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J. Biol. Chem., Vol. 276, Issue 45, 42514-42519, November 9, 2001

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Cytosolic Targeting Domains of  and  Calmodulin-dependent Protein Kinase II^*

Nicole Caran, Lesley D. Johnson, Kimberley J. Jenkins, and Robert M. Tombes

From the Departments of Biology and Biochemistry and Molecular Biophysics, Virginia Commonwealth University, Richmond Virginia 23284-2012

Received for publication, April 5, 2001, and in revised form, August 30, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Ca²⁺/calmodulin-dependent protein kinase II (CaMK-II) isozyme variability is the result of alternative usage of variable domain sequences. Isozyme expression is cell type-specific to transduce the appropriate Ca²⁺ signals. We have determined the subcellular targeting domain of _E CaMK-II, an isozyme that induces neurite outgrowth, and of a structurally similar isozyme, _C CaMK-II, which does not induce neurite outgrowth. _E CaMK-II co-localizes with filamentous actin in the perinuclear region and in cellular extensions. In contrast, _C CaMK-II is uniformly cytosolic. Constitutively active _E CaMK-II induces F-actin-rich extensions, thereby supporting a functional role for its localization. C-terminal constructs, which lack central variable domain sequences, can oligomerize and localize like full-length _E and _C CaMK-II. Central variable domains themselves are monomeric and have no targeting capability. The C-terminal 95 residues of  CaMK-II also has no targeting capability but can efficiently oligomerize. These findings define a targeting domain for  and  CaMK-IIs that is in between the central variable and association domains. This domain is responsible for the subcellular targeting differences between  and  CaMK-IIs.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The multifunctionality of the type II Ca²⁺/calmodulin-dependent protein kinase (CaMK-II)¹ family is reflected in the diversity of its gene products. CaMK-II isozyme variability is the result of alternative mRNA splicing from four genes (, , , and ) found on separate chromosomes in humans (1-7). Splicing occurs primarily in the central variable domain where up to seven alternative exons can be used in different combinations to yield over two dozen unique isozymes (8-10). An additional alternative splice domain is found only in  CaMK-II at the C-terminal tail (11, 12). Similar to other multifunctional protein kinase families, CaMK-II isozymes gain specificity by subcellular targeting to locations, such as the nucleus, the plasma membrane, the cytoskeleton, and specialized structures, such as post-synaptic densities or the sarcoplasmic reticulum (4, 10, 13-16). CaMK-II holoenzyme is normally dodecameric, and targeting can be influenced by the heterooligomerization of CaMK-II monomers (16-18). For example, cytosolic CaMK-IIs can redirect nuclear-targeted isozymes to the cytosol by heterooligomerization (13, 19). Properly targeted CaMK-IIs can influence cellular events unlike catalytically identical but mistargeted isozymes (20, 21). CaMK-II localization can respond to the activation state of CaMK-II (15, 22) or to phosphorylation by other kinases (14). Other than nuclear localization sequences, the targeting domains have not been defined for CaMK-II isozymes, particularly those encoded by the  and  genes.

Although  and  are the predominant CaMK-II genes transcribed in the central nervous system,  isozymes are the most common CaMK-II gene product in embryonic cells (6, 8, 10, 12). Mouse embryonic cells express some _e, _C, and _B gene products but primarily express _C CaMK-II (12, 21, 23). During early mouse development,  and  CaMK-II gene products are the primary gene products expressed throughout the embryo including the developing nervous system (6).  CaMK-II isozymes have been implicated in rodent neuronal and muscle differentiation (12, 21, 24-27). Human _E CaMK-II was originally cloned from neuroblastoma cells (8). _E is also known as ₁₀ or ₉ depending on the presence or absence of the C-terminal tail (11). _C CaMK-II was originally cloned from human T lymphocytes where it is particularly enriched (2), but its function is not known.

Both _C and _E CaMK-II encode 57-kDa proteins with one alternative exon in their central variable domain. Despite this structural similarity and the extranuclear location of both isozymes, only _E induces neurite outgrowth (21). Their catalytic properties are similar, suggesting that subtle targeting differences are responsible for their disparate functional roles. It is not known whether sequence differences in their one alternative exon are responsible for their targeting differences.

The intent of this work has been to define the targeting domain for both _C and _E CaMK-II. Green fluorescent protein (GFP)-linked deletion constructs of _C and _E CaMK-II were prepared, and their oligomeric nature and localization were evaluated. Our findings define a targeting domain that is located in between the central variable domain and the C-terminal association (oligomerization) domain. The localization of _E CaMK-II suggests that it promotes neurite outgrowth through the stabilization of the actin cytoskeleton.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cell lines and Culture-- NIH/3T3 mouse embryo fibroblasts were cultured on polystyrene dishes in Dulbecco's modified Eagle's medium (BioWhitaker, Walkersville, MD) with 10% fetal bovine serum (Life Technologies, Inc.), supplemented with penicillin and/or streptomycin in a 5% CO₂ humidified chamber at 37 °C.

cDNA Preparation-- The first 301 and the last 22 amino acids encoded by full-length wild type or constitutively active CaMK-II cDNAs used in this study were identical as described previously (21). Constitutively active mutants were prepared by point mutagenesis of Thr²⁸⁷ to Asp²⁸⁷, which renders CaMK-II active in the absence of Ca²⁺/CaM. Using polymerase chain reaction-mediated directional cloning, CaMK-II cDNAs were linked to sequences encoding enhanced green fluorescent protein (EGFP) by using the vector pEGFP-C1 (CLONTECH, Palo Alto, CA). EGFP is placed at the NH₂ terminus of CaMK-II with only one additional codon (encoding a glycine residue) separating the two sequences. Primers were synthesized containing restriction enzyme sites (BspE1 at the 5' end and BamHI at the 3' end) to amplify the desired cDNA fragment. They enabled the in-frame introduction of CaMK-II domains on the C-terminal side of EGFP. Proper clone construction was confirmed by DNA sequencing in both directions as described previously (21) by evaluation of enzymatic activity (for full-length CaMK-IIs) and by anti-GFP immunoblotting. Sequence analysis was performed using Gene Jockey II (Biosoft, Inc, Cambridge, United Kingdom).

Transfection of DNA into Mammalian Cells-- cDNAs encoding full-length CaMK-II isozymes were transfected into NIH/3T3 cells using LipofectAMINE PLUS (Life Technologies, Inc.) for 3 h followed by culture for at least an additional 18 h. Transfection efficiencies routinely exceeded 50%.

Whole Cell Lysate Preparation-- Transfected cells were grown for 1-2 days, harvested with trypsin-EDTA, and then washed with phosphate buffered saline (PBS) containing 2.5 mM EGTA. Pellets were resuspended in 3 volumes of ice-cold homogenization buffer, which consisted of 30 mM Hepes, pH 7.4, 2.6 mM EGTA, 20 mM MgCl₂, 80 mM -glycerol phosphate, 0.1 µM okadaic acid (Life Technologies, Inc.), 0.01 mg/ml each chymostatin, leupeptin, aprotinin, pepstatin, and soybean trypsin inhibitor (Sigma). Samples were then sonicated (three 5-s bursts on ice), centrifuged at 10,000 × g for 15 min at 4 °C and either assayed immediately or frozen and stored at 80 °C. Lysates prepared by sonication solubilized over 90% of the total CaMK-II activity as measured by solution assays and immunoblots (data not shown). Cytosolic fractions were diluted to 0.1-0.2 mg/ml protein in homogenization buffer, and 10 µl was assayed for CaMK-II activity as described previously (21).

Immunoblots-- Whole cell lysates were separated on 10% polyacrylamide gels using the Mini-Protean II gel electrophoresis system (Bio-Rad). Proteins were transferred to 0.45 µm of nitrocellulose sheets for 1 h at 100V and blocked with TBSTA containing 2.5% nonfat dry milk, 2.5% bovine serum albumin, and 2% normal goat serum for 1 h. The anti-GFP antibody was a mouse monoclonal IgG (CLONTECH). Primary antibodies were typically diluted to 0.5 µg/ml in 2% bovine serum albumin/TBSTA and incubated between 1 and 12 h with the nitrocellulose blot. Blots were washed three times with TBSTA and incubated for 1 h with 0.5 µg/ml alkaline phosphatase-coupled goat anti-mouse IgG (Kierkegaard Perry Labs, Gaithersburg, MD) in 2% bovine serum albumin/TBSTA. Blots were developed with 0.25 mg/ml 5-bromo-4-chloro-3-indolyl phosphate and 0.25 mg/ml nitro blue tetrazolium (Sigma) in 0.1 M Tris, 0.1 M NaCl, 5 mM MgCl₂, pH 9.4. Monomeric molecular weights of bands were interpolated from a linear plot of log M_r of standards versus R_F.

Native Molecular Weight Determinations-- Whole cell lysates were filtered through 0.45-µm syringe tip filters and then loaded onto a 40 × 1.0-cm Superose-12 gel filtration column (Amersham Pharmacia Biotech, Inc., Piscataway, NJ) in 50 mM Tris, pH 7.4, 150 mM NaCl, 0.1 mM dithiothreitol, 5% glycerol, and 0.001 mg/ml each chymostatin, leupeptin, aprotinin, pepstatin, and soybean trypsin inhibitor. Superose-12 was the matrix chosen, because it spanned predicted monomeric and oligomeric CaMK-II sizes. The reported exclusion limit for Superose-12 is 2 × 10⁶ (Amersham Pharmacia Biotech). Retention times of standards, which included thyroglobulin (M_r = 669,000), -amylase (M_r = 200,000), bovine serum albumin (M_r = 66,000), and carbonic anhydrase (M_r = 29,000), were determined from in-line absorbance at 280 nm. Sample fractions were assessed for GFP fluorescence using the Fluorstar fluorescence microtiter plate reader (B&L Systems, Maarsen, Holland) or with anti-GFP immunoblotting. Elution volumes (V_e) were plotted as a function of the void volume (V₀) and the total volume (V_T). V₀ was determined using blue dextran, and V_T was determined using glycine. Native molecular weights of samples were interpolated from a linear plot of log M_r of standards versus K_av (V_e  V₀/V_T  V₀) as described previously (28).

Immunohistochemistry and Microscopy-- Transfected fibroblasts were grown on polystyrene dishes or glass coverslips for 1-2 days after transfection. Cells were imaged either directly on a heated stage or after fixation. Fixation was routinely performed as follows. Coverslips were first rinsed in PBS, incubated with fresh 4% formaldehyde in PBS for 15 min, permeabilized with 0.05% Nonidet P-40 (Pierce) in PBS, and then post-fixed with 4% formaldehyde in PBS for 5 min. Cells were stained with 100 nM rhodamine-phalloidin (Cytoskeleton, Inc., Denver, CO) for actin, the AA2-purified mouse monoclonal antibody for total tubulin (courtesy of Dr. Anthony Frankfurter, University of Virginia) at 1 µg/ml, or the V9 mouse monoclonal antibody (Sigma) for vimentin. For the latter two, 1 µg/ml rhodamine-coupled goat anti-mouse IgG (Kierkegaard Perry Labs) in 2% bovine serum albumin/TBSTA was used as the secondary antibody. Cells were imaged using the Olympus Fastscan 2000 12-bit digital camera mounted on an Olympus IX70 fluorescent microscope (Olympus America, Melville, NY). Images were compiled using Photoshop 5.5 (Adobe Systems, Inc, San Jose, CA).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Expression and Characterization of CaMK-II Isozymes-- To characterize the minimal targeting domain of _C and _E CaMK-II, deletion constructs (Fig. 1, A-F) were linked at their NH₂ termini to EGFP. This and other labs have shown that the catalytic properties of GFP-linked full-length constructs (A) are not affected by an NH₂-terminal GFP domain (18, 21). C-terminal constructs (B) encode the variable and association domains over the last 185 (_C) or 182 (_E) residues. Variable domain constructs (C and D) begin with Ser³¹¹ (_C) or Thr³¹¹ (_E) and end 108-111 (C) or 51-54 (D) residues downstream. Association domain constructs (E and F) have a normal C terminus but begin with Thr³⁵⁴ (_C) or Thr³⁵¹ (_E) for construct E or with Ala³⁹⁸ for construct F as shown in Fig. 1. The sequence comprising these last 195 (_C) or 192 (_E) residues is shown with differences highlighted (Fig. 1).

View larger version (66K): [in this window] [in a new window]   Fig. 1.   Constructs used in this study. CaMK-II constructs used in this study have GFP (27 kDa) linked at the NH2 terminus to the constructs of C or E CaMK-II at the indicated residues. Approximate boundaries of the catalytic (residues 1-311), central variable (residues 312-361), and association (residues 362-492 or 365-495) domains are indicated by shading. C and E sequence alignment starts at residue 301 and is numbered according to C. Dots indicate identity, and bold residues in E CaMK-II represent differences. The C-terminal 22 residues of E CaMK-II was made identical to C CaMK-II to avoid complications of alternative C-terminal tails.

These 11-GFP-CaMK-II constructs were transfected into NIH/3T3 cells. After 2 days, cells were harvested and evaluated by anti-GFP immunoblots (Fig. 2). As expected, full-length _C and _E migrated at 84 kDa, which was 27 kDa larger than full-length CaMK-II alone (A). All other constructs were proportionally smaller as predicted. Lysates containing transfected full-length CaMK-II exhibited Ca²⁺/CaM-dependent catalytic activity, which exceeded total endogenous CaMK-II activity levels by at least 4-fold (21). Predicted and observed monomeric molecular weights (M_r), as determined by SDS-polyacrylamide gel electrophoresis, are summarized in Table I.

View larger version (11K): [in this window] [in a new window]   Fig. 2.   Anti-GFP immunoblot of constructs. 2 µg of protein lysate from NIH/3T3 cells transfected with the indicated constructs in Fig. 1 were separated by SDS-polyacrylamide gel electrophoresis and reacted with a monoclonal antibody directed against GFP.

Oligomerization Domain of  and  CaMK-II-- CaMK-II targeting may depend upon oligomerization. Therefore, we evaluated the native molecular weights of all expressed constructs. Previous studies have indicated that the minimal oligomerization domain of  CaMK-II begins approximately at Ala³⁸⁴ (17), which corresponds to Ala³⁹⁸ in _E CaMK-II. Other studies indicate that the  CaMK-II association domain comprises the last 135 amino acids but is most dependent upon the last 110 residues (18). The boundaries of the association domain had not yet been determined for  or  CaMK-IIs, but we expected constructs A, B, E, and possibly F to oligomerize based on homology to  CaMK-II.

To assess oligomerization, lysates of cells transiently transfected with _C and _E CaMK-II constructs were applied to a Superose-12 gel filtration column (see under "Experimental Procedures"). Anti-GFP immunoblots demonstrated that protein constructs remained intact through gel filtration and peaked at different elution volumes (Fig. 3, _E only). Quantitative profiles for all constructs were obtained by analyzing the GFP fluorescence of each fraction (Fig. 3, bottom panels). Elution profiles were plotted as a function of K_av, and native sizes were interpolated (Table I). For both _C and _E, constructs A, B, E, and F exhibited major peaks corresponding to oligomers of at least 9 subunits. Construct A had a substantial secondary peak corresponding to monomer and additional shoulders on both of these peaks. Constructs B, E, and F all had less relative contribution from their "monomeric" peaks and were, therefore, considered to have oligomerized more efficiently. Constructs C and D did not oligomerize whatsoever as they showed single peaks corresponding to their monomeric size. These results indicate that the C-terminal 142 residues of  and  CaMK-II can efficiently oligomerize, and that the C-terminal 95 amino acids of  CaMK-II are sufficient for oligomerization.

View larger version (80K): [in this window] [in a new window]   Fig. 3.   Native Mr of GFP-CaMK-II constructs. Superose-12 gel filtration fractions of representative E CaMK-II constructs A, B, C, E, and F were assessed by anti-GFP immunoblot analysis (top panel) and are shown as a function of Kav (Ve  V0/VT  V0). The immunoreactivity of the column starting material (SM) for each construct is shown in the last lane. Elution profiles of all constructs were also assessed by quantitative GFP fluorescence analysis (bottom plots). Native molecular masses were interpolated to the nearest 5 kDa (Table I). The elution position of standard protein peaks are shown with their molecular weights.

Localization of Full-length  and  CaMK-II Isozymes-- The localization patterns of full-length GFP-linked _C and _E CaMK-IIs were determined using conventional fluorescence microscopy of living cells 2 days after transfection. The populations of cells are shown at low magnification, and individual cells are shown at higher magnification using both phase-contrast and fluorescence illumination (Fig. 4). Note that these images demonstrate the high transfection efficiencies typically obtained in these experiments. Both GFP-linked CaMK-IIs demonstrated localization patterns that were similar to both the indirect immunofluorescence pattern of transfected non-GFP-linked CaMK-IIs (21) and to endogenous CaMK-II determined by using gene-specific antibodies (12). _C CaMK-II exhibited a dispersed and uniform distribution throughout the cytoplasm. In contrast, _E CaMK-II exhibited a distinctive perinuclear and cortical cytoplasmic staining as previously reported for endogenous  CaMK-II in rodent fibroblasts, astrocytes, and myoblasts (10, 12, 27).

View larger version (83K): [in this window] [in a new window]   Fig. 4.   Localization of full-length CaMK-II in living cells. Live NIH/3T3 cells were imaged under phase-contrast and fluorescent microscopy 2 days after transfection with full-length C and E CaMK-II (construct A). Exposure times were similar. Scale bar corresponds to 100 µm (Low Magnification) and 25 µm (High Magnification).

Minimal  and  CaMK-II Targeting Domain-- C-terminal construct B of _C (Fig. 5, top row) and _E (Fig. 5, bottom row) CaMK-II localized similar to full-length construct A. _C was uniformly dispersed throughout the cytoplasm, whereas _E showed cortical and perinuclear localization. High intensity fluorescent particles were occasionally seen with some constructs (see Fig. 5, _C constructs B and E and _E constructs E and F).

View larger version (57K): [in this window] [in a new window]   Fig. 5.   Localization of CaMK-II deletion constructs in living cells. Live NIH/3T3 cells were viewed under fluorescent microscopy 2 days after transfection with the indicated constructs B-F. Exposure times were similar for all constructs. Scale bar corresponds to 25 µm.

Constructs C and D span the entire variable domain. Construct C ends within the first third of the association domain, whereas construct D encodes a smaller segment that ends in a region that links the variable and association domains (see Fig. 1). Neither construct exhibited any targeting, as they were found throughout the cytoplasm and nucleus in an identical pattern to GFP alone (Fig. 1, GFP panel, top row). GFP is slightly more enriched in the nucleus but is not exclusively found in the nucleus.

Construct E has a normal C terminus but begins at either Thr³⁵⁴ () or Thr³⁵¹ () and thus lacks any variable domain sequences. Construct E is oligomeric (see Table I). Although it was lacking variable domain sequences, construct E localized much like constructs A or B, i.e. it exhibited the striking perinuclear distribution for  and the dispersed cytoplasmic localization for  CaMK-II.

Construct F was prepared for  CaMK-II only. This 95 amino acid domain begins at Ala³⁹⁹ and continues to the C terminus. This construct is synthesized at its predicted size and is efficiently oligomeric at 11 times its monomeric size (Fig. 3 and Table I).  Construct F, however, was not targeted like full-length _E constructs A, B, or E. Like GFP alone,  construct F was found throughout the cell in both the nucleus and the cytoplasm, but unlike GFP, it was oligomeric and often seen as small cytoplasmic fluorescent particles (Fig. 5).

These findings indicate that alternative exons in the central variable domain are not necessary for cytoplasmic targeting of  and  CaMK-IIs. Rather, the sequence that comprises the difference between constructs E and F is necessary for targeting. Further support for this conclusion comes from our observations that GFP-linked _C CaMK-II oligomerizes and localizes in an identical perinuclear fashion to _E. The _C gene product is a  CaMK-II isozyme, which naturally lacks alternative variable domain sequences (21).

Co-localization and Influence of  CaMK-IIs on the Actin Cytoskeleton-- Living cells transfected with full-length _E construct A or C-terminal constructs B and E often exhibited filaments in the perinuclear region. These filaments were more easily seen when cells were permeabilized and fixed (Fig. 6). _C CaMK-II constructs did not exhibit these filaments. The _E filamentous pattern was observed even when cells were detergent washed or extracted, which supports a co-localization with the cytoskeleton and not endomembranes. Therefore, fixed cells were counterstained with antibodies against tubulin or vimentin and were counterstained with phalloidin for actin. _E CaMK-II co-localized with F-actin around the nucleus and at the cell cortex. The arrows point out some of these co-localizing CaMK-II and actin fibers (Fig. 6). Vimentin and tubulin fibers were enriched in the perinuclear region but not in the same bundles or fibers as CaMK-II and actin. CaMK-II did not co-localize with actin stress fibers, the predominant actin structure in these cells. A particularly striking-merged color image of a _E CaMK-II pattern (green) with F-actin (red) reveals co-localizing yellow filaments passing around and above the nucleus and into a short actin-rich extension (Fig. 7). Actin stress fibers can be seen as fibers that run along the base of the cell and do not co-localize with CaMK-II.

View larger version (117K): [in this window] [in a new window]   Fig. 6.   Actin counterstaining of CaMK-II constructs in fixed cells. NIH/3T3 cells transfected with full-length or C-terminal constructs were fixed with formaldehyde after 2 days and counterstained with rhodamine-phalloidin. Arrows indicate co-localizing CaMK-II and actin fibers. Scale bar corresponds to 25 µm.

View larger version (30K): [in this window] [in a new window]   Fig. 7.   Wild-type E CaMK-II and actin co-localization. NIH/3T3 cells transfected with full-length E CaMK-II were fixed with formaldehyde after 2 days and counterstained with rhodamine-phalloidin. Images were combined into the color view with CaMK-II in green, actin in red, and coincident fluorescence in yellow. Scale bar corresponds to 25 µm.

From this actin co-localization and our previous finding that constitutively active GFP-linked _E CaMK-II can induce neurite outgrowth (21), we predicted that transfected constitutively active _E CaMK-II might induce actin polymerization and demonstrate a more pronounced co-localization with actin, and this is what we observed. _E CaMK-II co-localized with enhanced levels of F-actin along the entire length of the cellular extensions that it induced, particularly at the tip (Fig. 8, middle panel). Cells that were not transfected or were transfected with constitutively active _C CaMK-II exhibited normal actin patterns and showed no extensions (Fig. 8, top panel). Both of these constitutively active constructs were previously shown to exhibit high levels of autonomous (Ca²⁺/CaM-independent) activity (21). At a higher magnification, constitutively active _E CaMK-II could be seen at the tips of these extensions in a pattern that co-localized with bundled and cortical F-actin (Fig. 8, bottom panel). We interpret these results as indicating that cellular extensions are formed in the presence of constitutively active _E CaMK-II through either the induction or stabilization of actin fibers.

View larger version (86K): [in this window] [in a new window]   Fig. 8.   Constitutively active CaMK-II and actin co-localization. NIH/3T3 cells transfected with full-length constitutively active C and E CaMK-II were fixed with formaldehyde after 1 day and counterstained with rhodamine-phalloidin. Scale bar corresponds to 10 µm.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cytosolic CaMK-II activity is involved in the differentiation of adipocytes, myocytes, and pre-neuronal cells (24, 29-34). Of the four CaMK-II genes,  CaMK-II gene products are the most highly expressed in these cells and have been shown to directly induce neuritogenesis (12, 21, 25). Most CaMK-II gene products are strikingly similar in structural and kinetic features, leading to the conclusion that  CaMK-IIs, like other protein kinases, must be selectively targeted to substrates to influence events such as neurite outgrowth. In this study, we have demonstrated that _E CaMK-II, an isozyme independently discovered in heart and pre-neuronal cells (8, 11, 21), co-localizes with F-actin in the perinuclear region and at the cell periphery. This is the identical localization for endogenous  CaMK-II in mouse embryonic fibroblasts (12), rat myoblasts (27), and rat astrocytes (10). When _E CaMK-II is made constitutively active and is transfected, it induces cellular extensions (21), localizes along and at the tips of these cellular extensions, and increases filamentous F-actin. _C CaMK-II, a catalytically and structurally similar isozyme originally found at high levels in T lymphocytes (35), is found in the cytosol but does not induce neurite outgrowth and does not co-localize with F-actin.

We have also shown that _C and _E CaMK-II targeting is not dependent on the central variable domain. Constructs C and D target no differently than GFP alone, whereas C-terminal construct E, which lacks central variable domain sequences, targets like full-length CaMK-II. This finding was somewhat surprising, because there are  and  CaMK-II isozymes that have central variable domain (nuclear)-targeting sequences (10, 13, 14). Because  targeting does not require alternative exons in the variable domain, the _E CaMK-II targeting shown here represents the "default" targeting pattern for  gene products such as _C CaMK-II, which is the principal  CaMK-II isozyme expressed in embryonic cells (6, 8, 10, 12, 21). This conclusion is consistent with our finding that _C and _E CaMK-II show identical localization and are equally capable of inducing neurite outgrowth (21).

The variable domains of both _C and  CaMK-II are structurally similar (8), i.e. they lack alternative exons and are therefore the simplest products of their respective genes. Whereas _C CaMK-II has targeting sequences in the C-terminal domain,  CaMK-II can be targeted to the post-synaptic density by heterooligomerization with  CaMK-II (19) or to the sarcoplasmic reticulum by heterooligomerization with KAP (9). Although we have now shown that central variable domain sequences are not necessarily required for CaMK-II targeting, it is undoubtedly clear that proper targeting is necessary for CaMK-II function.

_C CaMK-II and _E CaMK-II targeting requires only the last 150 residues, which does not include the central variable domain. Our findings indicate that the first 50 residues of this C-terminal domain are necessary for targeting, and the last 95 residues are minimally necessary for oligomerization. Therefore, targeting is dependent upon oligomerization, but oligomerization alone does not result in targeting. The 50 amino acid domain corresponds to _E Thr³⁵¹-Ala³⁹⁹ (Fig. 1), which is analogous to  Thr³³⁷-Ala³⁸⁴. In a model of full-length oligomeric  CaMK-II, this domain is part of a linker between the NH₂-terminal peripheral catalytic domain and the C-terminal association domain, which form a central oligomeric core (36). This creates the structural potential for the interaction of this domain with other proteins. Oligomerization has also been reported as necessary for CaMK-II targeting to the NR2B subunit of the N-methyl, D-aspartate receptor, although the targeting sequences are found in the catalytic domain (37). Regardless of where the targeting domain resides, its juxtaposition as an oligomer may present a unique three-dimensional targeting site that is not present in monomeric CaMK-II.

Both _C and _E CaMK-II are composed of three separate exons in their variable region (8). This has been confirmed through the examination of the human  CaMK-II gene on chromosome four (4q25) and the human  CaMK-II gene on chromosome 10 (10q22) via sequence analysis through the National Center for Biotechnology Information. The second of these three exons (_E^329-342, EPQTTVIHNPDGNK) contains some unique and repeated sequence elements that are absent from _C (21). This exon, however, is not responsible for  CaMK-II targeting to the perinuclear region as described here. Although the function of this domain is not yet known in  CaMK-II, the analogous  domain (EPQTTVVHNATDGIK) is used in _A CaMK-II to regulate the targeting of a preceding nuclear targeting domain (10). However, no known  CaMK-II expresses this domain in combination with the nuclear targeting domain (8).

CaMK-II has been reported to form 100-nm "clusters" in cultured hippocampal neurons (38). It is not clear what constitutes the molecular basis of these clusters, but we do not believe that they are related to the much larger fluorescent particles as described here. The particles observed here were seen only with oligomeric constructs A, B, E, and F and, therefore, were not an artifact of EGFP by itself. They were most prominent with  construct F, which interestingly was the only oligomeric construct that lacked any subcellular targeting capability. We also observed these particles more often with _C than with _E oligomeric constructs. We suspect that when overexpressed oligomeric CaMK-IIs exceed the level of endogenous binding partners, they are more prone to cellular disposal pathways, appearing as accumulations of fluorescence.

In this study, we have shown that differentially effective  and  CaMK-II isozymes are differentially targeted. _E CaMK-II co-localizes with F-actin in the perinuclear region and the cellular cortex in a manner that is consistent with its role in neuritogenesis. Our evidence indicates that the targeting of _E CaMK-II is dependent on sequences residing between Thr³⁵¹ and Ala³⁹⁸. Targeting is also dependent upon oligomerization but not on the central variable domain.  CaMK-II oligomerization requires no more than the last 95 residues. Further identification of binding targets and substrates of  CaMK-II isozymes will help identify their precise locus of action and may account for known effects of Ca²⁺-dependent protein phosphorylation on neurite stabilization, outgrowth, and turning (39-41).

	ACKNOWLEDGEMENTS

This work is dedicated to Dr. G. Watson James, III. We are extremely grateful to Amanda Itnyre, H. Helen Han, and Jessica Myers for technical assistance and to Drs. Helen Fillmore, Ann Kwiatkowski, Richard Moran, Donald Porter, and Shirley Taylor for technical and editorial advice.

	FOOTNOTES

^* This work was supported by grants from the Kate and Thomas Jeffress Foundation Trust and the R. Clifton Brooks fund for Biomedical Research and by National Science Foundation Grant 9904765.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.

To whom correspondence should be addressed: Depts. of Biology and Biochemistry and Molecular Biophysics, Virginia Commonwealth University, P. O. Box 842012, Richmond, VA 23284-2012. Tel.: 804-827-0141; Fax: 804-828-0503; E-mail: rtombes@hsc.vcu.edu.

Published, JBC Papers in Press, September 4, 2001, DOI 10.1074/jbc.M103013200

	ABBREVIATIONS

The abbreviations used are: CaMK-II, Ca²⁺/CaM-dependent protein kinase type II; GFP, green fluorescent protein; EGFP, enhanced green fluorescent protein; PBS, phosphate-buffered saline; TBSTA, Tris-buffered saline, pH 7.4, 0.05% Tween 20, 0.05% sodium azide.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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