Induction of Alzheimer-specific Tau epitope AT100 in apoptotic human fetal astrocytes

In Alzheimer's and other neurodegenerative diseases, hyperphosphorylated tau accumulates in affected neuronal and glial cells in the form of paired helical filaments (PHFs). This tau binds antibody AT100, which recognizes the double phosphorylation site (Thr212/Ser214) that is not present in normal biopsy tau. In primary cultures, highly enriched (>98%) in astrocytes of human fetal brain, three polypeptides of 52, 64, and 70 kD showed immunoreactivity with tau antibodies against non-phosphorylated epitopes, accounting for 88, 12, and <1%, respectively, of the total reactivity. All three polypeptides were phosphorylated at the PHF-1 epitope but not at the epitopes Tau-1, 12E8, AT8, and AT100. Treatment of cultures with okadaic acid resulted in apoptosis characterized by the blebbing of the plasma membrane, condensation of nuclear chromatin, and fragmentation of the nucleus. This treatment also resulted in a 3- to 5-fold increase in the content of both tau protein and phosphorylation. The increases were observed in all phosphorylation sites examined, and included the AT100 site. The AT100 site has been proposed to be generated by protein kinase B/Akt and Cdc2. Since okadaic acid can induce an AD-like hyperphosphorylated state of normal tau in primary cultures of human brain cells, a simple cellular model is available permitting study of self-aggregation of tau and phosphorylation events characteristic of neurodegeneration.     

Changes in the phosphorylation of non-histone chromatin proteins during the cell cycle of HeLa S 3 cells

The phosphorylation of non-histone chromatin proteins in synchronized HeLa S₃ cells was studied in 5 phases of the cell cycle: mitosis, G₁, early and late S, and G₂. The rate of non-histone chromatin protein phosphorylation was found to be maximal during G₁ and G₂, somewhat decreased during S phase, and almost 90% depressed during mitosis. Analysis of the phosphorylated non-histone chromatin proteins by SDS-acrylamide gel electrophoresis showed a heterogeneous pattern of phosphorylation as measured by labeling with ³²P. Significant variations in the labeling pattern were seen during different stages of the cell cycle, and particular unique species appeared to be phosphorylated selectively during certain stages of the cycle.     

Discordance between phosphorylation and recruitment of 53BP1 in response to DNA double-strand breaks

During the DNA damage response (DDR), chromatin modifications contribute to localization of 53BP1 to sites of DNA double-strand breaks (DSBs). 53BP1 is phosphorylated during the DDR, but it is unclear whether phosphorylation is directly coupled to chromatin binding. In this study, we used human diploid fibroblasts and HCT116 tumor cells to study 53BP1 phosphorylation at Serine-25 and Serine-1778 during endogenous and exogenous DSBs (DNA replication and whole-cell or sub-nuclear microbeam irradiation, respectively). In non-stressed conditions, endogenous DSBs in S-phase cells led to accumulation of 53BP1 and γH2AX into discrete nuclear foci. Only the frank collapse of DNA replication forks following hydroxyurea treatment initiated 53BP1^Ser25 and 53BP1^Ser1778 phosphorylation. In response to exogenous DSBs, 53BP1^Ser25 and 53BP1^Ser1778 phosphoforms localized to sites of initial DSBs in a cell cycle-independent manner. 53BP1 phosphoforms also localized to late residual foci and associated with PML-NBs during IR-induced senescence. Using isogenic cell lines and small-molecule inhibitors, we observed that DDR-induced 53BP1 phosphorylation was dependent on ATM and DNA-PKcs kinase activity but independent of MRE11 sensing or RNF168 chromatin remodeling. However, loss of RNF168 blocked recruitment of phosphorylated 53BP1 to sites of DNA damage. Our results uncouple 53BP1 phosphorylation from DSB localization and support parallel pathways for 53BP1 biology during the DDR. As relative 53BP1 expression may be a biomarker of DNA repair capacity in solid tumors, the tracking of 53BP1 phosphoforms in situ may give unique information regarding different cancer phenotypes or response to cancer treatment.     

Phospho-eNOS Ser-114 in human mesenchymal stem cells: constitutive phosphorylation, nuclear localization and upregulation during mitosis

Activity of endothelial nitric oxide synthase (eNOS) is modulated by protein-protein interaction and phosphorylation at specific serine or threonine residues. Using immunofluorescence analysis we show here that proliferating mesenchymal stem cells (MSCs) derived from human bone marrow exhibit cytosolic and pronounced nuclear localization of eNOS. Examination of phosphorylated eNOS subspecies revealed that eNOS phosphorylated at Ser-114 is heavily enriched in the nucleus, whereas eNOS phosphorylated at Ser-1177 is localized at filamentous structures in the cytosol that are abundant in the perinuclear region. Phosphorylation of eNOS at Ser-114 but not at Ser-1177 was strongly increased in cells shortly before mitosis and decreased to normal level after completed cell division. Double immunofluorescence analysis revealed that subcellular localization of 8-hydroxyguanosine immunoreactivity was overlapping with eNOS phosphorylated at Ser-114 in human MSCs providing evidence that phosphorylation at this residue is linked to the generation of superoxide anions. As expected there was only a weak colocalization between eNOS phosphorylated at Ser-1177 and caveolin-1. Different from many other cell systems, human MSCs accumulate eNOS in the nucleus without an acute stimulus. eNOS constitutively phosphorylated at distinct amino acid residues is targeted to different subcellular compartments pointing to an important role of specific phosphorylation events in the life cycle of proliferating human MSCs.     

Regulation of merlin by protein phosphatase 1-TIMAP and EBP50 in endothelial cells

Merlin (moesin-ezrin-radixin like protein), the product of neurofibromatosis type 2 gene, was primarily recognized as a tumor suppressor, but it also functions as a membrane-cytoskeletal linker and regulator of multiple signaling pathways. The activity and localization of merlin is regulated by head to tail folding that is controlled by phosphorylation of the Ser518 side chain. Merlin localizes in the nucleus when the Ser518 side chain is not phosphorylated, while the phosphorylated form is present in the cytoplasm and the plasma membrane. In this work interactions and their impact on the subcellular localization and phosphorylation state of the Ser518 side chain of merlin were investigated in endothelial cells. It is shown that merlin (dephospho-Ser518 form) interacts in the nucleus of endothelial cells with the scaffolding protein EBP50, a member of the Na⁺/H⁺exchanger regulatory factor family. Upon EBP50 depletion, merlin translocated from the nucleus, suggesting that binding of merlin to EBP50 is critical in the nuclear localization of merlin. Along with the translocation, the phosphorylation level of phospho-Ser518-merlin was increased in EBP50 depleted cells. TIMAP (TGFβ-inhibited membrane-associated protein), a type 1 protein phosphatase (PP1) regulatory subunit, was newly recognized as an interacting partner for merlin. Domain mapping using truncated mutant forms in GST pull down revealed that the N-terminal half of TIMAP (aa 1-290) and the FERM domain of merlin are the regions responsible for the interaction.The catalytic subunit of PP1 (PP1c) was present in all merlin-TIMAP pull down or immunoprecipitation samples demonstrating that merlin actually interacts with the PP1c-TIMAP holoenzyme. On the other hand, from TIMAP depleted cells, without its targeting protein, PP1c could not bind to merlin. Also, when the phosphatase activity of PP1c-TIMAP was inhibited either with depletion of TIMAP or by treatment of the cells with specific PP1 inhibitor, there was an increase in the amount of phospho-Ser518 form of merlin in the membrane of the cells. These data strongly suggest that the PP1c-TIMAP- complex dephosphorylates phospho-Ser518-merlin. ECIS measurements indicate that phospho-merlin accelerates in vitro wound healing of the endothelial monolayer.In conclusion, in endothelial cells, EBP50 is required for the nuclear localization of merlin and the PP1c-TIMAP holoenzyme plays an important role in the dephosphorylation of merlin on its Ser518 side chain, which influence cell migration and proliferation.     

Cell cycle-dependent chromatin shuttling of HBO1–JADE1 histone acetyl transferase (HAT) complex

HAT HBO1 interacts with 2 isoforms of JADE1: JADE1S and JADE1L. JADE1 promotes acetylation of nucleosomal histones by HBO1. HBO1–JADE1 complex facilitates cell proliferation by unclear mechanisms. Here we report intracellular chromatin shuttling of HBO1–JADE1 complex during mitosis coupled to phosphorylation of JADE1. In interphase of dividing cells JADE1S was localized to the nucleus and associated with chromatin. As cells approached mitosis, specifically prophase, JADE1S dissociated from chromatin and associated with cytoplasm. JADE1S chromatin re-association began in telophase and paralleled nuclear envelope membrane reassembly. By early G₁, JADE1S was re-associated with chromatin and localized to the nucleus. Importantly, cytoplasmic but not chromatin-associated JADE1 protein was phosphorylated. Mass-Spectrometric analysis of JADE1S protein isolated from G₂/M-arrested cells identified 6 phosphorylated amino acid residues: S89, T92, S102, S121, S392, and T468, including 3 novel sites. Temporally, JADE1S phosphorylation and dephosphorylation during mitosis correlated with JADE1S chromatin dissociation and recruitment. JADE1S chromatin recruitment was accompanied by the global histone H4 acetylation. Pharmacological inhibitor of Aurora A kinase prevented JADE1S protein band shift and chromatin dissociation, suggesting regulatory function for phosphorylation. In vivo experiments supported our in vitro results. In mouse kidneys, JADE1S transiently accumulated in the cytoplasm of tubular epithelial cells during kidney regeneration. The transient increase in the number of cells with cytoplasmic JADE1S directly correlated with activation of tubular cell proliferation and inversely correlated with the number of cells with nuclear JADE1S staining, supporting biological role of HBO1–JADE1 shuttling during organ regeneration.     

Phosphorylation-dependent control of ZIPK nuclear import is species specific

ZIPK (zipper-interacting protein kinase) is a Ca²⁺-independent protein kinase that promotes myosin phosphorylation in both smooth muscle and non-muscle cells. A recent report attempted to clarify a debate over the subcellular localization of ZIPK in non-muscle cells (Shoval et. al. (2007) Plos Genetics. 3: 1884-1883). A species-specific loss of a key phosphorylation site (T299) in murine (mouse and rat) ZIPK seems to direct it to the nucleus, while the presence of the T299 site in human ZIPK correlates with cytoplasmic localization. T299 is immediately adjacent to a putative nuclear localization sequence (NLS) and may mask its function when phosphorylated, therefore explaining the species-specific dichotomy of intracellular localization. However, despite the murine ZIPK (mZIPK) lacking the T299 residue that is critical for controlling human ZIPK (hZIPK) subcellular localization, mutational analysis showed that this NLS control locus is nonfunctional in the murine context. A constitutively active Rho promoted the cytoplasmic retention of a human ZIPK mutant that would otherwise localize to the nucleus. Endogenous hZIPK showed sensitivity to the nuclear export inhibitor leptomycin B, suggesting a continuous shuttling between cytoplasm and nucleus that is dependent upon T299 dephosphorylation. Thus, the C-terminal domain of human and murine ZIPK demonstrated quite divergent nuclear import and export functionality. We conclude that in the case of ZIPK, studies between the species may not be directly comparable to each other given the gross differences in intracellular localization and movement.     

CDK-Dependent Nuclear Localization of B-Cyclin Clb1 Promotes FEAR Activation during Meiosis I in Budding Yeast

Cyclin-dependent kinases (CDK) are master regulators of the cell cycle in eukaryotes. CDK activity is regulated by the presence, post-translational modification and spatial localization of its regulatory subunit cyclin. In budding yeast, the B-cyclin Clb1 is phosphorylated and localizes to the nucleus during meiosis I. However the functional significance of Clb1''s phosphorylation and nuclear localization and their mutual dependency is unknown. In this paper, we demonstrate that meiosis-specific phosphorylation of Clb1 requires its import to the nucleus but not vice versa. While Clb1 phosphorylation is dependent on activity of both CDK and polo-like kinase Cdc5, its nuclear localization requires CDK but not Cdc5 activity. Furthermore we show that increased nuclear localization of Clb1 during meiosis enhances activation of FEAR (Cdc Fourteen Early Anaphase Release) pathway. We discuss the significance of our results in relation to regulation of exit from meiosis I.     

14-3-3 Protein mediates phosphorylation of microtubule-associated protein tau by serum- and glucocorticoid-induced protein kinase 1

The microtubule-associated protein, tau, is involved in numerous neuronal processes such as vesicle transport, microtubule-plasma membrane interaction and the intracellular localization of proteins. Tau is known to be phosphorylated by several kinases such as mitogen activated protein kinase, microtubule affinity regulating kinase, and protein kinase A. We found a putative serum- and glucocorticoid-induced protein kinase 1 (SGK1) phosphorylation site within the 207GSRSRTPSLP216 tau amino acid sequence. We report here that SGK1 phosphorylates Ser214 of Tau. Using a pull-down assay, we found that 14-3-3q interacts with SGK1 and tau to form a ternary protein complex that leads to phosphorylation of tau. 14-3-3 and phosphorylated tau were mainly co-localized in the nucleus of COS-1 cells. These results demonstrate that 14-3-3 scaffolds tau with SGK1 to facilitate the phosphorylation of tau at Ser214 and to regulate its subcellular localization.     

Interdependent Phosphorylation within the Kinase Domain T-loop Regulates CHK2 Activity

Chk2 is a critical regulator of the cellular DNA damage repair response. Activation of Chk2 in response to IR-induced damage is initiated by phosphorylation of the Chk2 SQ/TQ cluster domain at Ser¹⁹, Ser³³, Ser³⁵, and Thr⁶⁸. This precedes autophosphorylation of Thr³⁸³/Thr³⁸⁷ in the T-loop region of the kinase domain an event that is a prerequisite for efficient kinase activity. We conducted an in-depth analysis of phosphorylation within the T-loop region (residues 366–406). We report four novel phosphorylation sites at Ser³⁷², Thr³⁷⁸, Thr³⁸⁹, and Tyr³⁹⁰. Substitution mutation Y390F was defective for kinase function. The substitution mutation T378A ablated the IR induction of kinase activity. Interestingly, the substitution mutation T389A demonstrated a 6-fold increase in kinase activity when compared with wild-type Chk2. In addition, phosphorylation at Thr³⁸⁹ was a prerequisite to phosphorylation at Thr³⁸⁷ but not at Thr³⁸³. Quantitative mass spectrometry analysis revealed IR-induced phosphorylation and subcellular distribution of Chk2 phosphorylated species. We observed IR-induced increase in phosphorylation at Ser³⁷⁹, Thr³⁸⁹, and Thr³⁸³/Thr³⁸⁹. Phosphorylation at Tyr³⁹⁰ was dramatically reduced following IR. Exposure to IR was also associated with changes in the ratio of chromatin/nuclear localization. IR-induced increase in chromatin localization was associated with phosphorylation at Thr³⁷², Thr³⁷⁹, Thr³⁸³, Thr³⁸⁹, Thr³⁸³/Thr³⁸⁷, and Thr³⁸³/Thr³⁸⁹. Chk2 hyper-phosphorylated species at Thr³⁸³/Thr³⁸⁷/Thr³⁸⁹ and Thr³⁸³/Thr³⁸⁷/Thr³⁸⁹/Tyr³⁹⁰ relocalized from almost exclusively chromatin to predominately nuclear expression, suggesting a role for phosphorylation in regulation of chromatin targeting and egress. The differential impact of T-loop phosphorylation on Chk2 ubiquitylation suggests a co-dependence of these modifications. The results demonstrate that a complex interdependent network of phosphorylation events within the T-loop exchange region regulates dimerization/autophosphorylation, kinase activation, and chromatin targeting/egress of Chk2.     

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.     

Phosphorylation and Activation of RhoA by ERK in Response to Epidermal Growth Factor Stimulation

The small GTPase RhoA has been implicated in various cellular activities, including the formation of stress fibers, cell motility, and cytokinesis. In addition to the canonical GTPase cycle, recent findings have suggested that phosphorylation further contributes to the tight regulation of Rho GTPases. Indeed, RhoA is phosphorylated on serine 188 (¹⁸⁸S) by a number of protein kinases. We have recently reported that Rac1 is phosphorylated on threonine 108 (¹⁰⁸T) by extracellular signal-regulated kinases (ERK) in response to epidermal growth factor (EGF) stimulation. Here, we provide evidence that RhoA is phosphorylated by ERK on ⁸⁸S and ¹⁰⁰T in response to EGF stimulation. We show that ERK interacts with RhoA and that this interaction is dependent on the ERK docking site (D-site) at the C-terminus of RhoA. EGF stimulation enhanced the activation of the endogenous RhoA. The phosphomimetic mutant, GFP-RhoA S88E/T100E, when transiently expressed in COS-7 cells, displayed higher GTP-binding than wild type RhoA. Moreover, the expression of GFP-RhoA S88E/T100E increased actin stress fiber formation in COS-7 cells, which is consistent with its higher activity. In contrast to Rac1, phosphorylation of RhoA by ERK does not target RhoA to the nucleus. Finally, we show that regardless of the phosphorylation status of RhoA and Rac1, substitution of the RhoA PBR with the Rac1 PBR targets RhoA to the nucleus and substitution of Rac1 PBR with RhoA PBR significantly reduces the nuclear localization of Rac1. In conclusion, ERK phosphorylates RhoA on ⁸⁸S and ¹⁰⁰T in response to EGF, which upregulates RhoA activity.     

Phosphorylation of Maize Eukaryotic Translation Initiation Factor 5A (eIF5A) by Casein Kinase 2: IDENTIFICATION OF PHOSPHORYLATED RESIDUE AND INFLUENCE ON INTRACELLULAR LOCALIZATION OF eIF5A*

Maize eukaryotic translation initiation factor 5A (ZmeIF5A) co-purifies with the catalytic α subunit of protein kinase CK2 and is phosphorylated by this enzyme. Phosphorylated ZmeIF5A was also identified after separation of maize leaf proteins by two-dimensional electrophoresis. Multiple sequence alignment of eIF5A proteins showed that in monocots, in contrast to other eukaryotes, there are two serine/threonine residues that could potentially be phosphorylated by CK2. To identify the phosphorylation site(s) of ZmeIF5A, the serine residues potentially phosphorylated by CK2 were mutated. ZmeIF5A and its mutated variants S2A and S4A were expressed in Escherichia coli and purified. Of these recombinant proteins, only ZmeIF5A-S2A was not phosphorylated by maize CK2. Also, Arabidopsis thaliana and Saccharomyces cerevisiae eIF5A-S2A mutants were not phosphorylated despite effective phosphorylation of wild-type variants. A newly developed method exploiting the specificity of thrombin cleavage was used to confirm that Ser² in ZmeIF5A is indeed phosphorylated. To find a role of the Ser² phosphorylation, ZmeIF5A and its variants mutated at Ser² (S2A and S2D) were transiently expressed in maize protoplasts. The expressed fluorescence labeled proteins were visualized by confocal microscopy. Although wild-type ZmeIF5A and its S2A variant were distributed evenly between the nucleus and cytoplasm, the variant with Ser² replaced by aspartic acid, which mimics a phosphorylated serine, was sequestered in the nucleus. These results suggests that phosphorylation of Ser² plays a role in regulation of nucleocytoplasmic shuttling of eIF5A in plant cells.     

Evidence for cytosolic p27(Kip1) ubiquitylation and degradation during adipocyte hyperplasia

Objective: Subcellular localization has been shown to play an important role in determining activity and accumulation of p27 protein during cell cycle progression. The purpose of this study was to examine p27 localization and ubiquitylation in relation to E3 ligase expression during adipocyte hyperplasia. Research Methods and Procedures: This study used the murine 3T3‐L1 preadipocyte model to examine p27 regulation during synchronous cell cycle progression. Cell lysates were isolated over time after hormonal stimulation, fractionated to cytosolic and nuclear compartments, and immunoblotted for relative protein determinations. Results: Data presented in this study show that p27 was present in the cytosol and nucleus in density‐arrested preadipocytes and that abundance in both compartments decreased in a phase‐specific manner as preadipocytes synchronously re‐entered the cell cycle during early phases of adipocyte differentiation. Blocking CRM1‐mediated nuclear export did not prevent degradation, nor did it cause nuclear accumulation of p27, suggesting that distinct mechanisms mediating cytosolic and nuclear p27 degradation were involved. Treating preadipocytes with a potent and specific proteasome inhibitor during hormonal stimulation prevented Skp2 accumulation and p27¹⁸⁷ phosphorylation, which are essential events for SCF^Skp2 E3 ligase activity and nuclear p27 ubiquitylation during S/G₂ phase progression. Proteasome blockade also resulted in the first evidence of cytosolic p27 ubiquitylation during late G₁ phase as preadipocytes undergo the transition from quiescence to proliferation. Discussion: These data are consistent with the postulate that p27 is ubiquitylated and targeted for degradation by the 26S proteasome in a phase‐specific manner by distinct ubiquitin E3 ligases localized to the cytosol and nucleus during adipocyte hyperplasia.     

PKB/Akt mediates cell-cycle progression by phosphorylation of p27(Kip1) at threonine 157 and modulation of its cellular localization

We have shown a novel mechanism of Akt-mediated regulation of the CDK inhibitor p27(kip1). Blockade of HER2/neu in tumor cells inhibits Akt kinase activity and upregulates nuclear levels of the CDK inhibitor (Kip1). Recombinant Akt and Akt precipitated from tumor cells phosphorylated wild-type p27 in vitro. p27 contains an Akt consensus RXRXXT(157)D within its nuclear localization motif. Active (myristoylated) Akt phosphorylated wild-type p27 in vivo but was unable to phosphorylate a T157A-p27 mutant. Wild-type p27 localized in the cytosol and nucleus, whereas T157A-p27 localized exclusively in the nucleus and was resistant to nuclear exclusion by Akt. T157A-p27 was more effective than wild-type p27 in inhibiting cyclin E/CDK2 activity and cell proliferation; these effects were not rescued by active Akt. Expression of Ser(473) phospho Akt in primary human breast cancers statistically correlated with expression of p27 in tumor cytosol. These data indicate that Akt may contribute to tumor-cell proliferation by phosphorylation and cytosolic retention of p27, thus relieving CDK2 from p27-induced inhibition.     

Mediator subunit MED1 modulates intranuclear dynamics of the thyroid hormone receptor

The thyroid hormone receptors (TRs) mediate thyroid hormone (T₃)-dependent gene expression. The nuclear import and export signals that direct TR shuttling are well characterized, but little is known about factors modulating nuclear retention. We used fluorescence-based nucleocytoplasmic scoring and fluorescence recovery after photobleaching in transfected cells to investigate whether Mediator subunits MED1 and MED13 play a role in nuclear retention of TR. When MED1 was overexpressed, there was a striking shift towards a greater nuclear localization of TRβ1 and the oncoprotein v-ErbA, subtypes with cytosolic populations at steady-state, and TRβ1 intranuclear mobility was reduced. For TRα1, there was no observable change in its predominantly nuclear distribution pattern or mobility. Consistent with a role for MED1 in nuclear retention, the cytosolic TRα1 and TRβ1 population were significantly greater in MED1^−/− cells, compared with MED1^+/+ cells. Exposure to T₃ and epidermal growth factor, which induces MED1 phosphorylation, also altered TR intranuclear dynamics. Overexpression of miR-208a, which downregulates MED13, led to a more cytosolic distribution of nuclear-localized TRα1; however, overexpression of MED13 had no effect on TRβ1 localization. The known binding site of MED1 overlaps with a transactivation domain and nuclear export signal in helix 12 of TR's ligand-binding domain (LBD). Coimmunoprecipitation assays demonstrated that TR's LBD interacts directly with exportins 5 and 7, suggesting that binding of exportins and MED1 to TR may be mutually exclusive. Collectively, our data provide evidence that MED1 promotes nuclear retention of TR, and highlight the dual functionality of helix 12 in TR transactivation and nuclear export.     

Phosphorylation and acetylation of chromatin conjugate protein A24.

An analysis was made of the phosphorylation and acetylation of chromatin protein A24, a conjugate of histone 2A and ubiquitin. ³²P-orthophosphate was incorporated into phosphoserine of histone 2A and protein A24 in Novikoff hepatoma ascites cells in culture. The ratio of ³²P incorporation and the pattern of tryptic digestion of ³²P-labeled protein A24 indicated that the histone 2A component was phosphorylated and the ubiquitin component was not. Analysis of ε-N-acetyl lysine in protein A24, histone 2A and ubiquitin showed that while protein A24 and histone 2A were acetylated, ubiquitin was not. Apparently, even though it is conjugated with ubiquitin, the histone 2A portion of protein A24 has the same modifications as free histone 2A. The lack of modification of ubiquitin differs from that of high mobility group (HMG) non-histone chromatin proteins with which it is co-extracted from chromatin.