The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

The Journal of Immunology, 2008, 181, 5015 -5023
Copyright © 2008 by The American Association of Immunologists, Inc.
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Zhang, X.
Right arrow Articles by Rosengart, M. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Zhang, X.
Right arrow Articles by Rosengart, M. R.

Calcium/Calmodulin-Dependent Protein Kinase (CaMK) IV Mediates Nucleocytoplasmic Shuttling and Release of HMGB1 during Lipopolysaccharide Stimulation of Macrophages1

Xianghong Zhang2,*, David Wheeler2,*, Ying Tang*, Lanping Guo*, Richard A. Shapiro*, Thomas J. Ribar{dagger}, Anthony R. Means{dagger}, Timothy R. Billiar*, Derek C. Angus{ddagger} and Matthew R. Rosengart3,*,{ddagger}

* Department of Surgery, University of Pittsburgh, Pittsburgh, PA 15213; {dagger} Department of Pharmacology and Cancer Biology, Duke University, Durham, NC 27710; and {ddagger} Department of Critical Care Medicine, University of Pittsburgh, Pittsburgh, PA 15213


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The chromatin-binding factor high-mobility group box 1 (HMGB1) functions as a proinflammatory cytokine and late mediator of mortality in murine endotoxemia. Although serine phosphorylation of HMGB1 is necessary for nucleocytoplasmic shuttling before its cellular release, the protein kinases involved have not been identified. To investigate if calcium/calmodulin-dependent protein kinase (CaMK) IV serine phosphorylates and mediates the release of HMGB1 from macrophages (M{phi}) stimulated with LPS, RAW 264.7 cells or murine primary peritoneal M{phi} were incubated with either STO609 (a CaMKIV kinase inhibitor), KN93 (a CaMKIV inhibitor), or we utilized cells from which CaMKIV was depleted by RNA interference (RNAi) before stimulation with LPS. We also compared the LPS response of primary M{phi} isolated from CaMKIV+/+ and CaMKIV–/– mice. In both cell types LPS induced activation and nuclear translocation of CaMKIV, which preceded HMGB1 nucleocytoplasmic shuttling. However, M{phi} treated with KN93, STO609, or CaMKIV RNAi before LPS showed reduced nucleocytoplasmic shuttling of HMGB1 and release of HMGB1 into the supernatant. Additionally, LPS induced serine phosphorylation of HMGB1, which correlated with an interaction between CaMKIV and HMGB1 and with CaMKIV phosphorylation of HMGB1 in vitro. In cells, both HMGB1 phosphorylation and interaction with CaMKIV were inhibited by STO609 or CaMKIV RNAi. Similarly, whereas CaMKIV+/+ M{phi} showed serine phosphorylation of HMGB1 in response to LPS, this phosphorylation was attenuated in CaMKIV–/– M{phi}. Collectively, our results demonstrate that CaMKIV promotes the nucleocytoplasmic shuttling of HMGB1 and suggest that the process may be mediated through CaMKIV-dependent serine phosphorylation of HMGB1.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
High-mobility group box 1 (HMGB1)4 was initially identified as an architectural chromatin-binding factor that bends DNA and directs protein assembly on specific DNA targets. Recently it has been demonstrated to function as a proinflammatory cytokine and late mediator of mortality in murine endotoxemia and sepsis (1, 2, 3). HMGB1 appeared 8 h poststimulation and plateaued at 16–32 h, a time course very distinct from the acute rise and fall of early mediators of severe sepsis and septic shock (TNF-{alpha}, IL-1β) (3). Recombinant HMGB1 mimicked the lethality of high-dose LPS and induced the release of TNF-{alpha} by macrophages (M{phi}) (3). Anti-HMGB1 Abs conferred a dose-dependent protection in animal models of endotoxemia, even when the first dose of anti-HMGB1 Abs was delayed for at least 2 h (2, 3). Human studies have revealed that systemic HMGB1 levels are markedly elevated in patients that die of sepsis and during hemorrhagic shock (4, 5).

In the context of sepsis, current data indicate that HMGB1 is released by activated monocytes and M{phi} via an active process that involves shuttling the protein from nucleus to cytoplasm (3, 6, 7). Acetylation of HMGB1 and of the histones to which it binds appears to be essential for its release (8). HMGB1 contains two nuclear localization signals (NLS) and two putative nuclear export signals, suggesting that HMGB1 shuttles between the nucleus and cytoplasm through a tightly controlled mechanism (6). Recently, serine phosphorylation has been demonstrated to be a requisite step for this process, although neither the kinase nor the mechanisms controlling nucleocytoplasmic shuttling have been identified (9). Considering the data suggesting that HMGB1 may serve as a target to reduce mortality from sepsis, identifying the mechanisms responsible for inducing and controlling its release is important.

We have recently demonstrated the integral role for members of the family of multifunctional calcium/calmodulin-dependent protein kinases (CaMKI, II, IV) in the release of HMGB1 by hepatocytes subjected to oxidant stress and hepatocellular injury in an in vivo model of hepatic ischemia/reperfusion, although we have yet to identify the specific members involved (10). The multifunctional CaMKs are serine/threonine kinases sensitive to changes in intracellular [Ca2+] that coordinate a variety of cellular functions, including gene expression, cell cycle progression, apoptosis, differentiation, and ischemic tolerance (11, 12). Whereas isoforms of CaMKI and CaMKII are expressed in all mammalian cells, CaMKIV is present in only selective tissues, which include the bone marrow (13). CaMKIV is activated and translocates into the nucleus upon its phosphorylation by an upstream CaMKK in the cytoplasm (14, 15). The nuclear, autonomously active form of CaMKIV phosphorylates a number of proteins involved in the regulation of transcription (16). Additionally, it has recently been shown that CaMKIV is a component of a signaling cascade initiated by LPS activation of TLR4 that facilitates survival of dendritic cells by phosphorylating CREB and regulating expression of the Bcl-2 gene (17). These observations suggested to us that CaMKIV would be an attractive candidate kinase to phosphorylate HMGB1 in macrophages and facilitate its translocation from nucleus to cytoplasm in response to LPS.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Reagents

Escherichia coli 0111:B4 LPS was obtained from Sigma-Aldrich. KN93, obtained from Calbiochem, was dissolved in sterile DMSO at a concentration of 10 mM. STO690 was obtained from Calbiochem. STO609 is selective for CaMKK: it has an in vitro IC50 of 0.13–0.38 µM for CaMKK and 32 µM for CaMKII with little or no inhibition of CaMKI, CaMKIV, protein kinase A, protein kinase C, ERK, or myosin light chain kinase (18). mAb against autonomously active, Thr196-phosphorylated CaMKIV (anti-p-Thr196-CaMKIV) was the generous gift of Dr. Naohito Nozaki (Kanagawa, Japan). Abs against total CaMKIV and HMGB1 were obtained from Abcam. Ab against phosphoserine was obtained from Promega. Ab against FLAG epitope was obtained from Sigma-Aldrich. DAPI was obtained from Molecular Probes.

Cell isolation and treatment

Murine monocyte/macrophage-like cells (RAW 264.7, American Type Culture Collection) were grown in DMEM (BioWhittaker) supplemented with 10% FCS (Sigma-Aldrich), 50 U/ml penicillin, and 50 µg/ml streptomycin (Cellgro/Mediatech). Primary murine peritoneal M{phi} were isolated by lavaging the peritoneal cavity with five 3-ml aliquots of sterile PBS. After centrifugation at 300 x g for 10 min, the M{phi} were resuspended in RPMI 1640 with 10% FCS, 50 U/ml penicillin, and 50 µg/ml streptomycin. Selected cells were pretreated with varying concentrations of KN93 (5, 10, 20 µM) or STO609 (1, 2, 5, 10, 20 µM) for 1 h or subjected to RNA interference (RNAi) using nontarget or CaMKIV small interfering RNA (siRNA) (see below). Selected cells were then treated with increasing doses of LPS (1, 10, 100 ng/ml).

Transfection of fluorescein-labeled cyclophilin, nontarget, and CaMKIV siRNA

RAW 264.7 cells (2 x 104) or murine peritoneal M{phi} (1 x 105) were plated in 0.5 ml of growth medium (without antibiotics) in each well of a 24-well plate, resulting in 30% or 80% confluence, respectively. Fluorescein-labeled cyclophilin control siRNA, nontarget siRNA, and CaMKIV siRNA obtained from Dharmacon were added to 50 µl serum-free DMEM in a final concentration of 25 nM. We utilized the SMARTpool siRNA from Dharmacon that incorporates four separate siRNA sequences for CaMKIV: 5'-GAGAUCCUCUGGGCGAUUUUU3-', 5'-UCAAGGAAAUAUUCGAAACUU3-', 5'-GGUGCUACAUCCAUUGUGUUU3-', and 5'-GGGAUGAAGUGUCUUUAAAUU3-'. In a separate tube, 3 µl HiPerFect was diluted in 50 µl serum-free DMEM and incubated at room temperature for 5 min. These two solutions were combined, and the final transfection mixture was incubated for 20 min at room temperature. This transfection mixture was applied to each well and incubated for 6 h, after which it was replaced by 500 µl of cell medium and incubated for 72 h. Transfection efficiency was determined at 24 h by fluorescence microscopy. For each experiment at least three microscopic visual fields (x200 magnification) were counted to facilitate calculation of the ratio of fluorescent cyclophilin-expressing cells to nonfluorescent cells. Inhibition of each targeted protein was determined by immunoblot or RT-PCR. All experiments and cell number determinations were performed in triplicate.

Plasmid construction and transfection

Plasmids encoding a constitutively active CaMKIV (CaMKIV-dCT) or a kinase-inactive CaMKIV-dCTK75E mutant were the generous gifts of Dr. Douglas Black (19). CaMKIV-dCT contains a C-terminally truncated version of the human CaMKIV-encoding gene, truncated to Leu317, and an N-terminal FLAG epitope (19, 20). CaMKIV-dCTK75E was constructed by changing Lys75 to glutamate in CaMKIV-dCT, which negatively affects ATP binding at the catalytic site (19, 20). For transient transfection, RAW 264.7 cells were seeded in a 24-well plate at 3 x 105 cells/well. After 2 h of adhesion, M{phi} were transfected with 1 µg of plasmid CaMKIV-dCT or CaMKIV-dCTK75E using the Lipofectamine 2000 reagent according to the instructions specified by the manufacturer (Invitrogen). Following transfection, cells were handled as detailed in the figure legends.

Cellular protein extraction

Total cellular protein was extracted at 4°C in 500 µl of lysis buffer (20 mM Tris, 137 mM NaCl, 2 mM EDTA, 10% glycerol, 1% Triton X-100, 1 µM sodium orthovanadate, 100 µM DTT, 200 µM PMSF, 10 µg/ml leupeptin, 0.15 U/ml aprotinin, 50 mM sodium fluoride, 10 mM sodium pyrophosphate, 2.5 µg/ml pepstatin A, 1 mM benzamidine, and 40 mM {alpha}-glycerophosphate). Protein concentration was determined using a bicinchoninic acid protein assay (Pierce).

Nuclear and cytoplasmic protein isolation

Cells were harvested and washed with PBS, followed by centrifugation at 300 x g for 10 min. The cell pellet was lysed with NE-PER nuclear and cytoplasmic extraction reagent according to the instructions specified by the manufacturer (Thermo Scientific).

Immunoprecipitation

Equal amounts of cellular protein obtained from each experimental condition were used for immunoprecipitation. Five microliters of Ab was added to 500 µg of isolated cellular protein within lysis buffer and incubated at 4°C overnight on a rotator. Fifty microliters of 50% slurry of prewashed protein G-agarose beads (Abcam) were then added to each sample, followed by incubation for an additional 2 h at 4°C. The samples were spun briefly in a microcentrifuge at 14,000 rpm and washed four times in lysis buffer. Samples were then resuspended in 30 µl of lysis buffer for future analysis.

Immunoblots

Total cellular protein was electrophoresed in a 10% SDS-PAGE gel and transferred to a Hybond ECL nitrocellulose membrane (Amersham Pharmacia Biotech). The membrane was blocked for 1 h at room temperature with 5% milk and was then incubated with primary Ab for 12 h at 4°C. Blots were then incubated in a HRP-conjugated secondary Ab against the primary Ab at room temperature for 1 h. The blot was developed using the SuperSignal chemiluminescent substrate (Pierce) and exposed on KAR-5 film (Eastman Kodak). In addition to utilizing the primary anti-threonine phosphorylated (Thr196) CaMKIV Ab, the active threonine phosphorylated form of CaMKIV was determined using CaMK-immunoprecipitated protein. Densitometry was performed by the National Institutes of Health (Bethesda, MD) image program to quantify OD.

Immunocytochemistry and confocal microscopy

Cells were cultured on glass coverslips until 80% confluent. The coverslips were washed in PBS and fixed for 20 min in 4% paraformaldehyde in PBS at 4°C. Cells were permeabilized with 5% BSA, 0.1% Triton X-100 for 1 h at 4°C and then stained with the appropriate Ab (1 µg/ml) overnight at 4°C. Cells were further treated with anti-rabbit IgG conjugated with FITC (1/1000), anti-mouse IgG conjugated with tetramethylrhodamine isothiocyanate (1/1000), and DAPI (5 mg/ml) in 5% nonfat milk for an additional 2 h at room temperature. The cells were then washed four times with PBS, mounted with Gel/Mount (Biomeda) and examined by confocal microscopy.

In vitro kinase assay

Recombinant HMGB1 (1 µg) was incubated in the presence or absence of activated p-Thr196-CaMKIV (25 ng) for 10 min at 30°C with the following additions: 10 mM MgCl2, 0.2 mM ATP, 1 mM CaCl2, and 1 µM CaM. Reactions were terminated by boiling in SDS-2-ME dissociation solution, subjected to 10% SDS-PAGE, and probed with anti-phosphoserine, anti-CaMKIV, or anti-HMGB1 Ab.

Cell viability and morphologic features

Representative cell populations from each condition were examined under light microscopy. Cell viability was also confirmed by MTT assay. Cells were incubated in 96-well plates (Costar). After a 24-h incubation in 100 µl RPMI 1640 medium containing the stimulus, 50 µl of a 5 mg/ml MTT (Sigma-Aldrich) solution in PBS was added, and cells were incubated at 37°C for 2 h. The cells were then lysed by addition of 100 µl per well extraction buffer (20% (w/v) SDS, 50% (v/v) N,N-dimethyl formamide, 2% (v/v) acetic acid (pH 4.7)). After overnight incubation with extraction buffer, the OD at 562 nm was measured.

Statistic analysis

Values are expressed as means ± SEM. Groups are compared by Mann-Whitney. A p value of <0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
LPS activates CaMKIV

The phosphorylation of CaMKIV on Thr196 generates an autonomously active p-CaMKIV (11). We first established the time course for CaMKIV activation in RAW 264.7 cells following LPS stimulation. LPS activated CaMKIV, with Thr196 phosphorylation occurring at 15 min and returning toward baseline by 60 min after stimulation (Fig. 1A). Upstream inhibition of CaMKIV kinase with STO609 prevented CaMKIV phosphorylation (Fig. 1A). We did observe two bands in our assessment of CaMKIV phosphorylation, and two CaMKIV isoforms have been described (21). This time course of CaMKIV phosphorylation was consistent across all independent experiments, yielding a mean CaMKIV activation time course that peaked 15–30 min after LPS (Fig. 1B). We repeated these experiments with primary peritoneal M{phi}. LPS induced Thr196 phosphorylation of CaMKIV within 15–30 min (Fig. 1C). However, even at 60 min after LPS exposure, CaMKIV remained phosphorylated (Fig. 1, C and D). Again, CaMKIV activation was dependent upon CaMKIV kinase, as inhibition with STO609 markedly reduced the LPS-induced increase in p-Thr196-CaMKIV (data not shown).


Figure 1
View larger version (40K):
[in this window]
[in a new window]

  		FIGURE 1. LPS activates CaMKIV. A, RAW 264.7 cells were treated with LPS (10 ng/ml) for the durations of 15, 30, 60, and 120 min, at which time total cell lysate was harvested, subjected to immunoblot analysis, and probed for p-Thr196-CaMKIV. Parallel populations were treated with STO609 (5 µM) for 1 h before LPS stimulation and subjected to similar immunoblot analysis. Each blot was stripped and reprobed with anti-CaMKIV Ab to confirm equal loading. Representative blot of three individual experiments. B, Densitometry represents means ± SEM OD of individual p-Thr196-CaMKIV immunoblots (n = 3); *, p < 0.05 vs control, unstimulated cells. C, Murine peritoneal M{phi} were exposed to similar experimental conditions, and p-Thr196-CaMKIV and total CaMKIV were assessed by SDS-PAGE as previously described. Representative blot of three individual experiments. D, Densitometry represents means ± SEM OD of individual p-Thr196-CaMKIV immunoblots (n = 3); *, p < 0.05 vs control, unstimulated cells.

 
The CaM kinase cascade mediates LPS-induced HMGB1 release

Consistent with previous studies, we found that LPS induced the release of HMGB1 by RAW 264.7 cells, with detectable concentrations of supernatant HMGB1 occurring as early as 8 h (data not shown) and peaking at 16 h after LPS stimulation as shown (Fig. 2A, lanes 1 and 2). Incubation with STO609 markedly reduced supernatant HMGB1 concentration (Fig. 2A, lane 3). As shown in Fig. 2B, the HMGB1 release was not due to differential cell death since similar cell viability was observed for treated cell populations.


Figure 2
View larger version (27K):
[in this window]
[in a new window]

  		FIGURE 2. CaMK kinase cascade mediates LPS-induced HMGB1 release. A, RAW 264.7 cells were treated with LPS (100 ng/ml) for 16 h, at which time cellular supernatant was harvested, subjected to immunoblot analysis, and probed for HMGB1. Parallel populations were pretreated with STO609 (5 µM) for 1 h before LPS stimulation and subjected to similar immunoblot analysis. Representative blot of three individual experiments. B, RAW 264.7 cells were subjected to similar conditions as described above. Cell viability was evaluated by MTT assay and is shown as means ± SEM fold change relative to control, untreated cell population (n = 4). C, Murine peritoneal M{phi} were isolated and treated with LPS and STO609 as previously described. The supernatant was harvested, subjected to Western blot analysis, and probed for HMGB1. Representative blot of three individual experiments.

 
We confirmed that the CaM kinase cascade also mediated LPS-induced HMGB1 release in primary peritoneal M{phi}. Similar to RAW 264.7 cells, LPS induced HMGB1 release by peritoneal M{phi} that was inhibited by upstream CaM kinase inhibition with STO609 (Fig. 2C).

CaMKIV mediates LPS-induced HMGB1 release

The previous observations suggested that the CaM kinase cascade is integral to LPS-induced M{phi} HMGB1 release. We utilized RNAi to study the individual role of downstream CaMKIV in these events. As determined by fluorescent microscopy, cell transfection with fluorescent cyclophilin B siRNA was efficacious (Fig. 3A) and, as determined by SDS-PAGE, CaMKIV RNAi markedly reduced CaMKIV expression (Fig. 3B). Control, nontarget RNAi-treated cells released HMGB1 in response to LPS similar to untreated RAW 264.7 cells and peritoneal M{phi} (Fig. 3C, lanes 1 and 2). By contrast, CaMKIV RNAi almost completely inhibited LPS-induced HMGB1 release (Fig. 3C, lanes 3 and 4). The effects of CaMKIV RNAi were not related to cell death, as the MTT assay demonstrated similar viability for all cells regardless of how they were treated (Fig. 3D).


Figure 3
View larger version (27K):
[in this window]
[in a new window]

  		FIGURE 3. CaMKIV mediates LPS-induced HMGB1 release. A, RAW 264.7 cells were transfected with fluorescent cyclophilin B siRNA for 24 h and imaged by confocal microscopy. Transfection efficiency was determined by determining the ratio of fluorescent cells to total number of cells. B, RAW 264.7 cells were transfected with either nontarget (NT) or CaMKIV siRNA for 72 h, at which time total cell lysate was harvested, subjected to immunoblot analysis, and probed for CaMKIV. Representative blots of three individual experiments. C, RAW 264.7 cells were transfected with either NT or CaMKIV siRNA for 72 h and then stimulated with LPS (100 ng/ml) for 16 h. The supernatant was harvested, subjected to immunoblot analysis, and probed for HMGB1. Representative blots of three individual experiments. D, RAW 264.7 cells were subjected to similar conditions as described above. Cell viability was evaluated by MTT assay and is shown as means ± SEM fold change relative to control, untreated NT cell population (n = 4).

 
CaMKIV mediates nuclear export of HMGB1 after LPS stimulation

HMGB1 release is an active process by which HMGB1 is shuttled from nucleus to cytoplasm and then out of the cell. Because most of the identified functions of CaMKIV occur within the nucleus, we hypothesized that CaMKIV plays an integral role in the translocation of HMGB1 from nucleus to cytoplasm. We initially explored the spatial and temporal relationships of CaMKIV phosphorylation and HMGB1 release. As shown in Fig. 4A, HMGB1 (green) is predominantly intranuclear under basal, unstimulated conditions (Fig. 4A, top row). Within 120 min of LPS stimulation, HMGB1 is observed within the cytoplasm and continues to increase within the cytoplasm over the ensuing 8 h (Fig. 4A, top row). Unstimulated RAW 264.7 cells demonstrate minimal CaMKIV Thr196 phosphorylation (red), but within 15 min of LPS stimulation, phosphorylation increases and continues to increase with time, peaking at 120 min after LPS stimulation (Fig. 4A, middle row). Although some active CaMKIV is apparent within the cytoplasm, most of this p-Thr196-CaMKIV seems to be concentrated within the nucleus. This localization appears to result from translocation of active CaMKIV to the nucleus as demonstrated by the increased fluorescence of both p-Thr196-CaMKIV (Fig. 4A, middle row) and total CaMKIV (Fig. 4B, top row) within the nucleus after LPS stimulation. Inhibition of CaMKIV kinase with STO609 inhibits the Thr196 phosphorylation of CaMKIV and the translocation of HMGB1 to the cytoplasm (Fig. 4A, last column).


Figure 4
View larger version (47K):
[in this window]
[in a new window]

  		FIGURE 4. Spatial and temporal relationship of LPS-induced CaMKIV Thr196 phosphorylation and HMGB1 nucleocytoplasmic shuttling. A, RAW 264.7 cells were stimulated with LPS (100 ng/ml) for 15 min and 1, 2, and 8 h, at which time they were fixed, permeabilized, stained for HMGB1 (green) or p-Thr196-CaMKIV (red), and imaged by confocal microscopy at x630. Green, HMGB1; red, p-Thr196-CaMKIV; blue, DAPI. Representative image of three individual experiments. B, RAW 264.7 cells were subjected to similar conditions as described above, stained for total CaMKIV, and imaged by confocal microscopy at x630. Red, CaMKIV; blue, DAPI. Representative image of three individual experiments.

 
To confirm that this translocation process was CaMKIV dependent, we conducted similar experiments using cells treated with CaMKIV RNAi. RAW 264.7 cells transfected with control, nontarget siRNA demonstrated nucleocytoplasmic shuttling of HMGB1 (green) at 8 h after LPS stimulation (Fig. 5A, left). On the other hand, CaMKIV RNAi inhibited LPS-induced HMGB1 translocation with strong nuclear and minimal cytoplasmic staining of HMGB1 at 8 h after LPS stimulation (Fig. 5A, right). This inhibition was similar to that induced by pretreatment with the CaMKIV kinase inhibitor STO609 (Fig. 4A, last column).


Figure 5
View larger version (43K):
[in this window]
[in a new window]

  		FIGURE 5. CaMKIV mediates HMGB1 nucleocytoplasmic shuttling. A, RAW 264.7 cells underwent RNAi using nontarget (NT) siRNA or CaMKIV siRNA. Cells were stimulated with LPS (100 ng/ml) for 8 h and then fixed, permeabilized, stained for HMGB1 (green), and imaged by confocal microscopy at x630. Green, HMGB1; blue, DAPI. Representative image of three individual experiments. B, RAW 264.7 cells were subjected to STO609 (5 µM) or KN93 (10 µM) or underwent NT or CaMKIV RNAi before stimulation with LPS (100 ng/ml) as detailed above. Nuclear and cytoplasmic protein were isolated, subjected to immunoblot analysis, and probed for HMGB1. Representative blots of three separate experiments.

 
We confirmed these immunofluorescent microscopic observations by analyzing the HMGB1 concentration of nuclear and cytoplasmic protein isolated from M{phi} subjected to similar conditions as detailed above. As shown in Fig. 5B, LPS induced the translocation of HMGB1 from the nucleus to the cytoplasm. By comparison to LPS-stimulated M{phi}, there was a significant reduction in HMGB1 translocation to the cytoplasm in response to either KN93 (which inhibits CaMKIV) or STO609 (which inhibits CaMKIV kinase) (Fig. 5B). Similarly, by comparison to control, nontarget RNAi, CaMKIV RNAi reduced the LPS-induced nucleocytoplasmic shuttling of HMGB1 (Fig. 5B). These results confirm our idea that a CaMKK/CaMKIV cascade regulates LPS-induced nuclear-to-cytoplasmic translocation of HMGB1.

CaMKIV directly serine phosphorylates HMGB1 after LPS stimulation

Our results demonstrate that CaMKIV mediates LPS-induced HMGB1 release, in part, through processes guiding the nuclear exportation of HMGB1. Recently, HMGB1 translocation to the cytoplasm has been demonstrated to require serine phosphorylation within its NLSs (9). We first confirmed that LPS induced serine phosphorylation of HMGB1. As shown in Fig. 6A, stimulation of RAW 264.7 cells with LPS results in serine phosphorylation of HMGB1 within 2 h. Additionally, this phosphorylation event may require CaMKIV, as preincubation of primary peritoneal M{phi} with STO609 inhibited LPS-induced serine phosphorylation of HMGB1 (Fig. 6B). Similarly, CaMKIV RNAi treatment of M{phi} also reduced LPS-induced HMGB1 serine phosphorylation (Fig. 6B), and the mean degree of inhibition determined by densitometry is illustrated in Fig. 6C. Finally, we compared the degree of serine phosphorylated HMGB1 in peritoneal M{phi} isolated from either CaMKIV+/+ or CaMKIV–/– mice. As shown in Fig. 6D, control CaMKIV+/+ M{phi} show serine phosphorylation of HMGB1 after LPS stimulation, whereas the extent of this induced HMGB1 phosphorylation is reduced in CaMKIV–/– M{phi}.


Figure 6
View larger version (45K):
[in this window]
[in a new window]

  		FIGURE 6. CaMKIV mediates serine phorphorylation of HMGB1. A, RAW 264.7 cells were stimulated with LPS (100 ng/ml) for 15 min and 1, 2, and 4 h, at which point total cell lysate was harvested, immunoprecipitated for HMGB1, subjected to immunoblot analysis, and probed for phosphorylated serine residues. Each membrane was stripped and probed with anti-HMGB1 Ab to confirm equal loading. B, Murine peritoneal M{phi} were stimulated with LPS (100 ng/ml) for 2 h either in the presence or absence of STO609 (5 µM) or after CaMKIV RNAi. Total cell lysate was harvested, immunoprecipitated for HMGB1, subjected to immunoblot analysis, and probed for phosphorylated serine residues. Each membrane was stripped and probed with anti-HMGB1 Ab to confirm equal loading. C, Densitometry represents means ± SEM OD of individual phosphoserine HMGB1 immunoblots (n = 4); *, p < 0.05 vs LPS stimulated M{phi}; #, p < 0.05 vs LPS stimulated nontarget RNAi M{phi}. D, Peritoneal M{phi}, isolated from CaMKIV–/– or CaMKIV+/+ mice, were exposed to LPS 100 ng/ml for 2 h, at which point total cell lysate was harvested, immunoprecipitated for phosphoserine, subjected to immunoblot analysis, and probed for HMGB1.

 
Our immunocytochemistry data suggest colocalization of CaMKIV and HMGB1 after LPS stimulation, and thus we investigated whether CaMKIV interacts with HMGB1. As shown in Fig. 7A, LPS stimulation markedly increased the degree to which HMGB1 coimmunoprecipitated with CaMKIV. One hour after stimulation with LPS, CaMKIV and HMGB1 coimmunoprecipitation was markedly increased and peaked by 2 h. Reciprocal coimmunoprecipitation experiments yielded similar results (data not shown). These LPS-stimulated interactions likely require the phosphorylation and activation of CaMKIV, as preincubation of cells with STO609 before addition of LPS inhibited the coimmunoprecipitation of HMGB1 with CaMKIV (Fig. 7A).


Figure 7
View larger version (69K):
[in this window]
[in a new window]

  		FIGURE 7. CaMKIV directly serine phosphorylates HMGB1. A, RAW 264.7 cells were stimulated with LPS (100 ng/ml) either in the presence or absence of STO609 (5 µM) for 1, 2, and 4 h, at which point total cell lysate was harvested, immunoprecipitated for HMGB1, subjected to immunoblot analysis, and probed for CaMKIV. Representative blots of three separate experiments. B, Recombinant HMGB1 (1 µg) was incubated in the presence or absence of activated p-Thr196-CaMKIV (25 ng) for 10 min at 30°C with the following additions: 10 mM MgCl2, 0.2 mM ATP, 1 mM CaCl2, and 1 µM CaM. Reactions were terminated by boiling in SDS-2-ME dissociation solution, subjected to 10% SDS-PAGE, and probed with anti-phosphoserine Ab. Representative blots of three separate experiments.

 
To determine whether CaMKIV could directly serine phosphorylate HMGB1 we conducted in vitro kinase assays. Active p-Thr196-CaMKIV was unable to phosphorylate HMGB1 in the absence of Mg2+/ATP (Fig. 7B, lane 1). Similarly, in the absence of p-Thr196-CaMKIV, there was minimal serine phosphorylation of HMGB1 (Fig. 7B, lane 2). However, activated p-Thr196-CaMKIV, in combination with Mg2+/ATP, was able to serine phosphorylate HMGB1 (Fig. 7B, lane 4).

Active CaMKIV is sufficient for HMGB1 release

To assess whether active CaMKIV is sufficient for HMGB1 release, we transfected RAW 264.7 cells with plasmids expressing either a truncated, constitutively active form of CaMKIV (CaMKIV-dCT) or a similarly truncated kinase-inactive mutant of CaMKIV (CaMKIV-dCTK75E). Both plasmids were successfully transfected and expressed, although expression of the active CaMKIV-dCT was greater than that of CaMKIV-dCTK75E (Fig. 8A), confirming previously published observations that substitutions that disrupt kinase activity, including K75E, result in less protein expression in mammalian cells compared with wild-type CaMKIV-dCT (Fig. 8A) (20). As shown in Fig. 8B, expression of the active CaMKIV-dCT induced the secretion of HMGB1 from the cells, whereas CaMKIV-dCTK75E failed to do so (Fig. 8B). This 6-fold induction of HMGB1 release in response to CaMKIV-dCT was highly reproducible in three independent experiments (Fig. 8C).


Figure 8
View larger version (31K):
[in this window]
[in a new window]

  		FIGURE 8. Active CaMKIV is sufficient for HMGB1 release. A, RAW 264.7 cells were transfected with either a constitutively active CaMKIV-dCT or a kinase-dead mutant CaMKIV-dCTK75E. Eight hours later, total cell lysate or nuclear protein was isolated, resolved by SDS-PAGE, and probed for kinase expression using an anti-FLAG Ab. Representative blots of three separate experiments. B, RAW 264.7 cells were transfected with the indicated kinase construct as detailed above. After 24 h, the supernatant was harvested, resolved by SDS-PAGE, and probed with an anti-HMGB1 Ab. Representative blots of three separate experiments. C, Densitometry represents means ± SEM OD of individual supernatant HMGB1 immunoblots (n = 3); *, p = 0.03 vs CaMKIV-dCTK75E.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
During sepsis, perturbations in cellular calcium homeostasis are postulated to mediate the aberrant inflammation underlying organ dysfunction and death (22, 23). Recently, HMGB1, an architectural chromatin-binding factor that bends DNA and directs protein assembly on specific DNA targets, has been demonstrated to function as a late mediator of mortality in murine endotoxemia and sepsis (1, 2, 3). Monocytes and M{phi} have been demonstrated to be a primary source of HMGB1, and evidence is accumulating that production of this inflammatory mediator is Ca2+ dependent. Herein, we characterize that LPS-induced HMGB1 release is mediated by a Ca2+-dependent signaling cascade involving a CaMKIV kinase and CaMKIV. Our data suggest that this series of reactions may terminate in the nucleus where CaMKIV phosphorylates HMGB1, an event that is required to facilitate the translocation of HMGB1 from nucleus to cytoplasm (24).

Substantial work has been conducted to elucidate the mechanisms by which HMGB1 is released, in part, because interventions that inhibit systemic HMGB1 concentrations reduce mortality in murine models of sepsis (2). Current data support an active process initiated by HMGB1-histone disengagement, HMGB1 hyperacetylation, and shuttling of the protein from nucleus to cytoplasm (6). Recently, serine phosphorylation of HMGB1 has been demonstrated to be essential for this translocation event, although the kinase responsible had not been identified (9). We hypothesized that CaMKIV mediates this process, in part, because this enzyme has been shown to function downstream of TLR4 in the pathway by which LPS induces the Ca2+-dependent survival of dendritic cells (17). Additionally, prior studies demonstrated that activation of CaMKIV by Ca2+/CaM and threonine phosphorylation (Thr196 in mice, Thr200 in humans) by an upstream CaMK kinase generates increased and autonomous CaMKIV activity that enables the kinase to translocate to the nucleus where it modulates gene expression (11, 25, 26).

Indeed, our data demonstrate that LPS induces this Thr196-phosphorylated and autonomous species of CaMKIV in M{phi}, which then enters the nucleus where it interacts with and phosphorylates HMGB1. The evidence to support this contention is as follows: 1) CaMKIV translocation to the nucleus preceded HMGB1 translocation to the cytoplasm; 2) CaMKIV RNAi reduced cytoplasmic HMGB1 concentration by retaining HMGB1 in the nucleus; 3) CaMKIV coimmunoprecipitated with HMGB1 in response to LPS; and 4) activated CaMKIV directly phosphorylated HMGB1 in in vitro kinase assays. The modest degree of HMGB1 phosphorylation observed in our in vitro kinase assay suggests that, perhaps, additional protein components may be necessary to assemble a signaling complex that facilitates CaMKIV phosphorylation of HMGB1. The minimum consensus CaMKIV substrate recognition sequence Hyd-X-R-X-X-(S/T) provides only a rough template for CaMKIV-mediated phosphorylation. In HMGB1 only Ser39 and Ser46 in NLS1 meet the hydrophobic requirement at the –5 position amino acid, and Ser46 carries a lysine substitution at –3, which is also present in the well-characterized substrate oncoprotein 18 (27).

Interestingly, HMGB1 has two NLS sequences, and current evidence suggests that phosphorylation of both sequences is important for the cytoplasmic localization of HMGB1 (9). Our data support a role for CaMKIV in the mechanism that controls the nuclear export of HMGB1 (9). Regarding this mechanism, Bianchi et al. contend that the strength of HMGB1-histone binding is inversely related to the histone acetylation status, and that increased histone acetylation may enable HMGB1-histone disengagement and subsequent cellular release of HMGB1 (6, 8). The CaMKs have been demonstrated to modulate the nuclear export of various class II histone deacetylases HDAC4 and HDAC5, a process that could favor histone acetylation (28, 29, 30, 31). Additionally, HMGB1 requires direct hyperacetylation before export from the nucleus (8). Further investigation of how CaMKIV activation and histone and HMGB1 acetylation are related will be an important aspect of future studies on HMGB1 nuclear export.

Although LPS stimulation of TLR4 can clearly activate CaMKIV, as shown here and in a prior study, the signaling events that occur between receptor ligation and CaMKIV activation have yet to be determined (17). Prior studies have established the dependency of monocyte/M{phi} function on Ca2+ transients and CaM activation, and although it remains unclear precisely how the Ca2+ transients are generated, the multifunctional CaMKs respond to relatively small changes in intracellular [Ca2+] (32). Regarding CaMKIV activation and generation of autonomous activity, published data have established a requirement for binding of the Ca2+/CaM complex to both CaMKK and CaMKIV (11, 12, 25). We note the presence of nuclear and cytoplasmic activated CaMKIV in resting cells and an increasing concentration of activated and total CaMKIV within the nucleus after LPS stimulation. These observations support a LPS-mediated increase in the cytoplasmic Ca2+ signal that is sufficient to activate CaMKIV and induce its nuclear translocation. Alternatively, it is possible that CaMK may be activated by reactive oxygen species in the absence of a Ca2+ rise. For example, Howe et al. suggested that I{kappa}B phosphorylation in lymphocytes in response to hydrogen peroxide is mediated by Ca2+-independent activation of CaMKs (33, 34). Hence, additional studies are necessary to determine the spatiotemporal patterns of Ca2+ signaling and CaMKIV activation following LPS stimulation as well as the TLR4 dependence of such events.

In summary, CaMKIV kinase/CaMKIV mediates M{phi} LPS-induced HMGB1 production by translocating to the nucleus after activation and phosphorylating HMGB1 in a manner that enables nuclear export of HMGB1 that must occur in order for the protein to be secreted from the cell. The combined observations of investigations conducted by our laboratories and those of others are beginning to highlight the integral role of the CaMK transduction cascade in M{phi} function and cytokine production and inflammation. Future studies will elucidate the sites on both proteins that participate in the CaMKIV-HMGB1 interaction and serine phosphorylation of HMGB1. Interestingly, precedent exists for CaMKIV forming a stable complex with PP2A that is competitive with Ca2+/CaM and CaMKKβ, one of the two CaMKKs that has been shown to form stable complexes with CaMKIV and PP2A or AMPK (15, 35). In the latter but not the former case, the interaction requires the "RP" domain of CaMKKβ (35). Perhaps, HMGB1 will also utilize a unique sequence of CaMKKβ to form a complex. At any rate, these combined observations suggest that modulation of CaMKK and/or CaMKIV activity may be of potential utility in diseases characterized by heightened and aberrant inflammation.


   Acknowledgments
 
Special thanks to Dr. Naohito Nozaki, who provided the monoclonal anti-p-Thr196-CaMKIV Abs, and Dr. Douglas Black, who provided the CaMKIV-dCT and CaMKIV-dCTK75E plasmids. Without the gracious assistance of these individuals, a considerable portion of this work would not be possible.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflicts of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by Grant 1KL2 RR024154-01 (to M.R.R.) from the National Center for Research Resources (NCRR), a component of the National Institutes of Health (NIH), and the NIH Roadmap for Medical Research. This work was also supported by NIH grant DK074701 (to A.R.M.). Back

2 X.Z. and D.W. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. Matthew R. Rosengart, Department of Surgery, University of Pittsburgh, 200 Lothrop Street, Pittsburgh, PA 15213. E-mail address: rosengartmr{at}upmc.edu Back

4 Abbreviations used in this paper: HMGB1, high-mobility group box 1; CaMK, calcium/calmodulin-dependent protein kinase; M{phi}, macrophage; NLS, nuclear localization signal; RNAi, RNA interference; siRNA, small interfering RNA. Back

Received for publication October 15, 2007. Accepted for publication July 22, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Wang, H., H. Yang, C. J. Czura, A. E. Sama, K. J. Tracey. 2001. HMGB1 as a late mediator of lethal systemic inflammation. Am. J. Respir. Crit. Care Med. 164: 1768-1773. [Free Full Text]
  2. Yang, H., M. Ochani, J. Li, X. Qiang, M. Tanovic, H. E. Harris, S. M. Susarla, L. Ulloa, H. Wang, R. DiRaimo, et al 2004. Reversing established sepsis with antagonists of endogenous high-mobility group box 1. Proc. Natl. Acad. Sci. USA 101: 296-301. [Abstract/Free Full Text]
  3. Wang, H., O. Bloom, M. Zhang, J. M. Vishnubhakat, M. Ombrellino, J. Che, A. Frazier, H. Yang, S. Ivanova, L. Borovikova, et al 1999. HMG-1 as a late mediator of endotoxin lethality in mice. Science 285: 248-251. [Abstract/Free Full Text]
  4. Angus, D. C., L. Yang, L. Kong, J. A. Kellum, R. L. Delude, K. J. Tracey, L. Weissfeld. 2007. Circulating high-mobility group box 1 (HMGB1) concentrations are elevated in both uncomplicated pneumonia and pneumonia with severe sepsis. Crit. Care Med. 35: 1061-1067. [Medline]
  5. Ombrellino, M., H. Wang, M. S. Ajemian, A. Talhouk, L. A. Scher, S. G. Friedman, K. J. Tracey. 1999. Increased serum concentrations of high-mobility-group protein 1 in haemorrhagic shock. Lancet 354: 1446-1447. [Medline]
  6. Bonaldi, T., F. Talamo, P. Scaffidi, D. Ferrera, A. Porto, A. Bachi, A. Rubartelli, A. Agresti, M. E. Bianchi. 2003. Monocytic cells hyperacetylate chromatin protein HMGB1 to redirect it towards secretion. EMBO J. 22: 5551-5560. [Medline]
  7. Gardella, S., C. Andrei, D. Ferrera, L. V. Lotti, M. R. Torrisi, M. E. Bianchi, A. Rubartelli. 2002. The nuclear protein HMGB1 is secreted by monocytes via a non-classical, vesicle-mediated secretory pathway. EMBO Rep. 3: 995-1001. [Medline]
  8. Scaffidi, P., T. Misteli, M. E. Bianchi. 2002. Release of chromatin protein HMGB1 by necrotic cells triggers inflammation. Nature 418: 191-195. [Medline]
  9. Youn, J. H., J. S. Shin. 2006. Nucleocytoplasmic shuttling of HMGB1 is regulated by phosphorylation that redirects it toward secretion. J. Immunol. 177: 7889-7897. [Abstract/Free Full Text]
  10. Tsung, A., J. R. Klune, X. Zhang, G. Jeyabalan, Z. Cao, X. Peng, D. B. Stolz, D. A. Geller, M. R. Rosengart, T. R. Billiar. 2007. HMGB1 release induced by liver ischemia involves Toll-like receptor 4 dependent reactive oxygen species production and calcium-mediated signaling. J. Exp. Med. 204: 2913-2923. [Abstract/Free Full Text]
  11. Soderling, T. R.. 1999. The Ca-calmodulin-dependent protein kinase cascade. Trends Biochem. Sci. 24: 232-236. [Medline]
  12. Braun, A. P., H. Schulman. 1995. The multifunctional calcium/calmodulin-dependent protein kinase: from form to function. Annu. Rev. Physiol. 57: 417-445. [Medline]
  13. Kitsos, C. M., U. Sankar, M. Illario, J. M. Colomer-Font, A. W. Duncan, T. J. Ribar, T. Reya, A. R. Means. 2005. Calmodulin-dependent protein kinase IV regulates hematopoietic stem cell maintenance. J. Biol. Chem. 280: 33101-33108. [Abstract/Free Full Text]
  14. Lemrow, S. M., K. A. Anderson, J. D. Joseph, T. J. Ribar, P. K. Noeldner, A. R. Means. 2004. Catalytic activity is required for calcium/calmodulin-dependent protein kinase IV to enter the nucleus. J. Biol. Chem. 279: 11664-11671. [Abstract/Free Full Text]
  15. Anderson, K. A., P. K. Noeldner, K. Reece, B. E. Wadzinski, A. R. Means. 2004. Regulation and function of the calcium/calmodulin-dependent protein kinase IV/protein serine/threonine phosphatase 2A signaling complex. J. Biol. Chem. 279: 31708-31716. [Abstract/Free Full Text]
  16. Chow, F. A., K. A. Anderson, P. K. Noeldner, A. R. Means. 2005. The autonomous activity of calcium/calmodulin-dependent protein kinase IV is required for its role in transcription. J. Biol. Chem. 280: 20530-20538. [Abstract/Free Full Text]
  17. Illario, M., M. L. Giardino-Torchia, U. Sankar, T. J. Ribar, M. Galgani, L. Vitiello, A. M. Masci, F. R. Bertani, E. Ciaglia, et al 2008. Calmodulin-dependent kinase IV links Toll-like receptor 4 signaling with survival pathway of activated dendritic cells. Blood 111: 723-731. [Abstract/Free Full Text]
  18. Tokumitsu, H., H. Inuzuka, Y. Ishikawa, M. Ikeda, I. Saji, R. Kobayashi. 2002. STO-609, a specific inhibitor of the Ca2+/calmodulin-dependent protein kinase kinase. J. Biol. Chem. 277: 15813-15818. [Abstract/Free Full Text]
  19. Xie, J., D. L. Black. 2001. A CaMK IV responsive RNA element mediates depolarization-induced alternative splicing of ion channels. Nature 410: 936-939. [Medline]
  20. Chatila, T., K. A. Anderson, N. Ho, A. R. Means. 1996. A unique phosphorylation-dependent mechanism for the activation of Ca2+/calmodulin-dependent protein kinase type IV/GR. J. Biol. Chem. 271: 21542-21548. [Abstract/Free Full Text]
  21. Sun, Z., R. L. Means, B. LeMagueresse, A. R. Means. 1995. Organization and analysis of the complete rat calmodulin-dependent protein kinase IV gene. J. Biol. Chem. 270: 29507-29514. [Abstract/Free Full Text]
  22. Zaloga, G. P., B. Chernow. 1987. The multifactorial basis for hypocalcemia during sepsis: studies of the parathyroid hormone-vitamin D axis. Ann. Intern. Med. 107: 36-41. [Medline]
  23. Zaloga, G. P., A. Sager, K. W. Black, R. Prielipp. 1992. Low dose calcium administration increases mortality during septic peritonitis in rats. Circ. Shock 37: 226-229. [Medline]
  24. Sparatore, B., M. Passalacqua, M. Patrone, E. Melloni, S. Pontremoli. 1996. Extracellular high-mobility group 1 protein is essential for murine erythroleukaemia cell differentiation. Biochem. J. 320: 253-256. [Medline]
  25. Tokumitsu, H., T. R. Soderling. 1996. Requirements for calcium and calmodulin in the calmodulin kinase activation cascade. J. Biol. Chem. 271: 5617-5622. [Abstract/Free Full Text]
  26. Corcoran, E. E., A. R. Means. 2001. Defining Ca2+/calmodulin-dependent protein kinase cascades in transcriptional regulation. J. Biol. Chem. 276: 2975-2978. [Free Full Text]
  27. White, D. J., D. L. Maass, B. Sanders, J. W. Horton. 2002. Cardiomyocyte intracellular calcium and cardiac dysfunction after burn trauma. Crit. Care Med. 30: 14-22. [Medline]
  28. Miska, E. A., E. Langley, D. Wolf, C. Karlsson, J. Pines, T. Kouzarides. 2001. Differential localization of HDAC4 orchestrates muscle differentiation. Nucleic Acids Res. 29: 3439-3447. [Abstract/Free Full Text]
  29. Davis, F. J., M. Gupta, B. Camoretti-Mercado, R. J. Schwartz, M. P. Gupta. 2003. Calcium/calmodulin-dependent protein kinase activates serum response factor transcription activity by its dissociation from histone deacetylase, HDAC4: implications in cardiac muscle gene regulation during hypertrophy. J. Biol. Chem. 278: 20047-20058. [Abstract/Free Full Text]
  30. Chawla, S., P. Vanhoutte, F. J. Arnold, C. L. Huang, H. Bading. 2003. Neuronal activity-dependent nucleocytoplasmic shuttling of HDAC4 and HDAC5. J. Neurochem. 85: 151-159. [Medline]
  31. McKinsey, T. A., C. L. Zhang, J. Lu, E. N. Olson. 2000. Signal-dependent nuclear export of a histone deacetylase regulates muscle differentiation. Nature 408: 106-111. [Medline]
  32. Anderson, K. A., R. L. Means, Q. H. Huang, B. E. Kemp, E. G. Goldstein, M. A. Selbert, A. M. Edelman, R. T. Fremeau, A. R. Means. 1998. Components of a calmodulin-dependent protein kinase cascade: molecular cloning, functional characterization and cellular localization of Ca2+/calmodulin-dependent protein kinase kinase β. J. Biol. Chem. 273: 31880-31889. [Abstract/Free Full Text]
  33. Howe, C. J., M. M. LaHair, J. A. Maxwell, J. T. Lee, P. J. Robinson, O. Rodriguez-Mora, J. A. McCubrey, R. A. Franklin. 2002. Participation of the calcium/calmodulin-dependent kinases in hydrogen peroxide-induced I{kappa}B phosphorylation in human T lymphocytes. J. Biol. Chem. 277: 30469-30476. [Abstract/Free Full Text]
  34. Howe, C. J., M. M. Lahair, J. A. McCubrey, R. A. Franklin. 2004. Redox regulation of the calcium/calmodulin-dependent protein kinases. J. Biol. Chem. 279: 44573-44581. [Abstract/Free Full Text]
  35. Anderson, K. A., T. J. Ribar, F. Lin, P. K. Noeldner, M. F. Green, M. J. Muehlbauer, L. A. Witters, B. E. Kemp, A. R. Means. 2008. Hypothalamic CaMKK2 contributes to the regulation of energy balance. Cell Metab. 7: 377-388. [Medline]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
Y. J. Oh, J. H. Youn, Y. Ji, S. E. Lee, K. J. Lim, J. E. Choi, and J.-S. Shin
HMGB1 Is Phosphorylated by Classical Protein Kinase C and Is Secreted by a Calcium-Dependent Mechanism
J. Immunol., May 1, 2009; 182(9): 5800 - 5809.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
J.-H. Kim, S.-J. Kim, I.-S. Lee, M.-S. Lee, S. Uematsu, S. Akira, and K. I. Oh
Bacterial Endotoxin Induces the Release of High Mobility Group Box 1 via the IFN-{beta} Signaling Pathway
J. Immunol., February 15, 2009; 182(4): 2458 - 2466.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Zhang, X.
Right arrow Articles by Rosengart, M. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Zhang, X.
Right arrow Articles by Rosengart, M. R.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS