Cleavage of doublecortin-like kinase by calpain releases an active kinase fragment from a microtubule anchorage domain.

Doublecortin-like kinase (DCLK) is widely expressed in postmitotic neurons throughout the embryonic nervous system. DCLK consists of an N-terminal doublecortin domain, responsible for its localization to microtubules, and a C-terminal serine-threonine kinase domain. Here we report that DCLK is a physiological substrate for the cysteine protease calpain. Cleavage of DCLK by calpain severs the kinase domain from its microtubule anchorage domain and releases it into the cytoplasm. The isolated kinase domain retains catalytic activity and is structurally similar to CPG16, a second product of the DCLK gene expressed in the adult brain that lacks the doublecortin domain. We propose that in neurons cleavage of DCLK by calpain represents a calcium responsive mechanism to regulate localization of the DCLK kinase domain.

Doublecortin-like kinase (DCLK) is widely expressed in postmitotic neurons throughout the embryonic nervous system. DCLK consists of an N-terminal doublecortin domain, responsible for its localization to microtubules, and a C-terminal serine-threonine kinase domain. Here we report that DCLK is a physiological substrate for the cysteine protease calpain. Cleavage of DCLK by calpain severs the kinase domain from its microtubule anchorage domain and releases it into the cytoplasm. The isolated kinase domain retains catalytic activity and is structurally similar to CPG16, a second product of the DCLK gene expressed in the adult brain that lacks the doublecortin domain. We propose that in neurons cleavage of DCLK by calpain represents a calciumresponsive mechanism to regulate localization of the DCLK kinase domain.
During development neurons in the mammalian nervous system undergo large scale changes in morphology. Such changes occur during periods of cell division, migration, neurite outgrowth and pathfinding, synaptogenesis, and synaptic pruning. Cytoskeletal structures underlying cellular morphology are therefore expected to be maximally plastic during embryogenesis (1), and indeed many cytoskeletal proteins or protein splice forms are selectively transcribed in the brain during development (2,3), including drebrin (4), ␣-internexin (5), doublecortin (6,7), filamin (8), nestin (9), MAP2, and tau (10). It is therefore important to study the mechanisms that modulate cytoskeletal structure underpinning neuronal morphology.
We have previously demonstrated that the embryonally expressed protein, doublecortin-like kinase (DCLK) 1 is localized to neuronal growth cones where it is associated with the microtubule cytoskeleton (11). DCLK has a C-terminal serine/ threonine kinase domain and a N-terminal domain similar to the doublecortex syndrome disease gene, doublecortin (6,7). The N-terminal doublecortin-like domain is responsible for microtubule binding (11,12). The C-terminal kinase domain is also encoded by a distinct transcript, first cloned in a screen for activity-regulated transcripts in adult brain known as CPG16 (13,14). Thus, a microtubule-associated form of the kinase, DCLK, is mostly strongly expressed during embryogenesis while a cytoplasmic form of the kinase, CPG16, is expressed in the adult brain.
An important question is how the microtubule binding and kinase activities of DCLK are regulated. The catalytic core of DCLK shows strong amino acid similarity to members of the calcium-calmodulin family of protein serine-threonine kinases, though the calmodulin binding domain is not conserved in DCLK (14) and biochemical studies have indicated that calmodulin does not regulate the function of the DCLK kinase domain (13). Phosphorylated forms of both doublecortin and DCLK are associated with microtubules and may therefore contribute to regulation of their microtubule binding (15,16); however, DCLK autophosphorylation is not required for microtubule binding (11,17).
We and others have reported previously that overexpression of CPG16 or DCLK in cultured cells results in the appearance of truncated forms of the protein (13,14,17). In fact, many serine-threonine kinases have been shown to be susceptible to proteolytic processing by the ubiquitous calcium-activated cysteine protease calpain (18). Intriguingly, cytoskeletal proteins are also a prominent target for calpain (19), perhaps reflecting the partial association of -calpain to microtubules (20,21).
In this report we demonstrate that DCLK is a substrate for calpain, with proteolysis yielding an active kinase domain no longer anchored to the microtubule cytoskeleton binding domain. In neurons this appears to result in easily detectable differences in DCLK N-and C-terminal immunoreactivity within the soma, with the C-terminal kinase domain entering the nucleus following proteolysis. Since calpain is activated by calcium, we propose that this may be a calcium-responsive means for regulating the localization of DCLK kinase activity during embryonic development.

EXPERIMENTAL PROCEDURES
Cell Culture and Transfection-293-T cells were cultured at 37°C with 5% CO 2 and 95% air in Dulbecco's modified Eagle's medium with 10% fetal calf serum, 5 mM glutamine, 100 units/ml penicillin, and 0.1 mg/ml streptomycin. Transfections of plasmid DNA were carried out routinely using calcium phosphate precipitation (21), using 5 g of plasmid/6-cm plate or 15 g of plasmid/9-cm plate. Construction of FLAG-DCLK expression vector has been described previously (14). Calpeptin (Calbiochem, La Jolla, CA) was applied 2 days following transfection, at the indicated concentrations for 30 min before protein extraction. The calpastatin cDNA was kindly supplied by Dr. Piechaczyk (22) and subcloned into a pCDNA by Chava Gil-Henn 2 and cotransfected at the indicated amount with 2 g of FLAG-DCLK/ 6-cm plate. Primary neurons were prepared from corticostriatal tissue dissected from timed embryonic day 14.5 mouse embryos. Neurons were suspended in PBS ϩ 0.6% glucose (PBS-glu) and were gently titurated with a pipette, incubated with 0.003% DNase at 37°C for 10 min, then * This work was supported in part by Human Frontier Science Program Grant RG283199, by the Minerva Foundation, Germany, by Volkswagon-Stiftung, and by U.S.-Israel Binational Science Foundation Grant 97-00014. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
spun down and resuspended in PBS-glu three times and 1.5 ϫ 10 6 neurons plated on polyornithine-coated 3-cm plates or glass coverslips for culture (as described in Ref. 23).
In Vitro Proteolysis and Kinase Assay of DCLK-For preparation of recombinant GST-DCLK, DCLK open reading frame was subcloned in frame into pGEX-2T using BamHI and EcoRI sites from FLAG-DCLK. Plasmid was transfected into BL21(DE3)RIL bacteria (Stratagene, La Jolla, CA), which were grown in M9ZB ϩ 2% glucose at 37°C to an optical density of 0.6. Induction of protein expression was carried out using 0.2 mM IPTG at 18°C for 16 -24 h. Protein was extracted in NETN buffer (0.5% Nonidet P-40, 0.1 M NaCl, 1 mM EDTA, 20 mM Tris-HCl, pH 8.0) with sonication. Soluble fraction was bound to glutathione-agarose beads (Sigma) for 1 h and then washed extensively in NETN before elution from column in 10 mM Tris-HCl, pH 8.0, 0.15 M NaCl, 10 mM glutathione, 10% glycerol. For in vitro proteolysis, m-calpain (Sigma) was incubated with GST-DCLK at 30°C in the presence of 5 mM CaCl 2 for times indicated in the figure legends. Reactions were stopped by adding sample buffer supplemented with 10 mM EDTA, subjected to SDS-PAGE, and stained with Coomassie Blue or subjected to Western blot as described in the figure legends. For in vitro kinase assay following calpain cleavage, kinase reaction buffer containing 20 mM magnesium acetate, 20 M ATP, 100 mM NaCl, 100 mM Tris-HCl, pH 6.8, 1 mM ZnCl 2 , 2.5 Ci [␥-32 P]ATP, and 1 g of myelin basic protein (Sigma) was added after 1 h of in vitro proteolysis. As the kinase reaction contains zinc, a potent calpain inhibitor (24), there was no need to separately inactivate calpain. Kinase reaction was allowed to proceed for 30 min before termination by addition of sample buffer with 10 mM EDTA. Following separation by SDS-PAGE, gels were stained with Coomassie Blue and subjected to autoradiography. For assessment of the effect of calpain on DCLK activity, the kinase assay was allowed to proceed for multiple time points. Quantitation was accomplished by exposure of the dried gel to phosphoimager. To assess whether cleavage of DCLK dissociates the kinase domain from the microtubule domain, 5 g of GST-DCLK was bound to 10 l of glutathione-agarose beads and incubated in reaction buffer containing 50 mM Tris-HCl, pH 7.5, 1 mM CaCl 2 , 2 milliunits of m-calpain. After 60 min, the reaction buffer was removed, the beads washed twice with 50 mM Tris-HCl, pH 7.5, and resuspended in sample buffer. Both fractions were then subjected to SDS-PAGE and immunoblotting with anti-DCLK antibody.
Fractionation of Microtubule and Cytosolic Subcellular Fractions-Two days after transfection with FLAG-DCLK, 293-T cells were gently washed once with PBS and then overlaid with microtubule-stabilizing extraction buffer (50 mM MES, pH 6.8, 2.5 mM EGTA, 2.5 mM MgCl 2 supplemented with protease inhibitors as above) for 3 min at room temperature. The extraction buffer was removed and centrifuged at 500 ϫ g to discard detached cells. Cells remaining on the plate were then extracted in RIPA buffer as above. Both fractions were subjected to Western blot with anti-FLAG and anti-DCLK antibodies.

RESULTS
Truncated Forms of DCLK Are Observed in Primary Cultured Neurons and 293-T Cells-Immunoblot of extract from primary cultured neurons with the anti-DCLK C-terminal antibody gives an 85-kDa band corresponding to the predicted size of the DCLK protein and identical to that found in embryonic brain tissue (14). In addition, a less intense but reproducible band of 50 kDa is observed in these blots (Fig. 1, lane 1). We noted previously that, when DCLK was overexpressed in a variety of cell types, a 50-kDa specific immunoreactive band is recognized by the anti-DCLK C-terminal antibody in addition to the expected 85-kDa protein. We expressed an N-terminal FLAG epitope-tagged DCLK construct in 293-T cells to enable us to track both fragments produced by cleavage of DCLK. Western blot of FLAG-DCLK transfected cells with either anti-FLAG or anti-DCLK antibodies both revealed an 85-kDa band corresponding to the size of the FLAG-DCLK fusion protein ( Fig. 1, lanes 3-6). However, when cells were cultured at high confluence, each antibody recognized an extra band: for the anti-DCLK antibody, a 50-kDa band (Fig. 1, lane 3), and for the anti-FLAG antibody, a 35-kDa band (Fig. 1, lane 5). The appearance of a 50-kDa band in transfected 293-T cells that is of similar size to that found in cultured neurons suggests that the neuronal 50-kDa band is derived from DCLK and is not merely a cross-reactive protein. Furthermore, Mizuguchi et al. (25) noted a 35-kDa band cross reactive to their anti-DCLK Nterminal antibody in human fetal brain extracts. As the Cterminal 50-kDa band and the N-terminal 35-kDa band appear under the same conditions in 293-T cells and have a combined molecular mass the size of the full-length DCLK protein, we wondered whether these peptides may be derived by proteolytic cleavage of the FLAG-DCLK protein at the boundary of the doublecortin domain and the ST-rich domain (the cleavage A site in Fig. 2).
Calpain Inhibitors Abolish Cleavage of DCLK in 293-T Cells-The cysteine protease calpain has been implicated in proteolytic processing of many cytoskeletal proteins and serinethreonine kinases (18). As DCLK falls into both classes, we tested whether the degradation products observed in FLAG-DCLK-transfected cells were attributable to calpain. Following transfection of 293-T cells with FLAG-DCLK, we applied the cell-permeable calpain inhibitor calpeptin. Calpeptin inhibited the generation of both of the 50-kDa C-terminal and 35-kDa N-terminal bands and acted in a dose-dependant fashion (Fig.  3, A and B). In several transfection experiments, including this one, both antibodies recognized a third band. In these cases the C-terminal antibody revealed a 42-kDa band and the anti-FLAG anti-body a 43-kDa band. Although the C-terminal antibody reveals an ϳ40-kDa band in primary neuron extract (Fig. 1, lane 1), this band is distinctly smaller than that observed following transfection of 293-T cells. In addition, calpain cleavage of DCLK in embryonic brain lysate did not augment the 40-kDa band (Fig. 5). For these reasons we do not believe that the 40-kDa band in primary neuronal extract represents a calpain cleavage product of DCLK and may simply be a cross-reactive band. As the 42-kDa C-terminal and 43-kDa N-terminal bands appeared in the same transfection experiments and have a combined molecular mass the size of DCLK, we consider it likely that they are generated by proteolytic cleavage of DCLK at a second site (the cleavage B site in Fig. 2). Under these conditions the 42-kDa C-terminal and 43-kDa N-terminal bands are not lost, nor were they diminished by prolonged incubation with calpeptin, suggesting that they are very stable or are not products of proteolysis of DCLK by calpain.
Calpeptin is not absolutely specific for calpain. We therefore sought to test the effect of the potent and specific physiological calpain inhibitor calpastatin (26). Calpastatin was co-expressed with FLAG-DCLK in 293-T cells and completely eliminated the appearance of both the cleavage A products and the cleavage B products (Fig. 4). Only when minute amounts of calpastatin plasmid were used (30 ng compared with 2 g of FLAG-DCLK plasmid) were traces of the cleavage products visible. The high specificity of calpastatin strongly supports the contention that calpain constitutively cleaves DCLK overexpressed in 293-T cells. The elimination of the cleavage B site bands in this experiment probably reflects the potent inhibition of calpain by calpastatin before any cleavage of DCLK could occur. One caveat is that chronic expression of calpastatin may indirectly effect DCLK cleavage by another protease. However, the similar effects of acute calpeptin application and calpastatin co expression argue that calpain cleaves DCLK overexpressed in 293-T cells.
DCLK Is Cleaved by Calpain in Embryonic Brain Extract-To determine whether the 50-kDa band seen in neuronal cell extract could be produced by calpain, we studied cleavage of DCLK from embryonic mouse brain in vitro. Anti-DCLK C-terminal antibodies recognize a faint 50-kDa band in embryonic mouse brain extract, which was stable with prolonged incubation at 37°C (Fig. 5, lanes 1-3). However, on addition of calcium, DCLK was entirely cleaved to the 50-kDa C-terminal fragment within 90 min of incubation (Fig. 5, lanes 4 -6). Cleavage of DCLK could be entirely prevented by addition of calpeptin (Fig. 5, lanes 7-9). Together, the calcium dependence of this proteolytic event and its sensitivity to calpeptin indicate that DCLK is cleaved by calpain and supports the suggestion that the endogenous 50-kDa band in primary neuronal extract is also derived from proteolytic processing of DCLK by calpain.
Purified m-Calpain Cleaves Recombinant DCLK in Vitro-To directly demonstrate that calpain can cleave DCLK, we prepared recombinant GST fusion DCLK by glutathione affinity purification from bacteria. Coomassie Blue staining revealed that the full-length 110-kDa GST-DCLK fusion protein was copurified with several smaller bands mostly between 45 and 65 kDa (Fig. 6A, lane 1). Western analysis with anti-DCLK N-terminal antibody demonstrated that these bands contain the DCLK epitope recognized by this antibody, suggesting that they are either degradation products or abortive translation products (data not shown). As Western blot with anti-DCLK C-terminal antibody recognized only the 110-kDa band (Fig.  6B, lane 1) and no smaller fragments characteristic of degraded proteins, we favor the latter interpretation.
Purified m-calpain cleaved GST-DCLK in a concentrationdependent manner (Fig. 6A, lanes 3-6), requiring the presence of calcium (Fig. 6A, lanes 7 and 8). No degradation of GST-DCLK was seen following incubation with calcium and heat-inactivated calpain (Fig. 6A, lane 9). The cleavage products of ϳ28, 31, and 33 kDa (Fig. 6A, lanes 5 and 6) all belong to the GST protein (results from N-terminal sequencing; data not shown). Western blot using the C-terminal antibody revealed that DCLK was initially cleaved to a 50-kDa C-terminal band, which was further degraded to a ladder of bands between ϳ40 and 50 kDa, consistent with cleavage at various sites within and immediately after the ST-rich domain (Fig. 6B). This demonstrates that in vitro calpain directly processes DCLK into cleavage fragments similar to those observed in cultured cells. Calpain cleavage sites appear to reflect structural determinants on target proteins rather than precise amino acid motifs (27). The presence of a ladder of bands instead of discrete bands at 42 and 50 kDa, as seen in cell and tissue lysate, may reflect structural variations in recombinant GST-DCLK due to differential autophosphorylation of recombinant molecules or lack of normal DCLK association partners like microtubules. Alternatively, calpain may have degraded specificity due to in vitro conditions.
Proteolysis of DCLK Does Not Impair Kinase Activity-Cleavage of DCLK at the ST-rich domain would be predicted not to impair kinase activity of the fragment containing the catalytic domain because such a fragment is structurally very similar to another product of the DCLK gene, CPG16 (Fig. 2).  6 -10). In lane 9, calpain was heat-inactivated prior to addition (100°C, 1 min). Calcium was added at 5 mM, and EGTA at 10 mM where indicated. Following incubation, reactions were subjected to SDS-PAGE and Coomassie Blue stain. B, in vitro cleavage of DCLK releases an intact C-terminal domain. Following cleavage with calpain as above, Western blot (WB) analysis was carried out using the anti-DCLK C-terminal antibody. The single 110-kDa band representing GST-DCLK before cleavage was digested by calpain into several bands from 40 to 50 kDa in size.
We first assessed the ability of in vitro cleaved DCLK to autophosphorylate. Proteolysis initially resulted in the appearance of an autophosphorylating 50-kDa band (Fig. 7A, right panel,  lanes 3 and 4) similar to the 50-kDa C-terminal fragment detected in Fig. 6B; more extensive proteolysis resulted in loss of autophosphorylation (Fig. 7A, lane 5). Loss of autophosphorylation by the 40 -50-kDa C-terminal fragments detected in Fig. 6B could indicate abolition of kinase activity or loss of autophosphorylation sites. We therefore assayed kinase activity following DCLK cleavage using myelin basic protein as a substrate. No loss of kinase activity toward myelin basic protein was observed even after incubation of DCLK with large amounts of calpain (Fig. 7A, lane 5).
Cleavage of DCLK by Calpain Releases the Kinase Domain-Based on the sizes of the DCLK cleavage fragments, the calpain cleavage site is predicted to be in the ST-rich domain between the doublecortin domain and the kinase domain (Fig. 2). As the doublecortin domain is responsible for anchorage of the protein to the microtubule cytoskeleton, cleavage in this area could release the kinase domain from its microtubule localization. We therefore sought to confirm that the C-terminal cleavage fragment is released following cleavage by calpain. To determine whether the C-terminal fragment remains associated with the N-terminal domain after cleavage, we digested recombinant GST-DCLK bound to glutathione beads. Under these conditions, all of the C-terminal cleavage fragments were released from the beads (Fig. 8A). To determine whether the kinase domain is released from a microtubule localization following cleavage, we fractionated FLAG-DCLK-transfected 293-T cells into microtubule and cytosolic fractions. Under these extraction conditions in 293-T cells, the 50-kDa C-terminal fragment was not stable; however, the 42-kDa C-terminal fragment partitioned into the cytosolic fraction, whereas the 43-kDa N-terminal fragment containing the doublecortin microtubule binding domain partitioned into the microtubuleenriched fraction (Fig. 8B).
Perinuclear Staining of DCLK Is Enhanced by Calpain Inhibition-In primary corticostriatal neurons, DCLK C-terminal immunoreactivity is most marked on microtubules in growth cones, with weaker staining in the cell body (11). Interestingly, the anti-DCLK N-terminal antibody gives a similar staining of growth cones, but in the cell body, staining is concentrated in the perinuclear region rather than in the nucleus (Fig. 9, compare A and C). We wondered whether this difference may reflect differential localization of N-terminal and C-terminal fragments following proteolysis by calpain. Following overnight application of calpeptin, neurons were fixed and stained with both antibodies. Whereas the localization of the N-terminal fragment of DCLK was unaffected (Fig. 9, C and D), the C-terminal antibody immunostaining was strikingly altered, with staining now concentrated in a perinuclear localization similar to that of the N-terminal fragment. DISCUSSION The extensive modifications to neuritic architecture that occur during development imply the existence of mechanisms to selectively remodel the neuronal cytoskeleton. Proteolytic processing of the microtubule-associated protein DCLK may represent one such mechanism. In this report we present evidence that DCLK is a substrate for calpain. We demonstrate that a 50-kDa band in embryonic mouse brain extract and primary cultured neurons recognized by the anti-DCLK antibody is also generated in 293-T cells after transfection with full-length DCLK. In 293-T cells the 50-kDa band is eliminated by application of calpain inhibitors. In mouse brain extract, DCLK is completely cleaved to a 50-kDa product when calcium is added but not in the presence of the calpain inhibitor calpeptin. Calpain directly cleaves recombinant DCLK in vitro producing C-terminal fragments of 40 -50 kDa. Such an event may be physiologically significant, as we demonstrate that cleavage results in release of the fragment containing the kinase activity from microtubule anchorage into the cytosol. In primary neurons, DCLK C-terminal immunoreactivity in the cell soma is normally concentrated in the nucleus, with N-terminal staining highlighting the perinuclear region. However, inhibition of calpain causes C-terminal staining to closely resemble the Nterminal pattern of staining, suggesting that the C-terminal fragment enters the nucleus following cleavage.
Multiple roles for calpain in normal and pathological conditions have been posited including in promoting progression through the cell cycle and apoptosis (see Ref. 27 for a review). Loss of function mutations in a muscle-specific form of calpain, p94, cause limb girdle muscular dystrophy type 2A (28), whereas increased calpain activity has been linked with other muscular dystrophies, cataract formation, and Alzheimer's disease. Calpain promotes cell motility, by facilitating the elimination of focal adhesion complexes (29). In the adult central nervous system, calpain has been implicated in adult synaptic plasticity (see Ref. 30 for review) whereas, during development, calpain involvement has been suggested in regulation of neurite retraction (31), perhaps in the context of regulating connectivity during early postnatal development (32).
Cleavage of DCLK in transfected 293-T cells was prominent when cells were allowed to grow at high density. Although we did not study this phenomenon in detail, we believe it may relate to greater cell death at high density for two reasons. First, the physiological calpain inhibitor, calpastatin, has been shown to be a substrate for caspases (33,34); degradation of calpastatin during apoptosis could disinhibit calpain resulting in greater cleavage of DCLK. Second, we were able to stimulate production of the 50-kDa band in transfected 293-T cells by induction of apoptosis. The 50-kDa cleavage product observed in 293-T cells was rapidly degraded, disappearing once DCLK cleavage was inhibited. It has been suggested that proteolysis of muscle sarcomere proteins by calpain may promote ubiquitindependent degradation according to the N-end rule (35). DCLK contains three PEST domains (Fig. 2) (amino acid motifs enriched in proline, glutamic acid, serine, and threonine), which have been proposed to target proteins for rapid degradation. The 50-kDa cleavage product would retain all three PEST domains. Although the original PEST hypothesis implicated calpain in the degradative pathway (36) and many PEST-containing proteins are indeed calpain substrates, the calpain-PEST hypothesis is disputed (37). Splice variants of DCLK affect all three PEST domains, potentially modulating the susceptibility of DCLK to proteolysis by calpain. The anti-DCLK C-terminal antibody used in this study recognizes one of two C-terminal splice forms of DCLK (herein referred to as DCLK-␣). When we studied the fate of endogenous DCLK in embryonic brain lysate following calcium addition, all of the DCLK-␣ was digested by calpain (Fig. 5); however, the N-terminal antibody, which is insensitive to C-terminal splice variants, revealed that some DCLK remained intact (data not shown). Intriguingly, the other DCLK C-terminal splice form lacks a C-terminal PEST motif. We are currently investigating the susceptibility of different splice forms of DCLK to cleavage by calpain.
DCLK expression is widespread during the period of embryonic brain development and down-regulated thereafter (14,17,25). During this time, DCLK immunoreactivity is seen in neuronal cell bodies and neurites, but is strikingly enhanced in growth cones (11). The finding that DCLK is a substrate for calpain is pertinent in the light of studies demonstrating roles for calcium and calpain in regulating growth cone behavior. During neural development, molecular signals at axon guidance checkpoints cause calcium transients in growth cones, which induce growth cone stalling, and promote the elaboration of filopodia, which sample the local environment (38). Calcium fluxes in the growth cone are transduced by the calcium-calmodulin phosphatase calcineurin (39), Ca 2ϩ /calmodulin-dependent protein kinase II (40), and calpain (31). Calpain activation in response to calcium influx has been shown to block neurite outgrowth in SH-SY-5Y cells (41) and neuroblastoma cells (42) FIG. 9. Inhibition of calpain causes DCLK C-terminal immunoreactivity to resemble perinuclear N-terminal immunoreactivity. Following overnight incubation with Me 2 SO (DMSO; A and C) or 10 M calpeptin (B and D), neurons were fixed and stained with either anti-DCLK N-terminal antibody (C and D) or anti-DCLK C-terminal antibody (A and B). Following calpeptin incubation, staining with the C-terminal antibody shifts to resemble staining with the N-terminal antibody. We suggest this results from inhibition of calpain cleavage preventing generation of a C-terminal fragment, which normally enters the nucleus. and to be involved in neurite retraction (43,44). In non-neuronal cell types, calpain has been shown to be required for formation of filopodia and lammelipodia (45), and in cultured aplysia neurons, local calcium spikes trigger calpain activation and elaboration of morphologically complex growth cones (46). Activation of calpain in the growth cone by calcium influx may result in cleavage of DCLK, releasing the kinase activity from the cytoskeleton. At present we cannot distinguish between the possibilities that the DCLK doublecortin domain sequesters DCLK kinase activity from cytoplasmic or nuclear targets, and an alternative model in which cleavage serves to disengage the kinase activity from microtubule-associated targets. Models involving calpain-mediated release of an active fragment from an anchorage domain have been proposed for several proteins including the membrane-associated phosphatases STEP (47) and PTP-1B (48), the protein-tyrosine kinase Src (49), and the focal adhesion protein talin (50).
Immunostaining of primary neurons with anti-DCLK C-terminal antibody reveals partial nuclear localization (11). Interestingly, staining of primary neurons with an N-terminal antibody reproduces the growth cone pattern observed with the C-terminal antibody but stains the perinuclear region in the cell soma. Inhibition of calpain caused a shift in the localization of the C-terminal fragment within the cell soma, with immunoreactivity now concentrated in a perinuclear fashion similar to that observed with the N-terminal antibody. This hints that, in neurons, calpain cleaves the C-terminal kinase domain from its N-terminal microtubule anchorage domain, allowing it to enter the nucleus. Transfection of cultured cells with CPG16, which is similar to the 50-kDa C-terminal fragment of DCLK, results in mostly cytoplasmic localization. However, upon pharmacological elevation of cAMP, CPG16 moves into the nucleus (13). These two lines of evidence suggest that DCLK may have targets in the nucleus, and that the release of the kinase domain from the microtubule anchorage domain by calpain may represent a calcium-responsive means of regulating the localization of DCLK kinase activity.