Cdk5 Nuclear Localization Is p27-dependent in Nerve Cells

Antibodies and Chemical Regents

Antibodies against β-actin, Cdk4, Cdk5, hnRNP, lamin A/C, lamin B1, GFP,² Crm-1, HSP90, and hemagglutinin were from Santa Cruz Biotechnology (Santa Cruz, CA). Histone H3 was from Millipore (Billerica, MA). Rat-anti-BrdUrd was bought from Abcam (Cambridge, UK). Cleaved caspase-3 antibody was bought from Cell Signaling (Danvers, MA). Secondary antibodies used for immunocytochemistry were as follows: goat anti-mouse Alexa 488 and 594; goat anti-rat Alexa 488 and 594; goat anti-rabbit Alexa 488 and 594 (Invitrogen). All were used at a dilution of 1:1000. 4′,6′-Diamidino-2-phenylindole was used as a nuclear counterstain at 1 μg/ml. An Amaxa mouse neuron nucleofector kit was bought from Lonza (Köln, Germany).

Animals

A colony of Cdk5^−/+ mice were maintained on a mixed (C57BL/6Jx129/S1) background. Homozygous mutant embryos were produced by intercrossing heterozygous Cdk5^−/+ mice. Wild type C57BL/6J mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Timed pregnancies were established; the date of appearance of a vaginal plug was considered embryonic day 0.5. The embryos were taken at embryonic day 16.5 for either cortical cultures or histology. All of the animal procedures were carried out in accordance with Rutgers University Institutional Animal Care and Use Committee standards. The animal facilities at Rutgers University are fully Association for Assessment and Accreditation of Laboratory Animal Care accredited.

Constructs and Plasmids

Truncated Cdk5 fragments were synthesized by PCR and inserted into pEGFP-C1 or pGEX-4T-2. The fragments of Cdk5 (sequences encoding amino acids 64–83 and 128–147) with insert sites were synthesized and annealed to double-stranded DNA and then inserted into the pEGFP-C1 vector. The caspase-3-sensitive vector was constructed by inserting an NES-Caspase-3 fragment into DsRed-Nuc vector by AgeI/NheI restriction digestion and annealing. The cyclin D1 promoter-luciferase vector (40) was a generous gift of Dr Richard G. Pestell (Thomas Jefferson University), and the cyclin D1 promoter was subcloned into pd2EGFP vector (Clontech). The p27-HA vector was provided by Dr. Elizabeth Nabel (41). The HA-Crm-1 vector was obtained from Addgene.

Transfection

N2a cells were transfected with Lipofectamine 2000 following the instructions of the manufacturer. Primary mouse neurons were transfected with different vectors by the Amaxa mouse neuron nucleofector kit following the manufacturer's instructions.

Cell Culture and Synchronization

Neuroblastoma (Neuro-2a, N2a) and NIH 3T3 cells were purchased from ATCC. The NIH 3T3 (D51) cell line was kindly provided by Dr. Andrew Koff (Memorial Sloan-Kettering Cancer Center, New York). In this cell line, immortalized 3T3 cells were stably transfected with a p27 mutation, p27D51 that removes the N-terminal 51 amino acids of the protein preventing cyclin-Cdk interaction. HCT116-p21^−/− cells were kindly provided by Dr. Bert Vogelstein (Johns Hopkins University, Baltimore, Maryland). The cells were synchronized in the G₀ phase of the cell cycle by serum deprivation for 48–76 h in medium supplemented with 0.1% fetal bovine serum or 0.1% calf serum. To induce re-entry into the cell cycle, the cells were transferred to medium containing 10% fetal bovine serum or 10% calf serum.

Primary Neuronal Cultures

For Cdk5-deficient cultures, all of the embryos from a Cdk5^+/− × Cdk5^+/− mating were harvested and treated separately. Isolated embryonic day 16.5 embryonic cerebral cortices from control, Cdk5^−/−, or E2f1^−/− were treated with 0.25% trypsin-EDTA and dissociated into single cells by gentle trituration. The cells were suspended in neurobasal medium supplemented with B27 and 2 mm glutamine and then plated on coverslips coated with poly-l-lysine (0.05 mg/ml) and laminin (5 μg/ml). All of the cultures were grown for a minimum of 5 days in vitro before treatment. To monitor cultures during treatment, Cdk5^−/− or wild type neurons were cultured in glass-bottomed culture chambers (MatTek Corp.). After transfection or drug treatment, the dish to be monitored was placed into a CO₂ and temperature-controlled chamber mounted on the motorized stage of an inverted microscope (Leica LTM). Multiple neurons were monitored simultaneously using IP Lab software (BD Biosciences CA). GFP and DsRed were visualized with L5 and N3 filter sets, respectively.

Immunocytochemistry and BrdUrd Incorporation

At the appropriate time, the cultures were rinsed once with PBS and then exposed to 4% paraformaldehyde in 0.1 m phosphate buffer for 30 min at room temperature followed by three rinses with PBS. Immunohistochemistry of cell cultures was done without antigen retrieval. For BrdUrd labeling, the cells were cultured normally or serum-starved for 48 h followed by 12 h of serum add-back. Four hours before the end of the experiment, 10 μm BrdUrd was added to the medium. The cells were then fixed, and DNA was hydrolyzed by exposing the cells to 2 n HCl for 10 min. The specimens were then neutralized in 0.1 m sodium borate (pH 8.6) for 10 min and then rinsed extensively in PBS (three times) for 45 min before treatment with blocking reagent. Nonspecific antibody binding was blocked by exposing the fixed cells to 5% normal goat serum in 0.1% Triton X-100 for 1 h before application of the primary antibody.

Western Blotting and Co-immunoprecipitation

Dissected tissues or harvested cells were homogenized in 1:5 (w/v) ice-cold lysis buffer (1% Triton X-100, 20 mm Tris-HCl, pH 7.5), 150 mm NaCl, with protease inhibitor mix (Roche Applied Science). The samples were centrifuged at 12,000 × g for 20 min at 4 °C. The supernatant was collected, and the total protein levels were measured by a micro bicinchoninic acid protein assay kit (Pierce). Fractionation of cells into cytoplasmic and nuclear components was accomplished with an NER-mammalian kit according to the manufacturer's instructions (Pierce). For Western blots, the lysates were separated with SDS-PAGE and electrophoretically transferred onto nitrocellulose membranes. The membranes were blocked with 5% nonfat milk in TBST and probed with primary antibodies in blocking buffer, followed by treatment with horseradish peroxidase-linked secondary antibodies and ECL Western blotting detection reagents (Pierce). The intensity of immunoreactive bands was quantified using National Institutes of Health ImageJ. For immunoprecipitation, the cell lysates were incubated with immunoprecipitation antibody at 4 °C for 90 min, followed by additional incubation with protein G-Sepharose (GE Healthcare) for 90 min. The beads were washed five times with ice-cold PBS, and the bound proteins were analyzed by SDS-PAGE and immunoblot analysis.

Cdk5 Shuttles between Nucleus and Cytoplasm during the Cell Cycle

Although the levels of total Cdk5 do not change dramatically during the cell cycle, the nuclear/cytoplasmic ratio does (5). To further explore the movement of Cdk5 during the cell cycle, we used nocodazole to arrest cells at the G₂/M phase of the cell cycle, released them, and assayed the location of the Cdk5 at different times. The levels of nuclear Cdk5 show a wave-like pattern, which can be contrasted with nuclear Cdk4 whose levels do not change during the cell cycle (Fig. 1A). Proteins that shuttle between the nucleus and the cytoplasm are termed nucleocytoplasmic, and the movement of Cdk5 is consistent with its identity as a nucleocytoplasmic protein. Cdk5 would thus be predicted to have both an nuclear localization signal (NLS) and a nuclear export signal (NES) to regulate its subcellular localization. A scan of the primary amino acid sequence of Cdk5 did not reveal a canonical NLS. The closest match was found located at amino acids 33–36 (KRVR). This amino acid sequence is similar to the NLS of SV40 (KRKV), but it is unable to drive Cdk5 fragments containing it to the nucleus (supplemental Fig. S1). Without an endogenous NLS, the movement of Cdk5 into the nucleus must depend on binding to an accessory protein. Interestingly, in the course of our experiments, we found that the levels of nuclear Cdk5 during the cell cycle closely followed those of p27 (Fig. 1, A and B). We therefore considered the potential role for this Cdk inhibitor.

Nuclear Localization of Cdk5 Is Dependent on p27 Binding

In homozygous Cdk5^−/− brain (Fig. 1C) and Cdk5^−/− embryonic fibroblasts (Fig. 1D), the levels of p27 are dramatically reduced. This is significant because in Cdk5-deficient cells, cell cycle suppression is lost. To determine whether the two proteins physically interacted with each other, we performed co-immunoprecipitation experiments. Both full-length and GFP-Cdk5_N13–292 could be immunoprecipitated by HA-p27 (Fig. 2A; the subscript refers to the amino acid residues remaining in the Cdk5 construct). Successive removal of single amino acids revealed that when the threonine at position 17 was removed, the Cdk5/p27 interaction was eliminated; GFP-Cdk5_N18–292 no longer bound to HA-p27 (Fig. 2B, ninth lane). The physical interaction was also confirmed by glutathione S-transferase pulldown experiments (supplemental Fig. S2A). We note that the N-terminal residues around threonine 17 of Cdk5 are highly conserved in species from worm to human (supplemental Fig. S2C).

To determine the correlation between p27 binding and Cdk5 subcellular localization, N-terminal truncation mutations from N13 to N31 of Cdk5 were expressed as GFP fusion proteins, and their localization visualized by GFP fluorescence. Fig. 2C shows that truncations up to residue 17 localized throughout the whole cell; they were rarely excluded from the nucleus (<20%). By contrast, mutations that deleted amino acids Thr¹⁷ and beyond localized almost exclusively in cytoplasm (>60%). All of the fusion proteins were expressed at comparable levels (Fig. 2D). Fig. 2E shows representative images of various truncation mutations expressed as GFP fusion proteins in primary neurons (for additional mutations expressed in N2a cells, see supplemental Fig. S3). These data show that the N-terminal 17 residues of Cdk5 are crucial for its interaction with p27 and that the nuclear localization of Cdk5 depends on this interaction.

The Cytoplasmic Localization of Cdk5 Is Dependent on Its Intrinsic NES

The nuclear exclusion of the N-terminal deleted mutations suggested that the remaining Cdk5 fragment has an intrinsic NES. To determine its location, we designed the truncations shown in Fig. 3A. Wild type Cdk5 is localized throughout the cell. N-terminal truncations from residues 17 to 128 (Fig. 3A, constructs 2–6) localize primarily in the cytoplasm. Further truncations (Fig. 3A, construct 7) lose this cytoplasmic preference. This implies that an NES activity lies between residues 17 and 128. Three C-terminal truncation constructs were made: Cdk5(1–64), Cdk5(1–83), and Cdk5(1–147). Although Cdk5_N1–64 localized only in the nucleus; Cdk5_N1–83 was mostly nuclear but had some cytoplasmic localization as well; Cdk5_N1–147 localized through the cell, similar to full-length Cdk5 (Fig. 3, B and C; see also supplemental Fig. S4). This identified two potential NES, one between amino acids 64 and 83 and another between residues 128 and 147.

To validate the identification of these fragments as NES, we synthesized these fragments and inserted them into a GFP vector. As predicted, both fragments drove GFP into cytoplasm (Fig. 3D). Comparing the sequence of these fragments with the NES of MAPKKK reveals that fragments 64–83 and 128–147 both contain the typical sequence with multiple leucines but are missing the second of four predicted Leu residues (at Asp⁷⁴ and Asn¹³⁷; Fig. 3D). Nonetheless, Leu → Ala mutations of the remaining leucines abolished cytoplasmic localization (Fig. 3D). These data show that Cdk5 contains two NES-like signals, each made atypical by the absence of one leucine.

The binding of an NES to CRM1 leads to nuclear export (10), and we wished to determine whether Cdk5 used the CRM1 pathway. GFP-Cdk5 transfected N2a cells were treated with leptomycin B, an inhibitor of CRM1-dependent nuclear export (11, 12). Over 70% of the transfected cells accumulated GFP-Cdk5 in the nucleus (Fig. 3E). A direct interaction between CRM1 and Cdk5 was shown by co-immunoprecipitation (Fig. 3F), and Cdk5 fragments that lacked the NES regions, Cdk5_N1–64 and Cdk5_N170–292, did not interact with CRM1, nor did the Leu → Ala mutant. An empty GFP vector was used as a negative control.

Cytoplasmic Cdk5 Attenuates the Activation of Caspase-3 Induced by β-Amyloid

We have previously shown that nuclear Cdk5 functions as a cell cycle suppressor. This raises the question of what happens to Cdk5 once it is shuttled to the cytoplasm. We speculated that this fraction of Cdk5 might play a role in neuronal cell death. To explore this idea, we created a caspase-3-sensitive dsRed fluorescent protein vector in which a caspase-3 cleavage site was placed between an NES and the dsRed coding sequence that included an in-frame NLS. Because NES-based nuclear export is dominant over NLS-based import, a cell expressing our construct will have red fluorescence in the cytoplasm (Fig. 4A, top panel). With the activation of caspase-3, however, the NES is cleaved off. The NLS, now unopposed, moves the dsRed to the nucleus (Fig. 4A, bottom panel). The time required for complete caspase-3 cleavage is defined as the time when the dsRed marker is entirely nuclear. We tested the utility of this construct in Cdk5^−/− neurons; within 60 min of exposure to 10 μm fibrillar Aβ_1–42 peptide, the dsRed moves from cytoplasm to nucleus (Fig. 4B). The activation time of caspase-3 (dsRed translocation) was dependent on the concentration of Aβ (Fig. 4C).

Control GFP, GFP-Cdk5-NLS, or GFP-Cdk5-NES expressing constructs were co-transfected with the caspase-3-sensitive vector into Cdk5^−/− neurons. The next day, 10 μm Aβ_1–42 was added to the medium, and the location of dsRed was tracked. Although nuclear Cdk5 (GFP-Cdk5-NLS) had no effect on the rate of caspase-3 activation (Fig. 4, D and F), cytoplasmic Cdk5 (GFP-Cdk5-NES) slowed this process significantly (Fig. 4, E and F). These findings suggest that cytoplasmic Cdk5 is neuroprotective. This protection is dependent on kinase activity because both p35 binding-defective and kinase-dead Cdk5 mutants, even if expressed in the cytoplasm, were ineffective at delaying caspase-3 activation.

The experiments illustrated in Fig. 4 reflect the short term response of caspase-3 activation to Aβ exposure as measured by an exogenously applied caspase-3 detection peptide. To extend these observations, the endogenous level of cleaved capase-3 was investigated at longer survival times. GFP-NLS, GFP-Cdk5-NLS, GFP-Cdk5-NES, GFP-KDCdk5-NLS, and GFP-KDCdk5-NES were transfected into Cdk5^−/− neurons. Twelve hours after Aβ administration, cleaved capase-3 was detected by Western blotting (Fig. 5A). Kinase-active, cytoplasmic Cdk5 (Cdk5-NES) lowered the cleaved caspase-3 level induced by Aβ, whereas none of the other constructs tested (Cdk5-NLS, KDCdk5-NLS, and KDCdk5-NES) were found to have any effect (Fig. 5). The reduced activation of caspase-3 implies an anti-apoptotic action of Cdk5. Aβ is also reported to induce apoptosis through down-regulation of Bcl-2, and Cdk5 has been implicated in this action. Examining the levels of Bcl2 in the Aβ-treated neurons in our cultures revealed that of all of the constructs tested, only cytoplasmic Cdk5 overexpression prevented the decrease of the Bcl-2 level after Aβ administration.

Nuclear Cdk5 Blocks Cell Cycle Re-entry: p27 Dependence

Because nuclear Cdk5 has cell cycle blocking ability (5), and the nuclear localization of Cdk5 depends on its binding with p27, we wondered whether the cell cycle suppression ability of Cdk5 was also dependent on this interaction. To test this idea, two Cdk5 truncation mutations, Cdk5_N13–292 and Cdk5_N26–292, were forced into the nucleus of NIH 3T3 cells as GFP-NLS fusion proteins. After transfection, cell cycling was arrested by serum starvation followed by reinitiation in the presence of BrdUrd. Cdk5_N13–292, which retains p27 binding capacity, was capable of suppressing BrdUrd staining, but Cdk5_N26–292, which cannot bind p27, was inert, despite its NLS-directed nuclear location (Fig. 6, A and B). The requirement for p27 was further demonstrated by the inability of GFP-Cdk5-NLS-derived constructs to arrest the cell cycle in the NIH 3T3 p27(D51) cell line, in which a dominant negative p27 that is Cdk/cyclin binding-deficient is constitutively expressed (Fig. 6, A and C). We also investigated a possible role for p21, another member of the Kip/Cip Cdk inhibitor family. We transfected Cdk5-NLS and KDCdk5-NLS into the HCT116 p21^−/− cell line. All of the active Cdk5 and KDCdk5 species retain their cell cycle suppressor activity in the absence of p21 in contrast to the results with p27 deficiency (supplemental Fig. S5).

Cdk5 Is a Key Determinant of the Choice between Cell Cycle and Cell Death

It has been proposed that the death of neurons in several different neurodegenerative diseases is preceded by an unscheduled initiation of a cell cycle. We used two marker constructs to determine whether there was a consistent order to the initiation of these two processes in our system. To monitor cell cycle, we inserted the cyclin D promoter in front of a GFP construct. To monitor cell death activity, we used the caspase-3-sensitive vector described above. Cdk5^−/− neurons were treated with 1 μm β-amyloid after co-transfection of these two vectors. As shown in Fig. 7A, GFP fluorescence (cyclin D) was seen in neurons ∼3 h after Aβ_1–42 addition, marking this as the time of neuronal re-entry into the cell cycle. After 7 h, we observed high expression of cyclin D, and by this time, neurons had also begun the cell death process (nuclear dsRed). Thus cell cycle and cell death are closely linked in cultured neurons, and in this system cell cycle initiation precedes cell death. To validate this conclusion for endogenous Cdk5, neurons were double immunostained with BrdUrd and Cdk5 after β-amyloid exposure. Consistent with our results with the cyclin D-GFP and caspase-sensitive dsRed vector, neurons that had begun DNA synthesis (BrdUrd uptake) showed very low levels of nuclear Cdk5 staining (Fig. 7B) as well as low levels of nuclear p27 staining (Fig. 7C).

To further validate this ordering of events, GFP-Cdk5-NLS and GFP-Cdk5-NES were transfected into Cdk5^−/− neurons. After Aβ administration, BrdUrd incorporation and caspase-3 activation were detected by immunocytochemistry. Only 5% neurons with nuclear Cdk5 (GFP-Cdk5-NLS) incorporated BrdUrd, but more than 40% neurons with cytoplasmic Cdk5 (GFP-Cdk5-NES) incorporated BrdUrd (Fig. 8, A and B). Similarly, ∼50% of the neurons with nuclear Cdk5 was positive for cleaved-caspase-3, but only ∼15% neurons with cytoplasmic Cdk5 was cleaved caspase-3-positive (Fig. 8, C and D). Taken together these data show that Cdk5 is transported into the cytoplasm during or before cell cycle initiation and before caspase-3-dependent cell death begins.