steady-state amount of cyclin A, but not of cyclin E. A nuclear localization signal conserved in various species was identified in CDH1, and it sufficiently targets green fluorescent protein to the nucleus. Interestingly, a CDH1-4D mutant mimicking the hyperphosphorylated form was constitutively found in the cytoplasm. In further support of the notion that phosphorylation inhibits nuclear import, the nuclear localization signal of CDH1 with two phospho-accepting serine/threonine residues changed into aspartates was unable to drive heterologous protein into the nucleus. On the other hand, abolition of the cyclin-binding ability of CDH1 has no influence on its nuclear localization. Taken together, our findings document the phosphorylation-dependent localization of CDH1 in vertebrate cells.