TRANSLATIONAL PHYSIOLOGY
Nuclear CaMKII inhibits neuronal differentiation of PC12 cells without affecting MAPK or CREB activation

Louis W. Kutcher1, Shirelyn R. Beauman1, Eric I. Gruenstein2, Marcia A. Kaetzel1,3, and John R. Dedman1,3

Departments of 1 Molecular and Cellular Physiology, 2 Molecular Genetics, and 3 Genome Sciences, University of Cincinnati Medical School, Cincinnati, Ohio 45267


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Ca2+/calmodulin-regulated protein kinase II (CaMKII) mediates many cellular events. The four CaMKII isoforms have numerous splice variants, three of which contain nuclear localization signals. Little is known about the role of nuclear localized CaMKII in neuronal development. To study this process, PC12 cells were transfected to produce CaMKII targeted to either the cytoplasm or the nucleus and then treated with nerve growth factor (NGF). NGF triggers a signaling cascade (MAPK) that results in the differentiation of PC12 cells into a neuronal phenotype, marked by neurite outgrowth. The present study found that cells expressing nuclear targeted CaMKII failed to grow neurites, whereas cells expressing cytoplasmic CaMKII readily produced neurites. Inhibition of neuronal differentiation by nuclear CaMKII was independent of MAPK signaling, as sustained Erk phosphorylation was not affected. Phosphorylation of CREB was also unaffected. Thus nuclear CaMKII modifies neuronal differentiation by a mechanism independent of MAPK and CREB activation.

calcium/calmodulin-dependent protein kinase II; mitogen-activated protein kinase; calmodulin; neurite outgrowth; CaMKII isoforms; extracellular signal-regulated protein kinase; cAMP-response element-binding protein


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

WHEN THE LEVEL OF CA2+ INCREASES inside a cell, several Ca2+-regulated signal transduction cascades are initiated. One such pathway involves calmodulin (CaM), which activates several downstream targets, including calcium/calmodulin-regulated protein kinase II (CaMKII) (for reviews see Refs. 5 and 39). CaMKII regulates a wide variety of cellular events, from controlling cell cycle progression to regulating cellular differentiation and influencing apoptosis (1).

There are four isoforms of CaMKII, alpha , beta , gamma , and delta , each encoded by a separate gene but having very high sequence homology (1, 8, 46, 49). Each CaMKII isoform is composed of a catalytic domain, a regulatory domain, and an association domain. The association domain is responsible for 8-12 individual CaMKII molecules forming a holoenzyme complex that can be composed of one or several different isoforms (1, 24, 40). At the carboxyl end of the regulatory domain, there is a short sequence known as the variable region (1). Outside this variable region, the amino acid sequence homology is 80-90% (46, 49). The functional differences between isoforms provide unique developmental, regional, or subcellular expression patterns. The substrate specificity, kinetics, and calmodulin affinities are extremely similar (1, 3, 4, 25, 46).

Alternative splicing of these four genes gives rise to a large number of variants (for reviews see Refs. 1, 22, 24, 40). The alpha - and beta -isoforms are found in neuronal tissue, whereas the gamma - and delta -isoforms are more widely distributed (49); all cells express at least one gene type. The details of tissue distribution and developmental regulation of these splice variants are under investigation. Three of the isoforms, alpha B, gamma A, and delta B, contain nuclear localization signals (NLS) within the variable region (3, 10, 22). The nuclear localized delta B-isoform was first identified in rat hearts (10) and has subsequently been found in the rat cerebellum and, to a lesser extent, in the cerebrum (47). The nuclear targeted alpha B-isoform is found in astrocytes (46), whereas mRNA for it has been detected in the diencephalon and midbrain of rats but not in the hippocampus or cortex (3). For the alpha B- and delta B-isoforms, nuclear localization can be blocked by phosphorylation of a serine located immediately adjacent to the NLS (23), which decreases binding of CaMKII to a nuclear import receptor. Nuclear targeting may therefore be a dynamic event, changing as a result of several influences, potentially including kinases and phosphatases.

Studies with several cytoplasmic isoforms of CaMKII have shown them to be involved in neurite outgrowth, their exact role depending on both the cell type and the isoform studied. When the delta E-isoform (an embryonic mouse type) is expressed in NIH/3T3 cells, it locates in the perinuclear region and induces the spontaneous formation of processes coming from the cell (4, 25). This localization is in contrast to the delta C-isoform, which remains in the cytoplasm when expressed in NIH/3T3 cells and does not induce these extensions (4, 25). A constitutively active mutant of the delta C-isoform is capable of inducing neurite outgrowth in P19 cells (8). The role of the alpha -isoform in neurite outgrowth has been studied extensively. When overexpressed in CAD cells (9), Nb2a cells (15, 41), and NG108-15 cells (15), alpha -CaMKII causes spontaneous neurite outgrowth. However, when alpha -CaMKII is overexpressed in pheochromocytoma (PC12) cells, it has been shown to inhibit neurite outgrowth that is normally induced by either nerve growth factor (NGF) (31) or cAMP (48). These latter two studies are of particular interest, because they both employ PC12 cells, and they will be considered in more detail in DISCUSSION.

A further influence on the role of various CaMKII isoforms is the fact that their expression in a cell can change as a result of cellular development. For instance, a central nervous system (CNS) cell line, CAD cells, shows increased CaMKII activity on differentiation, as well as a shift in the predominant isoform expressed, from the cytoplasmic delta C (delta 2)-isoform in undifferentiated cells to another cytoplasmic isoform, delta D (delta 4), after differentiation (8). In P19 cells undergoing differentiation, expression of the delta C-isoform is decreased (8). However, this does not happen when PC12 cells are differentiated; rather, the delta D- and delta A (delta 1)-isoforms are increased, whereas the delta C remains constant (8). This has led Donai et al. (8) to suggest that the lineage and maturity of the cell may determine which CaMKII isoforms are expressed.

Because of the importance of the role of selected CaMKII isoforms in neurite outgrowth, the current study was undertaken to explore how the subcellular location of CaMKII influences NGF-induced neuronal differentiation. The similarity in kinetics, substrate specificity, and CaM-binding properties between the different CaMKII isoforms has allowed the alpha -isoform to be used in many studies of CaMKII function (9, 15, 31, 41, 48). Thus transgenes were constructed to target expression of alpha -CaMKII to either the nucleus or the cytoplasm, and these transgenes were transfected into rat PC12 cells. The effects of targeted CaMKII proteins on NGF-induced neurite outgrowth were then studied.

PC12 cells respond to NGF by elaborating neurites within a few days (18). This process requires both new RNA transcription and ongoing protein synthesis (17). The binding of NGF to its high-affinity receptor Trk A initiates several intracellular responses. Among these are an early, transient increase in intracellular Ca2+ concentration (33, 34) and the initiation of a signaling cascade leading to phosphorylation of the mitogen-activated protein kinases (MAPKs) extracellular signal-regulated kinases Erk1 and Erk2 (26, 36, 54). Several factors converge to maintain Erk phosphorylation and, hence, its activation over a sustained time period. The initial phosphorylation of Erk depends on CaM (11) and Ras (54), whereas at later time points it is mediated by PKA (53) and Rap1 (54). This sustained activation is necessary for neurite outgrowth in PC12 cells, since epidermal growth factor (EGF), which phosphorylates Erk for only a short time period, does not initiate neurites (26).

The experiments described here use the PC12 cell neuronal model, transfected with targeted alpha -CaMKII transgenes, to point to a role for nuclear localized CaMKII in controlling neuronal differentiation. Cell lines expressing CaMKII in the nucleus failed to grow neurites in response to NGF, whereas lines with cytoplasmic CaMKII responded to NGF similar to control cells.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Cell culture. Rat PC12 cells (18) were cultured in a 1:1 mix of DMEM-F12 medium supplemented with 10% fetal bovine serum, 15 mM HEPES, 100 U/ml penicillin G sodium, and 100 µg/ml streptomycin sulfate (all from GIBCO-BRL). For transfected cell lines, medium was supplemented with 500 µg/ml G418 antibiotic (BioWhittaker). Cells were grown on tissue culture plastic at 37°C in 5% CO2 and subcultured before reaching confluence. PC12 cells were a gift of Dr. Maria Czyzyk-Krzeska (University of Cincinnati) and were used between passages 7 and 20 of receipt. Transfected cell lines were used between passages 3 and 15 from initial selection. Stocks of all cell lines were kept on liquid N2 and thawed as needed. To assess the overall viability of transfected cell lines, the MTT assay for cell growth was used. Following the protocol of Loo and Rillema (30), cells were plated in one-half of a 96-well plate at 1.5 × 103 cells/well and grown under standard conditions in the presence of G418 antibiotic (if appropriate). On a given assay date, medium was removed from the wells and replaced with medium containing 1.25 mg/ml MTT reagent [3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] and incubated at 37°C for 2 h. An equal volume of extraction buffer (10% SDS dissolved in a 1:1 mix of H2O and N,N-dimethylformamide) was then added to each well, and the plates were incubated overnight. The optical density was measured at 570 nm and averaged over the 30 center wells of the 48 treated wells (omitting the outside edge wells). The experiment was repeated three to five times for each growth curve. These experiments showed that cell viability and proliferation rate were not changed in cell lines expressing either nuclear or cytoplasmic CaMKII (data not shown).

Neuronal differentiation with NGF. Lines of PC12 cells were plated at 5 × 103 cells/cm2 on glass coverslips coated with poly-L-lysine. A thin film of poly-L-lysine was applied to the coverslips in tissue culture dishes and allowed to dry for 2-4 h. Dishes were then washed four times with sterile water and once with complete medium. One day after cells were plated, medium was changed to serum-free medium with 50 ng/ml NGF (Alamone Laboratories) and 500 µg/ml G418 antibiotic for stable cell lines. After the given incubation time, the coverslips were fixed in 10% formalin and mounted on glass slides. To measure neurite outgrowth, six adjacent microscope fields were photographed from each coverslip and scanned (Nikon Coolscan II) as JPEG files along with an image of a stage micrometer. The JPEG file names were encoded so that images could be analyzed blinded as to treatment. Neurites were measured on enlarged photomicrographs with ImagePro software (Media Cybernetics), with spatial calibration taken from the stage micrometer. The criteria used to define a neurite-bearing cell required one process to extend >= 15 µm from the cell body. The length of the longest neurite coming from any given cell was also recorded. For branched neurites, the portion giving the greatest total length was used. In cases where the beginning point of a neurite could come from one of two cells, the neurite was arbitrarily assigned to one of the cells, and the other cell was scored as nonneurite bearing. Likewise, if two neurites overlapped and the end points could not be established, only one neurite and cell were included in the analysis. Because assigning individual neurites to their appropriate cell body was difficult in large clumps, groups of more than six cells were excluded from analysis. After all measurements for one experiment had been completed, the coding on the JPEG image files was broken, and the data were analyzed using an Excel (Microsoft) spreadsheet. For fluorescent photomicrographs, coverslips were fixed in formalin and then permeabilized in ice-cold acetone for 6-8 min and blocked with preimmune serum for a minimum of 30 min at 37°C. Coverslips were incubated with appropriate primary antibody either overnight at 4°C or for 1 h at room temperature. Coverslips were then washed four times with PBS and incubated with an FITC-conjugated secondary antibody at room temperature for 60 min. Coverslips were again washed four times with PBS and mounted in 90% glycerol on glass slides. Images were taken with Kodak film on a Nikon Optiphot inverted microscope.

Producing stable PC12 cell lines expressing targeted alpha -CaMKII. Constructs for targeted CaMKII are shown in Fig. 1. An epitope-specific sequence (the "FLAG tag," amino acids DYKDDDDK), used to identify the transgenic protein, and a Kozak sequence (nucleotides ACCACC) were added to the amino terminus of alpha -CaMKII DNA. Nuclear targeting was achieved by including an SV40 NLS (amino acids PKKKRKVE); this construct is designated NLS-CaMKII. The cytoplasmic targeting was achieved by leaving the NLS out; that construct is designated Cyto-CaMKII. Constructs were cloned into the HindIII/EcoR1 sites of the pcDNA3 mammalian expression vector. Transfast (Qiagen) reagents were used to cotransfect wild-type PC12 cells with a plasmid coding for neomycin resistance and either the cytoplasmic or nuclear localized, FLAG-tagged CaMKII. Cell lines were selected for antibiotic resistance with the neomycin analog G418 and then screened for properly targeted CaMKII expression by immunocytochemistry by use of a monoclonal anti-FLAG antibody (Eastman Kodak), as described. A cell line transfected to express neomycin resistance alone was also produced to be used as a mock transfected control. Three independent clones of the Cyto-CaMKII transfected cells (Cyto-A2, Cyto-I2, and Cyto-B4), and three clones of NLS-CaMKII transfected cells (NLS-TC2, NLS-KA1, and NLS-KB5) were chosen for further analysis.


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Fig. 1.   Targeted expression of alpha -isoform of Ca2+/calmodulin-regulated protein kinase II (alpha -CaMKII) in PC12 cells. A: schematic diagram of the DNA constructs used to produce targeted CaMKII. The Kozak sequence and a coding sequence for an antibody-specific epitope ("FLAG tag") were added to the 5' end of alpha -CaMKII DNA. For the nuclear localized CaMKII, the nuclear localization signal (NLS) was inserted between the Kozac sequence and the FLAG tag. B: fluorescent micrographs of PC12 cells transfected with the transgenes in A. Left: cytoplasmic expression of alpha -CaMKII; right: nuclear expression when the NLS is included. C: immunoblot screening for alpha -CaMKII protein. PC12 cells were cotransfected with the constructs in A, and a neomycin resistance plasmid (G418) and clonal lines were selected. The alpha -CaMKII protein in 3 independent lines expressing the nuclear targeted form (NLS-TC2, NLS-KB5, and NLS-KA1) migrates slightly higher then the cytoplasmic ones, consistent with its extra 8 amino acids. The lines expressing cytoplasmic alpha -CaMKII are designated Cyto-A2, Cyto-I2, and Cyto-B4.

Measuring CaMKII activity. The CaMKII activity assay followed the protocol of Hanson and Schulman (21). This assay measures CaMKII activity on the basis of the incorporation of 32P into an artificial CaMKII substrate, autocamtide III. Control or transfected PC12 cell lines were plated at 1-2 × 106 cells in a 60-mm dish on the day before assay. Cells were rinsed with PBS and lysed 2 min before the beginning of the autocamtide phosphorylation reaction by use of an extraction buffer containing PIPES (20 mM), EGTA (0.5 mM), leupeptin (10 µg/µl), Na-pyrophosphate (10 mM), ammonium molybdate (0.4 mM), Triton X-100 (0.1%), and dithiothreitol (20 µM). Cell lysate (20 µl) was then added to a reaction tube containing ATP (0.05 mM), PIPES (50 mM), BSA (0.2 mg/ml), MgCl2 (20 mM), and ~0.2-0.25 µCi of [gamma -32P]ATP. To measure CaMKII activity in the lysate, CaCl2 (1 mM), calmodulin (20 µg/ml), and autocamtide III (15 µM) were included; for Ca2+-independent activity, the CaCl2 and CaM were replaced with EGTA (1 mM). The reaction mix was incubated at 30°C for 2 min and then quenched by adding ice-cold trichloroacetic acid (3% final) and storing on ice. Proteins were pelleted, and 20 µl of supernatant were spotted onto phosphocellulose paper. Radioactivity, corresponding to incorporation of 32P into autocamtide III by CaMKII, was measured in a scintillation counter. All assays were performed in duplicate and averaged for one experiment. Three to fourteen experiments were performed on each cell line. For CaMKII assays in subcellular fractions, the nuclear and cytoplasmic fractions were separated (see next section), and the CaMKII activity assay was run on samples of each, as above.

Separation of nuclear and cytoplasmic fractions. The protocol of Kroll et al. (29) was used to isolate nuclear and cytoplasmic fractions from PC12 cells. Control or transfected PC12 cell lines were plated at ~1 × 106 cells in 60-mm tissue culture dishes 1-2 days before fractions were separated. Cells were scraped off three to four dishes, combined, and pelleted. The cells were resuspended in ice-cold PBS and repelleted. The plasma membrane was disrupted by resuspending cells in a 10 mM Tris buffer containing NaCl (10 mM), MgCl2 (3 mM), and NP-40 (0.5%) and incubating for 1 min on ice before centrifuging. Nuclei were pelleted, and the supernatant (cytoplasmic fraction) was removed. Nuclei were washed once in ice-cold PBS with EGTA (1 mM) and then resuspended in the NP-40-containing buffer and sonicated to disrupt nuclear membrane. Debris was pelleted and the supernatant (nuclear fraction) removed.

Immunoblots. Transfected or wild-type cell lines were plated at 8 × 105 cells in 35-mm dishes the day before protein collection. Plates were rinsed once in PBS and then scraped in the presence of warm lysis buffer. Lysates were heated to 95-100°C for 5 min and triturated through a 22-gauge × 0.5-in. needle five to six times to shear the DNA before it was aliquoted and stored at -80°C. Total protein content of lysates was measured using a Lowry protocol modified to accommodate a 96-well format (see Ref. 43). Equal protein loads (usually 20 µg of total protein) were subjected to SDS-PAGE separation in a 12% gel followed by transfer to nitrocellulose (Schleicher & Schuell). The transfer was confirmed with Ponceau S staining and the position of molecular weight markers identified. Membranes were blocked in TBS containing 5% nonfat dry milk and 0.1% Tween 20 (Bio-Rad Laboratories) for 1-2 h and then incubated with primary antibody overnight at 4°C. Membranes were rinsed three times in blocking solution for 5-15 min each, followed by incubation in the appropriate horseradish peroxidase-conjugated secondary antibody for 1-2 h at room temperature. Blots were developed with a chemiluminescence reagent kit (Amersham) and exposed to film. The density of protein bands was quantified by backlighting the film and using ImagePro software (Media Cybernetics) to measure the integrated optical density (IOD) of a fixed rectangular area corresponding to the largest band measured on a given film. The IOD of an identically sized adjacent background area was subtracted from the band IOD, and data were expressed as either total or ratioed IOD, as noted.

Reagents and antibodies. All chemicals were from Sigma unless noted otherwise. Media and cell culture supplies were from GIBCO-BRL unless noted otherwise. Antibodies to Erk, phospho-Erk, cAMP-response element-binding protein (CREB), and phospho-CREB Ser133 were from Cell Signaling Technologies. The anti-FLAG antibody was from Sigma, and the alpha -CaMKII antibody was from Zymed. The antibody to phospho-CREB Ser142 was the kind gift of Drs. Michael Greenberg and Jon Kornhauser (Harvard Medical School).

Statistics. Data were averaged using an Excel spreadsheet (Microsoft). Unless noted otherwise, the two-tailed Student's paired t-test was performed. A significant difference is defined as P < 0.05; a highly significant difference is defined as P < 0.01. Error bars in graphs are ±1 SE.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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Subcellular targeting of CaMKII in PC12 cells. To investigate the role that subcellular localization of CaMKII plays in neuronal differentiation, PC12 cells were transfected with transgenes (Fig. 1A) to produce an epitope-tagged alpha -CaMKII targeted to concentrate in either the cytoplasm (Cyto-CaMKII) or the nucleus (NLS-CaMKII). Cytoplasmic localization is conferred by the native alpha -CaMKII sequence, whereas nuclear localization was achieved by adding an SV40 NLS. To confirm proper targeting of the protein, transfected cells were fixed on glass coverslips and analyzed by immunofluorescence microscopy with an anti-FLAG primary antibody. This revealed that the protein produced by the Cyto-CaMKII construct retains its cytoplasmic localization, whereas the protein produced by the NLS-CaMKII construct accumulates exclusively in the nucleus (Fig. 1B). To generate stable cell lines expressing these proteins, the appropriate CaMKII plasmid was cotransfected into PC12 cells along with a neomycin resistance plasmid. Colonies were selected with G418 antibiotic and screened for appropriately targeted FLAG-tagged protein production by immunocytochemistry (as in Fig. 1B). Several colonies from each construct were identified for further analysis based on the presence of correctly targeted anti-FLAG staining. The identity of this protein as alpha -CaMKII was confirmed by immunoblot analysis using an anti-alpha -CaMKII antibody (Fig. 1C). PC12 cells lack the alpha -isoform of CaMKII, expressing primarily the gamma - and delta -isoforms (48). The absence of alpha -CaMKII in control PC12 cells is shown in lanes 1 (untransfected) and 2 (mock transfected) of Fig. 1C. Lanes 3-5 show cell lines with nuclear targeted alpha -CaMKII, which migrates slightly higher than cytoplasmic CaMKII, consistent with its extra eight amino acids coded for by the NLS. Lanes 6-8 show alpha -CaMKII in cell lines with cytoplasmic targeting. These data confirm that the transfected PC12 cells express a properly localized, FLAG-tagged alpha -CaMKII protein. Three independent clonal cell lines expressing cytoplasmic CaMKII (Cyto-A2, Cyto-I2, and Cyto-B4) and three lines expressing nuclear CaMKII (NLS-TC2, NLS-KB5, and NLS-KA1) were selected for further analysis.

The functional integrity of the alpha -CaMKII transgenic protein was assessed by measuring the level of kinase activity in these transfected cell lines by use of an assay system based on the phosphorylation of an idealized CaMKII substrate, autocamtide III. When CaMKII becomes activated by Ca2+/CaM, members of the holoenzyme complex cross-phosphorylate each other, allowing the CaMKII to remain partially active even after Ca2+ levels fall and CaM dissociates (1, 2, 24, 37). This ability of CaMKII to become Ca2+ independent is thought to be important in allowing CaMKII to detect oscillations in intracellular Ca2+ concentration (22, 40). Therefore, both the total CaMKII activity and the percentage of that activity that was Ca2+ independent were measured. Untransfected and mock transfected control cell lines show high levels of kinase activity from endogenous CaMKII isoforms, with 2.8-4% of that activity being Ca2+ independent (Fig. 2A). CaMKII activity is significantly increased above endogenous levels in cell lines expressing cytoplasmic alpha -CaMKII. The increase is from 88.4 ± 8.6 pmol/min (untransfected cells) to a maximum of 235 ± 46.6 pmol/min (Cyto-A2 line, Fig. 2A), but the percentage of Ca2+-independent activity is unchanged from controls (Fig. 2A). In two of the nuclear CaMKII-expressing cell lines (NLS-TC2 and NLS-KB5), the percentage of CaMKII activity that was Ca2+ independent was significantly greater than in control cells (Fig. 2A). However, the NLS-CaMKII cell lines do not show an increase in total CaMKII activity in whole cell lysate above control levels despite the fact that alpha -CaMKII protein is being expressed in all of these lines (see Fig. 1C). One hypothesis to explain this discrepancy is that the initial CaMKII activity assay, using a pestle tissue grinder to break the cells apart, did not effectively lyse the nuclei; thus nuclear CaMKII might not have been accessible to the autocamtide III substrate. Alternatively, if the nuclei were lysed, it is possible that the CaMKII released may not have contributed significantly to the total cellular CaMKII pool. Because PC12 cells have significant endogenous gamma - and delta -CaMKII isoforms (48, 50) the nuclear targeted CaMKII, although potentially high in the microdomain of the nucleus, may have contributed little to the overall CaMKII activity. To address these issues, nuclear and cytoplasmic fractions of PC12 cells were prepared (see MATERIALS AND METHODS), and CaMKII kinase activity was assayed on each fraction separately. To verify isolation of the nuclear fraction, cells were incubated with Hoechst 33342 (to stain nuclear DNA) and visually inspected during the isolation procedure. A suspension of intact cells showed large, round, phase bright cells with well-defined nuclei (Fig. 2B, top). After the first separation step, which disrupted the plasma membrane releasing the cytoplasmic contents, only the nuclei were visible (Fig. 2B, middle). The nuclear fraction was then completely disrupted by brief sonication. An immunoblot probing with an antibody to alpha -tubulin, a cytosolic structural protein that has been used as a marker for subcellular fractionation protocols, showed good separation of the two fractions (Fig. 2C). This ensured that the contents of the nuclei were fully released to the assay mix and that the CaMKII activity could be assayed in the microdomain of the nucleus. By use of this protocol, the level of total CaMKII activity (nuclear plus cytoplasmic fractions; data not shown) in all cell lines was consistent with the activity seen in the CaMKII assay run on whole cell lysate. For the untransfected control cells, 82% of that total CaMKII activity was found in the cytoplasmic fraction (Fig. 2D). In the cells expressing Cyto-CaMKII, the total CaMKII activity was significantly higher than in untransfected controls, consistent with the activity seen in unfractionated whole cell lysate. This increased activity was distributed similarly to the activity in control cells, with 76% in the cytoplasm and 24% in the nucleus (Fig. 2D). For the NLS-CaMKII cells, the total CaMKII activity was comparable to that of control cells, but the distribution was shifted to the nucleus. Over 40% of the activity was in that fraction, a significantly higher percentage than the activity in the nuclear fraction of untransfected control cells (Fig. 2D). This demonstrates that CaMKII activity can be increased in specific subcellular domains by expressing a targeted alpha -CaMKII protein.


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Fig. 2.   CaMKII activity in the cell lines expressing targeted alpha -CaMKII. A: CaMKII activity assayed in whole cell lysate from control cells (untransfected or mock transfected), cell lines expressing nuclear CaMKII (NLS-TC2, NLS-KB5, and NLS-KA1), and cell lines expressing cytoplasmic CaMKII (Cyto-A2, Cyto-I2, and Cyto-B4). Total CaMKII activity is graphed against the left axis, whereas Ca2+-independent activity is graphed against the right axis. Data are averages of 3-8 independent experiments run in duplicate; error bars, SE. B and C: nuclear/cytoplasmic separation. B: photomicrographs of PC12 cells treated with Hoechst 33342 to visualize the nucleus. Photographs at left are phase contrast, those in middle use an epifluorescent microscope with a UV filter, and those at right are a merge of the 1st two. Top: intact PC12 cells scraped off their tissue culture dish; bottom: isolated nuclei after treatment with an extraction buffer to break apart the plasma membrane. Cytoplasmic fraction was the supernatant of this pellet. Nuclear pellet was then washed and sonicated to disrupt nuclei, producing a nuclear fraction. Scale bar, 30 µm. C: immunoblots to confirm separation of nuclear and cytoplasmic fractions by using an antibody to the cytoplasmic structural protein, alpha -tubulin. W, whole cell lysate; C, cytoplasmic fraction; N, nuclear fraction. D: CaMKII activity in subcellular fractions of representative cell lines NLS-TC2 and Cyto-A2 (expressing nuclear and cytoplasmic CaMKII, respectively). Fractions were prepared as in B, and CaMKII activity was assayed in each fraction as in A. Activity is expressed as percent total CaMKII activity (cytoplasmic + nuclear fractions) measured. Data are averages of 4 independent experiments run in duplicate. Error bars, SE.

Effect of targeted CaMKII on neuronal differentiation. NGF initiates differentiation of PC12 cells into a neuron-like phenotype characterized by a decrease in proliferation, an increase in catecholamine synthesis, and the appearance of long, branching neurites (18). To assess the role of targeted CaMKII in this differentiation process, cells expressing CaMKII in either the nucleus or the cytoplasm were treated with 50 ng/ml NGF for 6-7 days, and the extent of neurite formation was measured (see representative photomicrographs in Fig. 3A). The concentration of NGF chosen (50 ng/ml) causes ~50% of control cells to respond with neurite outgrowth; thus either an increase or decrease in neuronal differentiation could be detected. Untransfected and mock transfected PC12 cells were used as controls. To quantify neurite outgrowth, computer-aided image analysis (ImagePro) was used on enlarged photomicrographs to measure the single longest identifiable process coming from a given cell. By tracing along the neurite path, the computer-aided system allowed measurement of actual neurite length, which was taken from the cell body to the tip of the growth cone. Processes of 15 µm or longer were scored as neurites (see MATERIALS AND METHODS for full criteria of inclusion). Thus not only was the percentage of neurite-bearing cells measured but the lengths of neurites were also compared. After 6-7 days of NGF treatment, 52% of untransfected control cells and 63% of mock transfected cells had at least one defined neurite (Fig. 3B). All of the cell lines expressing Cyto-CaMKII had a slight, but not statistically significant, increase in the percentage of neurite-bearing cells (62-66%) compared with untransfected controls (52%). In contrast, the cells expressing NLS-CaMKII had very few neurites. Of the three independent cell lines studied, from 4 to 25% of the cells had any processes of 15 µm or longer (Fig. 3B), which was significantly fewer than controls. Analyzing the length of neurites, when formed, gives a similar picture: Cyto-CaMKII-expressing cells have longer neurites than controls, whereas NLS-CaMKII-expressing cells have significantly shorter neurites. Figure 3C shows histograms of combined neurite length data. Figure 3C, top, shows neurite length of control cell lines (untransfected and mock transfected combined). A total of 2,528 cells were analyzed, of which 1,502 cells (59%) had neurites. The largest percentage of cells (mode) had neurites of 22 ± 1 µm in length, whereas the average length was 41.2 ± 3.5 µm. Many cells had neurites up to, and occasionally longer than, 100 µm. Figure 3C, middle, shows neurites from all cell lines expressing Cyto-CaMKII. A total of 1,025 cells were analyzed, with 682 (66%) being neurite bearing. The mode of neurite length for these lines is slightly longer than controls, at 26-27 µm, whereas the average neurite length, at 48.6 ± 3.7 µm, was statistically increased over control cells (P < 0.001, Fig. 3C). Figure 3C, bottom, shows the combined data for cell lines expressing NLS-CaMKII. A total of 1,982 cells were analyzed, with 298 (15%) having neurites. When cells did grow neurites, these neurites generally just made the cutoff, with a mode of 15 µm and an average length of 30.7 ± 3.7 µm, significantly (P < 0.001) shorter than controls; very few neurites grew longer than 50 µm.


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Fig. 3.   Nerve growth factor (NGF)-induced neuronal differentiation in cell lines expressing targeted alpha -CaMKII. A: typical representative phase-contrast photomicrographs of control or transfected cell lines treated with NGF in serum-free medium. Untreated cells (column at left) were maintained in complete medium containing 10% fetal bovine serum but no NGF. Scale bar, 30 µm. B: quantification of neurite outgrowth, shown as percent neurite-bearing cells. Photomicrographs from 6 adjacent fields were taken for each cell line in each experiment. The longest individual process from each cell was measured and a cell defined as neurite bearing if it had >= 1 process of >= 15 µm in length. Data are averages of 3-7 independent experiments (indicated at base of each bar). Error bars, SE. C: histograms showing no. of cells with neurites of a given length as a percentage of all cells analyzed in a given group. Top: grouped data from untransfected and mock transfected cells; middle: data grouped from Cyto-A2, Cyto-I2, and Cyto-B4 cell lines; bottom: data from NLS-TC2, NLS-KA1, and NLS-KB5 cell lines. Average is mean of all cells ± SE. Statistical significance is established by 2-way ANOVA on log transformed data.

Thus, on the basis of the percentage of neurite-bearing cells, NGF-treated PC12 cells with nuclear CaMKII exhibit less neuronal differentiation than control cells. In addition, when these NLS-CaMKII cells do differentiate, they produce shorter neurites than controls. In contrast, treating cells that express Cyto-CaMKII with NGF did not decrease the percentage of neurite-bearing cells but did allow the formation of longer neurites.

Modulation of the MAPK pathway. Decreased neurite outgrowth in the NLS-CaMKII cells could be caused by a disruption in the integrity of the NGF signal transduction pathways, which are characterized by several parallel signaling cascades (36, 42, 53, 54). One effect of NGF binding to its high-affinity receptor (Trk A) on PC12 cells is the initiation of signaling cascades leading to the sustained (>2 h) phosphorylation of the MAPKs Erk1 and Erk2 (Ref. 36 and Fig. 5). The sustained activation of this pathway has been shown to be necessary for neuronal differentiation and neurite outgrowth in PC12 cells (26, 36). To address the integrity of this MAPK pathway, PC12 cells were cultured overnight in low-serum medium (to reduce background Erk phosphorylation) and then stimulated with NGF for 10-120 min before proteins were extracted. Immunoblots were run, probing for both total Erk level and the presence of Erk phosphorylated at Thr183 and Tyr185. Untransfected control cells display rapid initial phosphorylation of Erk (10 min), followed by a sustained phosphorylation that does not fully decay over the 2-h time period studied (Fig. 4). This same phosphorylation pattern is seen in cell lines expressing either nuclear or cytoplasmic CaMKII (Fig. 4), indicating that this MAPK-signaling pathway remains intact and unmodified. Thus the lack of NGF-induced neuronal differentiation in cells expressing NLS-CaMKII cannot be accounted for by changes in the phosphorylation of Erk1 and Erk2.


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Fig. 4.   Phosphorylation of p44/42 MAPKs extracellular signal-regulated kinase Erk1 and Erk2. PC12 cells were gown in low-serum (0.5%) medium overnight and then stimulated with NGF (50 ng/ml in low-serum medium) for 10-120 min before proteins were extracted. A: immunoblots from representative cell lines, as in Fig. 2, using an antibody to either total Erk (top row) or to Erk phosphorylated at Thr183 and Tyr185 (pErk; bottom row). Equivalent amounts of total protein (20 µg) were loaded into each lane. B: quantification of Erk phosphorylation expressed as a ratio of the integrated optical density (OD) of the pErk doublet to the doublet for total Erk, as in A. Data are the average of 3 experiments. Error bars, SE.

Expression and phosphorylation of CREB. The transcription factor CREB is rapidly phosphorylated at Ser133 after a number of different extracellular stimuli. This phosphorylation is accomplished by several intracellular signaling pathways (see reviews in Refs. 12 and 38) including two MAPK cascades in PC12 cells (52) and by PKA after increases in intracellular Ca2+ concentration (19). Phosphorylation at Ser133 is a necessary step for CREB activation, and activated CREB is important in the maintenance of neuronal cell populations (12). Because NGF can activate CREB through several different signaling pathways, the hypothesis was tested that either the total amount of CREB present or its phosphorylation at Ser133 in response to NGF was modified by targeted expression of alpha -CaMKII. Control and CaMKII-transfected cell lines were exposed to medium with or without NGF (50 ng/ml) for 15 min before cells were lysed and proteins extracted. Immunoblots using antibodies to total CREB or phospho-CREB Ser133 were run on these proteins (Fig. 5A). Quantification of band density from the blots shows that the basal level of CREB is unchanged in the cell lines studied (Fig. 5B). There is a uniform, slight decrease in band density of CREB after NGF treatment (Fig. 5B, left), but because of the rapid time frame involved (15 min) this may indicate a slight decrease in antibody affinity for the phosphorylated form of CREB rather than an actual decrease in CREB content. The degree of Ser133 phosphorylation was evaluated in parallel blots by use of a phosphospecific antibody to Ser133. Treating untransfected control cells with NGF for 15 min led to a robust increase in phospho-CREB Ser133 (Fig. 5, A and B), as others have reported (14). In the Cyto-CaMKII and NLS-CaMKII cell lines, treatment with NGF also led to rapid phosphorylation of CREB Ser133 (Fig. 5, A and B), which was not statistically different from controls. These data show that the inhibition of neuronal differentiation in cells expressing NLS-CaMKII did not arise from changes in either total CREB or phospho-CREB Ser133.


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Fig. 5.   Expression and phosphorylation of transcription factor cAMP response element-binding protein (CREB). Control (untransfected) PC12 cells or representative cell lines (as in Fig. 2) expressing targeted alpha -CaMKII were exposed to medium with or without NGF (50 ng/ml) for 15 min before proteins were extracted. A: immunoblot using antibodies to total CREB (left) or to CREB phosphorylated at Ser133 (right). B: quantification of band density from immunoblots, as in A. Left: integrated OD of CREB bands with and without NGF treatment. Right: integrated OD for CREB phosphorylated at Ser133. Data are average integrated OD from bands on 3 independent immunoblots. Error bars, SE. C: immunoblot using antibody to CREB phosphorylated at Ser142. Cortical neurons were from 6-day-old primary cultures of rat embryonic neurons. Cells were untreated (C) or treated with KCl (K) for 15 min to stimulate Ca2+ influx or treated with NGF (N). This blot is representative of 3 similar experiments.

CREB can also be phosphorylated by CaMKII at Ser142 (28, 44), which may decrease CREB transcriptional activity in some contexts (13, 28, 51). A relevant hypothesis, then, is that nuclear CaMKII could phosphorylate CREB at Ser142 and thus block neuronal differentiation by inhibiting transcription of some CREB-responsive genes. To test this, proteins from control PC12 cells or PC12 cells with nuclear CaMKII were probed in an immunoblot for this phosphorylation (Fig. 5C). Rat cortical neurons, used as a positive control, show robust CREB Ser142 phosphorylation after 15 min of depolarization with KCl (Fig. 5C). Very little basal phosphorylation of CREB at Ser142 was observed in untransfected, NLS-CaMKII-transfected, or Cyto-CaMKII-transfected PC12 cells. Application of KCl to these cell lines appears to give a slight increase in CREB Ser142 phosphorylation, particularly in the NLS-CaMKII-expressing cells, indicating that this serine can be phosphorylated, given the correct stimulus. However, treating these cell lines with NGF (50 ng/ml for 15 min) failed to change CREB Ser142 phosphorylation consistently. Thus, although phosphorylation of Ser142 may be a critical step in regulating CREB activity in some contexts, the decreased neurite outgrowth in NLS-CaMKII PC12 cells is probably not do to an inhibitory phosphorylation at this site.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

CaMKII mediates many signal transduction systems and has been found in virtually every cell type examined (for reviews see Refs. 1 and 24). It is particularly abundant in neuronal populations, where it has been implicated in such diverse functions as promoting neuronal survival and regulating long-term potentiation. Although it appears necessary for proper neuronal development, the exact mechanisms of CaMKII's actions are still being defined.

In the area of neurite outgrowth, results of several studies have been inconclusive. A CNS cell line, CAD cells, which normally differentiate on serum withdrawal, underwent spontaneous neuronal differentiation after being transfected to produce CaMKII (9). In two neuroblastoma cell lines, overexpressing CaMKII led to an increase in both the percentage of cells with neurites and the neurite length (15). However, when CaMKII was overexpressed in PC12 cells, which were then stimulated with either NGF (31) or cAMP (48), neurite formation was inhibited. These latter two studies are similar to the experiments performed here and will be discussed separately.

Perhaps one reason for the incomplete picture arises from differences in the subcellular locations of various CaMKII splice variants. At least three CaMKII splice variants, alpha B, gamma A, and delta B, contain NLSs (3, 10, 46). Some progress has been made in determining tissue distribution of these isoforms (10, 47). Two of the variants, alpha B and delta B, have been shown to concentrate in the nucleus of astrocytes (46), whereas the delta B-isoform has also been seen in cerebellar granule cells (47). Little is known about the role that these isoforms play in normal cellular function. It is known that expression levels of various isoforms can change for a given cell type, depending on the stimuli to which the cells are exposed or the stage in their development (3, 8). Therefore, this study has set about specifically to investigate the role of nuclear targeted vs. cytoplasmic targeted CaMKII in neuronal differentiation with the PC12 cell line as a test system.

PC12 cells were transfected with an alpha -CaMKII gene, either containing or omitting an NLS. The CaMKII protein produced by these constructs is efficiently expressed in one of two subcellular locations, either the cytoplasm (Cyto-CaMKII) or the nucleus (NLS-CaMKII). Furthermore, the activity of CaMKII was increased in the appropriate compartment relative to either untransfected controls or cell lines with the opposite CaMKII targeting. These experiments demonstrate that functional CaMKII can be targeted to mimic various endogenous isoforms.

Control or transfected cell lines expressing Cyto-CaMKII or NLS-CaMKII were treated with a submaximal concentration of NGF (one that leads to ~50% of control cells differentiating and growing neurites within 6-7 days), and the percentage of neurite-bearing cells and neurite length were measured as an index of neuronal differentiation. In three independent clonal cell lines expressing Cyto-CaMKII, the percentage of neurite-bearing cells was similar to the percentage of neurite-bearing cells in untransfected or mock transfected PC12 cells. In addition, the length of neurites formed was increased compared with the control cell lines. These data indicate that an increase in cytoplasmic CaMKII above the endogenous levels found in PC12 cells does not inhibit neurite outgrowth.

This result is consistent with studies done on several neuroblastoma cell lines. When Nb2A (15, 41), NG108-15 (15), or CAD cells (9) are transfected with an alpha -CaMKII gene, the extent of neurite outgrowth after appropriate stimulation is markedly increased over untransfected control cells. Masse and Kelly (31), however, produced PC12 cell lines expressing alpha -CaMKII and showed a decrease in the percentage of neurite-bearing cells after NGF treatment. Several differences were observed in cellular growth properties, and one important difference in technique was noted between that study and this one. Their cell lines transfected with alpha -CaMKII showed a decreased initial replication rate compared with controls. The CaMKII-transfected cells studied here all proliferated similarly to untransfected controls (data not shown). In addition, Masse and Kelly reported increased cell-to-substrate adhesion in transfected cells compared with controls, something not seen in the cell lines used for the present study (data not shown). Experimentally, the culture medium used by Masse and Kelly to maintain their PC12 cells contained 10% horse serum and 5% fetal calf serum. In the present study, 10% FBS was used and no horse serum. Because serum is an undefined component known to contain numerous cytokines and growth factors, it seems reasonable that this difference in sera may have influenced the basal state of the cells. Therefore, the interaction of NGF signaling and CaMKII could be occurring in distinct intracellular contexts. These differences seem to include changes in cell-to-substrate adhesion, an important parameter for neurite growth.

Another group that has overexpressed alpha -CaMKII in the cytoplasm of PC12 cells, Tashima et al. (48), used cAMP to stimulate neurite outgrowth. They also saw a decrease in neurite-bearing cells in lines expressing alpha -CaMKII (48). However, when a different pathway from NGF is used, cAMP induces neurite outgrowth; the neurites appear much more rapidly and exhibit a different morphology (20, 35, 48). A similar effect to that of Tashima et al. was seen in the Cyto-CaMKII cells in our study. Neurite outgrowth was greatly reduced in Cyto-CaMKII cells following cAMP treatment compared with untransfected control cells (Kutcher LW, unpublished observation).

A novel feature of this study is employing an NLS to target CaMKII to the nucleus. When alpha -CaMKII is targeted to the nucleus of PC12 cells, a different picture emerges. In contrast to the Cyto-CaMKII-expressing cells, three independent clonal cell lines expressing NLS-CaMKII all showed significantly fewer neurite-bearing cells after NGF stimulation. When neurites did form in the NLS-CaMKII-transfected cells, they were shorter than the neurites formed in either control or Cyto-CaMKII-expressing cells. Thus, on the basis of the percentage of neurite-bearing cells and the length of neurites formed, nuclear targeted CaMKII inhibits NGF-induced neuronal differentiation in PC12 cells.

Several steps in the NGF-signaling cascade were evaluated to investigate the mechanisms leading to this phenomenon. NGF initiates neurite outgrowth in PC12 cells by a complex signaling pathway, with many sites of potential regulation (reviewed in Refs. 6, 27, and 45; see also Ref. 54). Binding of NGF to its high-affinity receptor, Trk A, initiates several intracellular events, including a transient increase in intracellular Ca2+ concentration (33, 34) and the sustained phosphorylation of the MAPKs Erk1 and Erk2 (36, 53). This Erk activation is mediated via Ras (36, 54) and Rap1 (54) and is necessary for neurite outgrowth (26). Stimuli other than NGF, such as EGF and activation of CaM (11), can transiently phosphorylate Erk without initiating neurite outgrowth, suggesting that the sustained phosphorylation of Erk is critical to neurite induction. To evaluate the integrity of this MAPK-signaling cascade, both the total amount of Erk and its phosphorylation in response to NGF were evaluated in Cyto-CaMKII and NLS-CaMKII cell lines. These cell lines show robust phosphorylation of Erk after 10 min of NGF, which is maintained at levels similar to controls for 120 min. Therefore, neither immediate nor longer-term Erk phosphorylation is affected by the presence of either NLS-CaMKII or Cyto-CaMKII. Thus the observed differences in neuronal differentiation are not related to this NGF-stimulated pathway, at least to the point of Erk phosphorylation.

NGF-signaling cascades ultimately activate the transcription factor CREB. Several kinases, such as PKA, PKC, RSK2, and CaM kinases, phosphorylate CREB at Ser133. This phosphorylation recruits coactivators into a signaling complex capable of driving transcription of genes that have the cAMP response element (CRE) in their promoters (see reviews in Refs. 7, 32, and 38). Xing et al. (52) have determined that NGF leads to the phosphorylation of CREB at Ser133 by two distinct mechanisms. The first is via the sustained activation of Erk (discussed above), which activates the kinases RSK1, -2, and -3; blocking this pathway decreases, but does not eliminate, CREB Ser133 phosphorylation. A second MAPK pathway, involving p38 MAPK activation of MAPK-activated protein kinase 2, is also initiated by NGF and leads to Ser133 phosphorylation (52). In hippocampal cells, the upstream activator of CaMKII, CaM, can translocate to the nucleus, where it correlates with increased CREB phosphorylation (6). It has been suggested that a nuclear targeted isoform of CaMKII, delta 3, may be involved in CREB regulation (47). Therefore, the hypothesis was tested that either total CREB or phospho-CREB Ser133 was altered in NLS-CaMKII cell lines. Immunoblots probing for these proteins in NLS-CaMKII, Cyto-CaMKII, and control cell lines all show equivalent amounts of CREB, and the cell lines respond to NGF with equivalent levels of Ser133 phosphorylation. Thus the inhibited neuronal differentiation in NLS-CaMKII cells does not arise from differences in either total CREB or its ability to be phosphorylated at a site critical for activation, Ser133.

CREB can be phosphorylated at sites in addition to Ser133. Phosphopeptide mapping shows that Ser142 (28, 44) and Ser143 (28) can both be phosphorylated by CaMKII. In vitro assays indicate that phosphorylation of Ser142 inhibits dimerization of CREB and subsequent binding of the CREB-binding protein (CBP) (51). Because CBP is critical to the activated CREB complex (32, 38), phosphorylation at Ser142 by CaMKII would be expected to inhibit CREB activity, which seems to happen in vitro. In cortical neurons stimulated with KCl (28) and in GH3 cells (44), full activity of a CREB/Gal4 reporter gene requires that the Ser142 of CREB be mutated to an alanine.

Recent in vivo work, however, indicates that the picture may be more complex; CREB Ser142 phosphorylation may actually increase gene transcription in certain contexts (13, 28). Gau et al. (13) produced transgenic mice with a Ser142-to-Ala mutation in CREB. These animals had alterations in their circadian clock that correlated with decreased expression of c-Fos (13), a transcription factor controlled by CREB (14).

Because of the emerging role of Ser142 phosphorylation in CREB activity, an antibody to phospho-CREB Ser142 (28) was used to probe for this phosphorylation in rat cortical neurons, control PC12 cells, and NLS-CaMKII- and Cyto-CaMKII-expressing PC12 cells. Immunoblots of primary cortical neurons treated for 15 min with depolarizing concentrations of KCl show robust phosphorylation of CREB Ser142 similar to that seen by Kornhauser et al. (28). In control or transfected PC12 cells, the basal level of CREB Ser142 phosphorylation was very low, equivalent to untreated cortical neurons. There was no increase in this phosphorylation when the cells were treated with NGF and only a slight increase when they were depolarized with KCl. The data shown here indicate that, in PC12 cells, CREB is not phosphorylated at Ser142 in response to NGF. Furthermore, the presence of a nuclear localized CaMKII is not sufficient to drive that phosphorylation either with or without treatment. In addition, NGF-stimulated c-Fos expression was not decreased by either NLS-CaMKII or Cyto-CaMKII (data not shown), indicating that CREB remains fully competent to drive gene expression in these cell lines. Therefore, the decreased neuronal differentiation in NLS-CaMKII-expressing PC12 cells does not seem to be due to an inhibitory phosphorylation of CREB at Ser142.

In conclusion, this study has used transgenes encoding targeted alpha -CaMKII to elevate CaMKII kinase activity in specific subcellular compartments and has shown that nuclear CaMKII inhibits NGF-induced neuronal differentiation of PC12 cells, whereas cytoplasmically localized CaMKII does not. The inhibition of differentiation is independent of MAPK activation, which is required for neurite outgrowth. The inhibited neurite outgrowth is also independent of the transcription factor CREB, as total CREB remained constant in cells with NLS-CaMKII compared with controls, nor was phosphorylation at Ser133 or Ser142 changed. Thus it would appear that the role of CaMKII in promoting or inhibiting neuronal differentiation depends on the subcellular location of the expressed CaMKII isoform. As more is learned about which neuronal populations express nuclear localized CaMKII isoforms and at what developmental stages these expressions occur, a more complete picture of the role for nuclear CaMKII in neurons will emerge.


    ACKNOWLEDGEMENTS

We thank Dr. Maria Czyzyk-Krzeska (University of Cincinnati) for the gift of PC12 cells and Drs. Michael Greenberg and Jon Kornhauser (Harvard Medical School) for the anti-phospho-CREB Ser142 antibody. We also thank Dr. Linda Levin (University of Cincinnati) for expert advice with the statistical analysis.


    FOOTNOTES

This work was partially funded by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-46433 to J. R. Dedman.

Address for reprint requests and other correspondence: J. Dedman, Univ. of Cincinnati College of Medicine, 231 Albert Sabin Wy, Cincinnati, OH 45267 (E-mail: John.Dedman{at}UC.edu).

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

First published February 5, 2003;10.1152/ajpcell.00510.2002

Received 5 November 2002; accepted in final form 21 January 2003.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Braun, AP, and Schulman H. The multifunctional calcium/calmodulin-dependent protein kinase: from form to function. Annu Rev Physiol 57: 417-445, 1995[ISI][Medline].

2.   Brickey, DA, Bann JG, Fong YL, Perrino L, Brennan RG, and Soderling TR. Mutational analysis of the autoinhibitory domain of calmodulin kinase II. J Biol Chem 269: 29047-29054, 1994[Abstract/Free Full Text].

3.   Brocke, L, Srinivasan M, and Schulman H. Developmental and regional expression of multifunctional Ca2+/calmodulin-dependent protein kinase isoforms in rat brain. J Neurosci 15: 6797-6808, 1995[ISI][Medline].

4.   Caran, N, Johnson LD, Jenkins KJ, and Tombes RM. Cytosolic targeting domains of gamma and delta calmodulin-dependent protein kinase II. J Biol Chem 276: 42514-42519, 2001[Abstract/Free Full Text].

5.   Dedman, JR, and Kaetzel MA. Calcium as an intracellular second messenger: mediation by calcium binding proteins. In: Cell Physiology Source Book. New York: Academic, 1995, p. 128-136.

6.   Deisseroth, K, Heist EK, and Tsien RW. Translocation of calmodulin to the nucleus supports CREB phosphorylation in hippocampal neurons. Nature 392: 198-202, 1998[ISI][Medline].

7.   Deisseroth, K, and Tsien RW. Dynamic multiphosphorylation passwords for activity-dependent gene expression. Neuron 34: 179-182, 2002[ISI][Medline].

8.   Donai, H, Murakami T, Amano T, Sogawa Y, and Yamauchi T. Induction and alternative splicing of delta isoform of Ca(2+)/calmodulin-dependent protein kinase II during neural differentiation of P19 embryonal carcinoma cells and during brain development. Brain Res Mol Brain Res 85: 189-199, 2000[ISI][Medline].

9.   Donai, H, Nakamura M, Sogawa Y, Wang JK, Urushihara M, and Yamauchi T. Involvement of Ca2+/calmodulin-dependent protein kinase II in neurite outgrowth induced by cAMP treatment and serum deprivation in a central nervous system cell line, CAD derived from rat brain. Neurosci Lett 293: 111-114, 2000[ISI][Medline].

10.   Edman, CF, and Schulman H. Identification and characterization of delta B-CaM kinase and delta C-CaM kinase from rat heart, two new multifunctional Ca2+/calmodulin-dependent protein kinase isoforms. Biochim Biophys Acta 1221: 89-101, 1994[ISI][Medline].

11.   Egea, J, Espinet C, Soler RM, Peiro S, Rocamora N, and Comella JX. Nerve growth factor activation of the extracellular signal-regulated kinase pathway is modulated by Ca(2+) and calmodulin. Mol Cell Biol 20: 1931-1946, 2000[Abstract/Free Full Text].

12.   Finkbeiner, S. CREB couples neurotrophin signals to survival messages. Neuron 25: 11-14, 2000[ISI][Medline].

13.   Gau, D, Lemberger T, von Gall C, Kretz O, Le Minh N, Gass P, Schmid W, Schibler U, Korf HW, and Schutz G. Phosphorylation of CREB Ser142 regulates light-induced phase shifts of the circadian clock. Neuron 34: 245-253, 2002[ISI][Medline].

14.   Ginty, DD, Bonni A, and Greenberg ME. Nerve growth factor activates a Ras-dependent protein kinase that stimulates c-fos transcription via phosphorylation of CREB. Cell 77: 713-725, 1994[ISI][Medline].

15.   Goshima, Y, Ohsako S, and Yamauchi T. Overexpression of Ca2+/calmodulin-dependent protein kinase II in Neuro2a and NG108-15 neuroblastoma cell lines promotes neurite outgrowth and growth cone motility. J Neurosci 13: 559-567, 1993[Abstract].

16.   Green, SH. The use of PC12 cells for the study of the mechanism of action of neurotrophic factors: signal transduction and programmed cell death. In: Methods: A Companion to Methods in Enzymology. New York: Academic, 1995, p. 222-237.

17.  Greene LA. The importance of both early and delayed responses in the biological actions of nerve growth factor. Trends Neurosci: 91-94, 1984.

18.   Greene, LA, and Tischler AS. Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor. Proc Natl Acad Sci USA 73: 2424-2428, 1976[Abstract].

19.   Grewal, SS, Fass DM, Yao H, Ellig CL, Goodman RH, and Stork PJ. Calcium and cAMP signals differentially regulate cAMP-responsive element-binding protein function via a Rap1-extracellular signal-regulated kinase pathway. J Biol Chem 275: 34433-34441, 2000[Abstract/Free Full Text].

20.   Gunning, PW, Landreth GE, Bothwell MA, and Shooter EM. Differential and synergistic actions of nerve growth factor and cyclic AMP in PC12 cells. J Cell Biol 89: 240-245, 1981[Abstract].

21.   Hanson, PI, and Schulman H. Inhibitory autophosphorylation of multifunctional Ca2+/calmodulin-dependent protein kinase analyzed by site-directed mutagenesis. J Biol Chem 267: 17216-17224, 1992[Abstract/Free Full Text].

22.   Heist, EK, and Schulman H. The role of Ca2+/calmodulin-dependent protein kinases within the nucleus. Cell Calcium 23: 103-114, 1998[ISI][Medline].

23.   Heist, EK, Srinivasan M, and Schulman H. Phosphorylation at the nuclear localization signal of Ca2+/calmodulin-dependent protein kinase II blocks its nuclear targeting. J Biol Chem 273: 19763-19771, 1998[Abstract/Free Full Text].

24.   Hook, SS, and Means AR. Ca(2+)/CaM-dependent kinases: from activation to function. Annu Rev Pharmacol Toxicol 41: 471-505, 2001[ISI][Medline].

25.   Johnson, LD, Willoughby CA, Burke SH, Paik DS, Jenkins KJ, and Tombes RM. Delta Ca(2+)/calmodulin-dependent protein kinase II isozyme-specific induction of neurite outgrowth in P19 embryonal carcinoma cells. J Neurochem 75: 2380-2391, 2000[ISI][Medline].

26.   Klesse, LJ, Meyers KA, Marshall CJ, and Parada LF. Nerve growth factor induces survival and differentiation through two distinct signaling cascades in PC12 cells. Oncogene 18: 2055-2068, 1999[ISI][Medline].

27.   Klesse, LJ, and Parada LF. Trks: signal transduction and intracellular pathways. Microsc Res Tech 45: 210-216, 1999[ISI][Medline].

28.   Kornhauser, JM, Cowan CW, Shaywitz AJ, Dolmetsch RE, Griffith EC, Hu LS, Haddad C, Xia Z, and Greenberg ME. CREB transcriptional activity in neurons is regulated by multiple, calcium-specific phosphorylation events. Neuron 34: 221-233, 2002[ISI][Medline].

29.   Kroll, SL, Paulding WR, Schnell PO, Barton MC, Conaway JW, Conaway RC, and Czyzyk-Krzeska MF. Von Hippel-Lindau protein induces hypoxia-regulated arrest of tyrosine hydroxylase transcript elongation in pheochromocytoma cells. J Biol Chem 274: 30109-30114, 1999[Abstract/Free Full Text].

30.   Loo, DT, and Rillema JR. Measurement of cell death. Methods Cell Biol 57: 251-264, 1998[ISI][Medline].

31.   Masse, T, and Kelly PT. Overexpression of Ca2+/calmodulin-dependent protein kinase II in PC12 cells alters cell growth, morphology, and nerve growth factor-induced differentiation. J Neurosci 17: 924-931, 1997[Abstract/Free Full Text].

32.   Mayr, B, and Montminy M. Transcriptional regulation by the phosphorylation-dependent factor CREB. Nat Rev Mol Cell Biol 2: 599-609, 2001[ISI][Medline].

33.   Nikodijevic, B, and Guroff G. Nerve growth factor-induced increase in calcium uptake by PC12 cells. J Neurosci Res 28: 192-199, 1991[ISI][Medline].

34.   Pandiella-Alonso, A, Malgaroli A, Vicentini LM, and Meldolesi J. Early rise of cytosolic Ca2+ induced by NGF in PC12 and chromaffin cells. FEBS Lett 208: 48-51, 1986[ISI][Medline].

35.   Pollock, JD, Krempin M, and Rudy B. Differential effects of NGF, FGF, EGF, cAMP, and dexamethasone on neurite outgrowth and sodium channel expression in PC12 cells. J Neurosci 10: 2626-2637, 1990[Abstract].

36.   Qiu, MS, and Green SH. PC12 cell neuronal differentiation is associated with prolonged p21ras activity and consequent prolonged ERK activity. Neuron 9: 705-717, 1992[ISI][Medline].

37.   Rich, RC, and Schulman H. Substrate-directed function of calmodulin in autophosphorylation of Ca2+/calmodulin-dependent protein kinase II. J Biol Chem 273: 28424-28429, 1998[Abstract/Free Full Text].

38.   Shaywitz, AJ, and Greenberg ME. CREB: a stimulus-induced transcription factor activated by a diverse array of extracellular signals. Annu Rev Biochem 68: 821-861, 1999[ISI][Medline].

39.   Soderling, TR. The Ca-calmodulin-dependent protein kinase cascade. Trends Biochem Sci 24: 232-236, 1999[ISI][Medline].

40.   Soderling, TR, Chang B, and Brickey D. Cellular signaling through multifunctional Ca2+/calmodulin-dependent protein kinase II. J Biol Chem 276: 3719-3722, 2001[Free Full Text].

41.   Sogawa, Y, Yoshimura Y, Otaka A, and Yamauchi T. Ca(2+)-independent activity of Ca(2+)/calmodulin-dependent protein kinase II involved in stimulation of neurite outgrowth in neuroblastoma cells. Brain Res 881: 165-175, 2000[ISI][Medline].

42.   Stephens, RM, Loeb DM, Copeland TD, Pawson T, Greene LA, and Kaplan DR. Trk receptors use redundant signal transduction pathways involving SHC and PLC-gamma 1 to mediate NGF responses. Neuron 12: 691-705, 1994[ISI][Medline].

43.   Stoscheck, CM. Quantitation of protein. Methods Enzymol 182: 50-68, 1990[ISI][Medline].

44.   Sun, P, Enslen H, Myung PS, and Maurer RA. Differential activation of CREB by Ca2+/calmodulin-dependent protein kinases type II and type IV involves phosphorylation of a site that negatively regulates activity. Genes Dev 8: 2527-2539, 1994[Abstract].

45.   Szeberenyi, J. Gene activation pathways of nerve growth factor signaling: a minireview. Neurobiology (Bp) 4: 1-11, 1996[Medline].

46.   Takeuchi, Y, Yamamoto H, Fukunaga K, Miyakawa T, and Miyamoto E. Identification of the isoforms of Ca(2+)/calmodulin-dependent protein kinase II in rat astrocytes and their subcellular localization. J Neurochem 74: 2557-2567, 2000[ISI][Medline].

47.   Takeuchi, Y, Yamamoto H, Matsumoto K, Kimura T, Katsuragi S, Miyakawa T, and Miyamoto E. Nuclear localization of the delta subunit of Ca2+/calmodulin-dependent protein kinase II in rat cerebellar granule cells. J Neurochem 72: 815-825, 1999[ISI][Medline].

48.   Tashima, K, Yamamoto H, Setoyama C, Ono T, and Miyamoto E. Overexpression of Ca2+/calmodulin-dependent protein kinase II inhibits neurite outgrowth of PC12 cells. J Neurochem 66: 57-64, 1996[ISI][Medline].

49.   Tobimatsu, T, and Fujisawa H. Tissue-specific expression of four types of rat calmodulin-dependent protein kinase II mRNAs. J Biol Chem 264: 17907-17912, 1989[Abstract/Free Full Text].

50.   Tombes, RM, Mikkelsen RB, Jarvis WD, and Grant S. Downregulation of delta CaM kinase II in human tumor cells. Biochim Biophys Acta 1452: 1-11, 1999[ISI][Medline].

51.   Wu, X, and McMurray CT. Calmodulin kinase II attenuation of gene transcription by preventing cAMP response element-binding protein (CREB) dimerization and binding of the CREB-binding protein. J Biol Chem 276: 1735-1741, 2001[Abstract/Free Full Text].

52.   Xing, J, Kornhauser JM, Xia Z, Thiele EA, and Greenberg ME. Nerve growth factor activates extracellular signal-regulated kinase and p38 mitogen-activated protein kinase pathways to stimulate CREB serine 133 phosphorylation. Mol Cell Biol 18: 1946-1955, 1998[Abstract/Free Full Text].

53.   Yao, H, York RD, Misra-Press A, Carr DW, and Stork PJ. The cyclic adenosine monophosphate-dependent protein kinase (PKA) is required for the sustained activation of mitogen-activated kinases and gene expression by nerve growth factor. J Biol Chem 273: 8240-8247, 1998[Abstract/Free Full Text].

54.   York, RD, Yao H, Dillon T, Ellig CL, Eckert SP, McCleskey EW, and Stork PJ. Rap1 mediates sustained MAP kinase activation induced by nerve growth factor. Nature 392: 622-626, 1998[ISI][Medline].


Am J Physiol Cell Physiol 284(6):C1334-C1345
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