Ferritoid,a tissue-specific nuclear transport protein for ferritin in corneal epithelial cells

Previously we reported that ferritin in corneal epithelial (CE) cells is a nuclear protein that protects DNA from UV damage. Since ferritin is normally cytoplasmic, in CE cells, a mechanism must exist that effects its nuclear localization. We have now determined that this involves a nuclear transport molecule we have termed ferritoid. Ferritoid is specific for CE cells and is developmentally regulated. Structurally, ferritoid contains multiple domains, including a functional SV40-type nuclear localization signal and a ferritin-like region of approximately 50% similarity to ferritin itself. This latter domain is likely responsible for the interaction between ferritoid and ferritin detected by co-immunoprecipitation analysis. To test functionally whether ferritoid is capable of transporting ferritin into the nucleus, we performed cotransfections of COS-1 cells with constructs for ferritoid and ferritin. Consistent with the proposed nuclear transport function for ferritoid, co-transfections with full-length constructs for ferritoid and ferritin resulted in a preferential nuclear localization of both molecules; this was not observed when the nuclear localization signal of ferritoid was deleted. Moreover, since ferritoid is structurally similar to ferritin, it may be an example of a nuclear transporter that evolved from the molecule it transports (ferritin).     

Phosphorylation of CLIP-170 by Plk1 and CK2 promotes timely formation of kinetochore�Cmicrotubule attachments

CLIP‐170 is implicated in the formation of kinetochore–microtubule attachments through direct interaction with the dynein/dynactin complex. However, whether this important function of CLIP‐170 is regulated by phosphorylation is unknown. Herein, we have identified polo‐like kinase 1 (Plk1) and casein kinase 2 (CK2) as two kinases of CLIP‐170 and mapped S195 and S1318 as their respective phosphorylation sites. We showed that a CK2 unphosphorylatable mutant lost its ability to bind to dynactin and to localize to kinetochores during prometaphase, indicating that the CK2 phosphorylation of CLIP‐170 is involved in its dynactin‐mediated kinetochore localization. Furthermore, we provide evidence that Plk1 phosphorylation of CLIP‐170 at S195 enhances its association with CK2. Finally, we detected defects in the formation of kinetochore fibres in cells expressing the CLIP‐S195A and ‐S1318A, but not the CLIP‐S195E and ‐S1318D, confirming that Plk1‐ and CK2‐associated phosphorylations of CLIP‐170 are involved in the timely formation of kinetochore–microtubule attachments in mitosis.     

RPAP3 interacts with Reptin to regulate UV‐induced phosphorylation of H2AX and DNA damage

We have previously reported that Monad, a novel WD40 repeat protein, potentiates apoptosis induced by tumor necrosis factor‐α and cycloheximide. By affinity purification and mass spectrometry, RNA polymerase II‐associated protein 3 (RPAP3) was identified as a Monad binding protein and may function with Monad as a novel modulator of apoptosis pathways. Here we report that Reptin, a highly conserved AAA + ATPase that is part of various chromatin‐remodeling complexes, is also involved in the association of RPAP3 by immunoprecipitation and confocal microscopic analysis. Overexpression of RPAP3 induced HEK293 cells to death after UV‐irradiation. Loss of RPAP3 by RNAi improved HeLa cell survival after UV‐induced DNA damage and attenuated the phosphorylation of H2AX. Depletion of Reptin reduced cell survival and facilitated the phosphorylation on H2AX. These results suggest that RPAP3 modulates UV‐induced DNA damage by regulating H2AX phosphorylation. J. Cell. Biochem. 106: 920–928, 2009. © 2009 Wiley‐Liss, Inc.     

Dimerization of DNA methyltransferase 1 is mediated by its regulatory domain

DNA methylation is a major epigenetic modification and plays a crucial role in the regulation of gene expression. Within the family of DNA methyltransferases (Dnmts), Dnmt3a and 3b establish methylation marks during early development, while Dnmt1 maintains methylation patterns after DNA replication. The maintenance function of Dnmt1 is regulated by its large regulatory N‐terminal domain that interacts with other chromatin factors and is essential for the recognition of hemi‐methylated DNA. Gelfiltration analysis showed that purified Dnmt1 elutes at an apparent molecular weight corresponding to the size of a dimer. With protein interaction assays we could show that Dnmt1 interacts with itself through its N‐terminal regulatory domain. By deletion analysis and co‐immunoprecipitations we mapped the dimerization domain to the targeting sequence TS that is located in the center of the N‐terminal domain (amino acids 310–629) and was previously shown to mediate replication independent association with heterochromatin at chromocenters. Further mutational analyses suggested that the dimeric complex has a bipartite interaction interface and is formed in a head‐to‐head orientation. Dnmt1 dimer formation could facilitate the discrimination of hemi‐methylated target sites as has been found for other palindromic DNA sequence recognizing enzymes. These results assign an additional function to the TS domain and raise the interesting question how these functions are spatially and temporarily co‐ordinated. J. Cell. Biochem. 106: 521–528, 2009. © 2009 Wiley‐Liss, Inc.     

14‐3‐3γ mediates Cdc25A proteolysis to block premature mitotic entry after DNA damage

14‐3‐3 proteins control various cellular processes, including cell cycle progression and DNA damage checkpoint. At the DNA damage checkpoint, some subtypes of 14‐3‐3 (β and ζ isoforms in mammalian cells and Rad24 in fission yeast) bind to Ser345‐phosphorylated Chk1 and promote its nuclear retention. Here, we report that 14‐3‐3γ forms a complex with Chk1 phosphorylated at Ser296, but not at ATR sites (Ser317 and Ser345). Ser296 phosphorylation is catalysed by Chk1 itself after Chk1 phosphorylation by ATR, and then ATR sites are rapidly dephosphorylated on Ser296‐phosphorylated Chk1. Although Ser345 phosphorylation is observed at nuclear DNA damage foci, it occurs more diffusely in the nucleus. The replacement of endogenous Chk1 with Chk1 mutated at Ser296 to Ala induces premature mitotic entry after ultraviolet irradiation, suggesting the importance of Ser296 phosphorylation in the DNA damage response. Although Ser296 phosphorylation induces the only marginal change in Chk1 catalytic activity, 14‐3‐3γ mediates the interaction between Chk1 and Cdc25A. This ternary complex formation has an essential function in Cdc25A phosphorylation and degradation to block premature mitotic entry after DNA damage.     

Phorbol Esters Increase Dopamine Transporter Phosphorylation and Decrease Transport Vmax

Abstract: Sodium- and chloride-coupled transport of dopamine from synapses into presynaptic terminals plays a key role in terminating dopaminergic neurotransmission. Regulation of the function of the dopamine transporter, the molecule responsible for this translocation, is thus of interest. The primary sequence of the dopamine transporter contains multiple potential phosphorylation sites, suggesting that the function of the transporter could be regulated by phosphorylation. Previous work from this laboratory has documented that phorbol ester activation of protein kinase C (PKC) decreases dopamine transport V _max in transiently expressing COS cells. In the present report, we document in vivo phosphorylation of the rat dopamine transporter stably expressed in LLC-PK₁ cells and show that phosphorylation is increased threefold by phorbol esters. Dopamine uptake is also regulated by phorbol esters in these cells; phorbol 12-myristate 13-acetate (PMA) reduces transport V _max by 35%. Parallels between the time course, concentration dependency, and staurosporine sensitivity of alterations in transporter phosphorylation and transporter V _max suggest that dopamine transporter phosphorylation involving PKC could contribute to this decreased transporter function. Phosphorylation of the dopamine transporter by PKC or by a PKC-activated kinase could be involved in rapid neuroadaptive processes in dopaminergic neurons.     

Phosphorylation of the nuclear transport machinery down-regulates nuclear protein import in vitro

We have examined whether signal-mediated nucleocytoplasmic transport can be regulated by phosphorylation of the nuclear transport machinery. Using digitonin-permeabilized cell assays to measure nuclear import and export, we found that the phosphatase inhibitors okadaic acid and microcystin inhibit transport mediated by the import receptors importin beta and transportin, but not by the export receptor CRM1. Several lines of evidence, including the finding that transport inhibition is partially reversed by the broad specificity protein kinase inhibitor staurosporine, indicate that transport inhibition is due to elevated phosphorylation of a component of the nuclear transport machinery. The kinases and phosphatases involved in this regulation are present in the permeabilized cells. A phosphorylation-sensitive component of the nuclear transport machinery also is present in permeabilized cells and is most likely a component of the nuclear pore complex. Substrate binding by the importin alpha.beta complex and the association of the complex with the nucleoporins Nup358/RanBP2 and Nup153 are not affected by phosphatase inhibitors, suggesting that transport inhibition by protein phosphorylation does not involve these steps. These results suggest that cells have mechanisms to negatively regulate entire nuclear transport pathways, thus providing a means to globally control cellular activity through effects on nucleocytoplasmic trafficking.     

Matrin 3: Chromosomal distribution and protein interactions

Matrin 3 (matr3), an abundant protein of the internal nuclear matrix, has been linked to a variety of functional events. As a step toward defining its multifunctional nature, we have studied the association of matr3 with chromosome territories and identified potential interacting proteins. A similar staining pattern of matr3 was observed in fixed WI38 fibroblast cells and in live HeLa cells using a matr3‐GFP construct. Matr3 was detected throughout autosomal and the active X chromosome territories. Conversely, matr3 was strikingly excluded from the inactive X chromosome as well as within both the perinuclear and perinucleolar heterochromatin. Yeast two hybrid analysis identified matr3 interactions with 33 unique nuclear localized proteins and also revealed its propensity for self association. A majority of these proteins are involved in RNA metabolism and chromatin remodeling while others function in protein translation, DNA replication/repair and apoptosis. Further analysis of a selection of these proteins and scaffold attachment factor A (SAFA) by co‐localization and co‐immunoprecipitation experiments using HeLa cells confirmed their interactions with matr3. J. Cell. Biochem. 108: 125–133, 2009. © 2009 Wiley‐Liss, Inc.     

Artemis phosphorylated by DNA-dependent protein kinase associates preferentially with discrete regions of chromatin

Artemis is a nuclear phosphoprotein required for genomic integrity whose phosphorylation is increased subsequent to DNA damage. Artemis phosphorylation by the DNA-dependent protein kinase (DNA-PK) and the association of Artemis with DNA-PK catalytic subunit (DNA-PKcs) have been proposed to be crucial for the variable, diversity, joining (V(D)J) reaction, genomic stability and cell survival in response to double-stranded DNA breaks. The exact nature of the effectors of Artemis phosphorylation is presently being debated. Here, we have delimited the interface on Artemis required for its association with DNA-PKcs and present the characterization of six DNA-PK phosphorylation sites on Artemis whose phosphorylation shows dependence on its association with DNA-PKcs and is induced by double-stranded DNA damage. Surprisingly, DNA-PKcs Artemis association appeared to be dispensable in a V(D)J recombination assay with stably integrated DNA substrates. Phosphorylation at two of the sites on Artemis, S516 and S645, was verified in vivo using phosphospecific antibodies. Basal Artemis S516 and S645 phosphorylation in vivo showed a significant dependence on DNA-PKcs association. However, regardless of its association with DNA-PKcs, phosphorylation of Artemis at both S516 and S645 was stimulated in response to the double-stranded DNA-damaging agent bleomycin, albeit to a lesser extent. This suggests that additional factors contribute to promote DNA damage-induced Artemis phosphorylation. Intriguingly, pS516/pS645 Artemis was concentrated in chromatin-associated nuclear foci in na?ve cells. These foci were maintained upon DNA damage but failed to overlap with the damage-induced gammaH2AX. These results provide the expectation of a specific role for DNA-PK-phosphorylated Artemis in both na?ve and damaged cells.     

Structural basis for recruitment of BRCA2 by PALB2

The breast cancer 2, early onset protein (BRCA2) is central to the repair of DNA damage by homologous recombination. BRCA2 recruits the recombinase RAD51 to sites of damage, regulates its assembly into nucleoprotein filaments and thereby promotes homologous recombination. Localization of BRCA2 to nuclear foci requires its association with the partner and localizer of BRCA2 (PALB2), mutations in which are associated with cancer predisposition, as well as subtype N of Fanconi anaemia. We have determined the structure of the PALB2 carboxy‐terminal β‐propeller domain in complex with a BRCA2 peptide. The structure shows the molecular determinants of this important protein–protein interaction and explains the effects of both cancer‐associated truncating mutants in PALB2 and missense mutations in the amino‐terminal region of BRCA2.     

Activation of Src kinase by protein–tyrosine phosphatase–PEST in osteoclasts: Comparative analysis of the effects of bisphosphonate and protein–tyrosine phosphatase inhibitor on Src activation in vitro

PTP–PEST is involved in the regulation of sealing ring formation in osteoclasts. In this article, we have shown a regulatory role for PTP–PEST on dephosphorylation of c‐Src at Y527 and phosphorylation at Y418 in the catalytic site. Activation of Src in osteoclasts by over‐expression of PTP–PEST resulted in the phosphorylation of cortactin at Y421 and WASP at Y294. Also enhanced as a result, is the interaction of Src, cortactin, and Arp2 with WASP. Moreover, the number of osteoclasts displaying sealing ring and bone resorbing activity was increased in response to PTP–PEST over‐expression as compared with control osteoclasts. Cells expressing constitutively active‐Src (527YΔF) simulate the effects mediated by PTP–PEST. Treatment of osteoclasts with a bisphosphonate alendronate or a potent PTP inhibitor PAO decreased the activity and phosphorylation of Src at Y418 due to reduced dephosphorylation state at Y527. Therefore, Src‐mediated phosphorylation of cortactin and WASP as well as the formation of WASP·cortactin·Arp2 complex and sealing ring were reduced in these osteoclasts. Similar effects were observed in osteoclasts treated with an Src inhibitor PP2. We have shown that bisphosphonates could modulate the function of osteoclasts by inhibiting downstream signaling mediated by PTP–PEST/Src, in addition to its effect on the inhibition of the post‐translational modification of small GTP‐binding proteins such as Rab, Rho, and Rac as shown by others. The promising effects of the inhibitors PP2 and PAO on osteoclast function suggest a therapeutic approach for patients with bone metastases and osteoporosis as an alternative to bisphosphonates. J. Cell. Physiol. 220: 382–393, 2009. © 2009 Wiley‐Liss, Inc.     

Involvement of the JNK activation in terbinafine‐induced p21 up‐regulation and DNA synthesis inhibition in human vascular endothelial cells

Previously, we demonstrated that the extracellular signal‐regulated kinase (ERK)‐mediated pathway contributes to the terbinafine (TB)‐induced increases of p21 and p53 protein level as well as decrease of DNA synthesis in human umbilical venous endothelial cells (HUVEC). The aim of this study is to examine the involvement of c‐Jun NH2‐terminal kinase (JNK) in the TB‐induced increase of p21 protein level and DNA synthesis inhibition. Western blot analysis and kinase assay demonstrated that TB treatment increased both the protein level and the kinase activity of JNK1/2 in HUVEC. Transfection of HUVEC with JNK1 dominant negative (DN‐JNK1) prevented the TB‐induced increases of p21 and p53 protein level and decrease of DNA synthesis, suggesting that JNK1/2 activation is involved in the TB‐induced cell cycle arrest in HUVEC. Moreover, over‐expression of mitogen‐activated protein kinase (MEK)‐1 prevented the TB‐induced increase of JNK1/2 protein levels, suggesting that MEK‐1 is an upstream inhibitor of JNK. Transfection of HUVEC with DN‐JNK1 prevented the TB‐induced inhibition of ERK phosphorylation, suggesting that JNK1/2 might serve as a negative regulator of ERK. Taken together, our results suggest that JNK activation is involved in the TB‐induced inhibition of ERK phosphorylation, p53 and p21 up‐regulation and DNA synthesis inhibition in HUVEC. J. Cell. Biochem. 108: 860–866, 2009. © 2009 Wiley‐Liss, Inc.     

Modulation of nucleocytoplasmic trafficking by retention in cytoplasm or nucleus

Nuclear protein transport processes have largely been studied using in vitro semi‐intact cell systems where high concentrations of nuclear localizing substrates are used, and cytoplasmic components such as the microtubule (MT) network, are either absent or damaged. Here we use the fluorescence recovery after photobleaching (FRAP) technique to analyze the nucleocytoplasmic flux of distinct fluorescently tagged proteins over time in living cultured cells. FRAP was performed in different parts of the cell to analyze the kinetics of nucleocytoplasmic trafficking and intranuclear/cytoplasmic mobility of the tumor suppressor Rb protein and a SV40 large tumor antigen (T‐ag) derivative containing the nuclear localization sequence (NLS), both fused to green fluorescent protein (GFP). The results indicate that proteins carrying the T‐ag NLS are highly mobile in the nucleus and cytoplasm. Rb, in contrast, is largely immobile in both cellular compartments, with similar nuclear import and export kinetics. Rb nuclear export was CRM‐1‐mediated, with its reduced mobility in the cytoplasm in part due to association with MTs. Overall our results show that nuclear and cytoplasm retention modulates the rates of nuclear protein import and export in intact cells. J. Cell. Biochem. 107: 1160–1167, 2009. © 2009 Wiley‐Liss, Inc.     

Effect on the Ras/Raf signaling pathway of post‐translational modifications of neurofibromin: In silico study of protein modification responsible for regulatory pathways

Mapping and chemical characterization of post‐translational modifications (PTMs) in proteins are critical to understand the regulatory mechanisms involving modified proteins and their role in disease. Neurofibromatosis type 1 (NF‐1) is an autosomal dominantly inherited disorder, where NF1 mutations usually result in a reduced level of the tumor suppressor protein, neurofibromin (NF). NF is a multifunctional cytoplasmic protein that regulates microtubule dynamics and participates in several signaling pathways, particularly the RAS signaling pathway. NF is a Ras GTPase‐activating protein (GAP) that prevents oncogenesis by converting GTP‐Ras to GDP‐Ras. This function of NF is regulated by phosphorylation. Interplay of phosphorylation with O‐GlcNAc modification on the same or vicinal Ser/Thr residues, the Yin Yang sites, is well known in cytoplasmic and nuclear proteins. The dynamic aspects of PTMs and their interplay being difficult to follow in vivo, we undertook this in silico work to predict and define the possible role of Yin Yang sites in NF‐1. Interplay of phosphorylation and O‐GlcNAc modification is proposed as a mechanism controlling the Ras signaling pathway. J. Cell. Biochem. 108: 816–824, 2009. © 2009 Wiley‐Liss, Inc.     

S6K1 is a multifaceted regulator of Mdm2 that connects nutrient status and DNA damage response

p53 mediates DNA damage‐induced cell‐cycle arrest, apoptosis, or senescence, and it is controlled by Mdm2, which mainly ubiquitinates p53 in the nucleus and promotes p53 nuclear export and degradation. By searching for the kinases responsible for Mdm2 S163 phosphorylation under genotoxic stress, we identified S6K1 as a multifaceted regulator of Mdm2. DNA damage activates mTOR‐S6K1 through p38α MAPK. The activated S6K1 forms a tighter complex with Mdm2, inhibits Mdm2‐mediated p53 ubiquitination, and promotes p53 induction, in addition to phosphorylating Mdm2 on S163. Deactivation of mTOR‐S6K1 signalling leads to Mdm2 nuclear translocation, which is facilitated by S163 phosphorylation, a reduction in p53 induction, and an alteration in p53‐dependent cell death. These findings thus establish mTOR‐S6K1 as a novel regulator of p53 in DNA damage response and likely in tumorigenesis. S6K1–Mdm2 interaction presents a route for cells to incorporate the metabolic/energy cues into DNA damage response and links the aging‐controlling Mdm2–p53 and mTOR‐S6K pathways.     

Chromatin PTEN is involved in DNA damage response partly through regulating Rad52 sumoylation

A pool of PTEN localizes to the nucleus. However, the exact mechanism of action of nuclear PTEN remains poorly understood. We have investigated PTEN’s role during DNA damage response. Here we report that PTEN undergoes chromatin translocation after DNA damage, and that its translocation is closely associated with its phosphorylation on S366/T370 but not on S380. Deletional analysis reveals that the C2 domain of PTEN is responsible for its nuclear translocation after exposure to genotoxin. Both casein kinase 2 and GSK3β are involved in the phosphorylation of the S366/T370 epitope, as well as PTEN’s association with chromatin after DNA damage. Significantly, PTEN specifically interacts with Rad52 and colocalizes with Rad52, as well as γH2AX, after genotoxic stress. Moreover, PTEN is involved in regulating Rad52 sumoylation. Combined, our studies strongly suggest that nuclear/chromatin PTEN mediates DNA damage repair through interacting with and modulating the activity of Rad52.