Cyclin-dependent Kinase-5 Is Involved in Neuregulin-dependent Activation of Phosphatidylinositol 3-Kinase and Akt Activity Mediating Neuronal Survival*

The phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway plays an important role in mediating survival signals in wide variety of neurons and cells. Recent studies show that Akt also regulates metabolic pathways to regulate cell survival. In this study, we reported that cyclin-dependent kinase-5 (Cdk5) regulates Akt activity and cell survival through the neuregulin-medi-ated PI 3-kinase signaling pathway. We found that brain extracts of Cdk5 (cid:1) / (cid:1) mice display a lower PI 3-kinase activity and phosphorylation of Akt compared with that in wild type mice. Moreover, we demonstrated that Cdk5 phosphorylated Ser-1176 in the neuregulin receptor ErbB2 and phosphorylated Thr-871 and Ser-1120 in the ErbB3 receptor. We identified the Ser-1120 sequence RSRSPR in ErbB3 as a novel phosphorylation consensus sequence we Ser/Thr phosphorylation of receptor in the domain required activate the ErbBs. This suggests that neuregulin-dependent activation of Akt-enhanced survival of cortical neurons deprived of could be inhibited by roscovitine, a specific Cdk5 inhibitor. These results implicate that Cdk5 is involved in neuregulin-dependent activation of the PI3K/Akt neuronal survival pathway by regulating the phosphorylation of ErbB2/ErbB3.

Cyclin-dependent kinase 5 (Cdk5) 1 is a serine/threonine kinase, which is predominantly expressed in postmitotic neurons (1). Cdk5 kinase activity requires association with its neuronspecific activators, p35 and p39. Cdk5 kinase activity is essential for neuronal migration, neurite outgrowth, and laminar configuration of the cerebral cortex (2)(3)(4). Recently, Cdk5 has been suggested as contributing to the control of neuronal positioning in Reelin signaling during neural development (5) and to mediate neuronal guidance by regulating semaphorin-3A with Fyn kinase (6). Cdk5 and p35 have recently also been shown to regulate presynaptic and postsynaptic activity by phosphorylating Munc-18, amphiphysin, and the NR2A subunit of the N-methyl-D-aspartate receptor (7)(8)(9)(10)(11). Cdk5 activity also regulates dopamine signaling by phosphorylating DARPP-32 protein (12) and has been shown to up-regulate the expression of acetylcholine receptor at the neuromuscular junction (13). These studies indicate that Cdk5 is a multifunctional protein kinase in the central nervous system.
Cdk5 has been implicated in both cell survival and programmed cell death in neuronal and nonneuronal systems (14,15). Cells induced to apoptosis by exogenous signals such as staurosporin display increased Cdk5 activity (16,17). Recently, Cdk5 in association with p25, a truncated form of p35, has been thought to be deregulated to produce hyperphosphorylated tau protein and to disrupt the neuronal cytoskeleton, and it may be involved in neurodegenerative diseases such as Alzheimer's disease, amyotrophic lateral secrosis, Parkinson's disease, and Niemann-Pick disease (18 -23). On the other hand, Cdk5 may also be involved in neuronal survival. Cdk5 knockout mice exhibit a unique phenotype with perinatal mortality, associated with extensively disrupted cerebral cortical layering due to abnormal neuronal migration, absence of cerebella foliation, and degeneration of neurons in the brain stem and the spinal cord (24,25). The p35 knockout mice are viable and fertile (26,27), with fewer severe abnormalities of laminar structures in the cerebral cortex, compared with Cdk5 null mice (28). Recent studies on Cdk5 null mice expressing transgenic Cdk5, regulated by a p35 promoter to ensure specific expression in brain neurons (TgKO), were rescued and exhibited the wild type phenotype (29). This suggests that neuronal survival (and host survival) is dependent upon Cdk5 activity. In addition, our recent studies demonstrated that Cdk5 prevents neuronal apoptosis by negative regulation of c-Jun N-terminal kinase 3 in cortical neurons (30). These studies indicate that the role of Cdk5 activity in neuronal survival may depend on the specific cell type as well as its site of action in survival and/or apoptotic signal pathways.
A clue as to other possible sites of Cdk5 activity in regulating neuronal survival is suggested by the observation that Cdk5 activity, mediated by the neuregulin/ErbB pathway, up-regulates acetylcholine receptor expression at the neuromuscular junction in embryonic myotubes and adult muscle (13). The neuregulins (NRGs) are a class of epidermal growth factor-like molecules; a multigene family encodes them. All NRG proteins contain an extracellular epidermal growth factor-like domain, which is essential for their function (31)(32)(33)(34)(35)15). The NRG-1/ * 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. neuregulin family includes heregulin, acetylcholine receptorinducing activity, neu differentiation factor, and glial growth factor (36,37). Neuregulin proteins mediate their action through the ErbB family of receptor tyrosine kinases, including ErbB2, ErbB3, and ErbB4 (36,38). NRG receptors differ in kinase activity and substrate selectivity. Each ErbB protein has an extracellular ligand binding domain, a single transmembrane domain, a short intracellular juxtamembrane region, a tyrosine kinase domain, and a protein-rich carboxylterminal tail (36). NRG-1 binds to ErbB3 and induces the formation of heterodimers between ErbB2 and ErbB3 and thus activates the receptor (36). Targeted disruptions of the NRG-1 gene as well as the neuregulin receptors demonstrate that neuregulins are essential for the formation of the heart and nervous system (39,40). Neuregulins have been implicated in a number of events in cell survival, mitosis, migration, and differentiation (41)(42)(43)(44). The survival of Schwann cells, for example, is mediated by neuregulin signaling through the phosphoinositide 3-kinase (PI3K)/Akt pathway, a critical survival pathway in neurons and in most cell types (45). Accordingly, it seemed possible that an alternative site for Cdk5 regulation of neuronal cell survival might be the neuregulin-mediated PI3K/ Akt signaling pathway. To explore this possibility, we took advantage of the Cdk5 null mouse, which exhibited no Cdk5 activity (24), and examined the activities of PI3K and Akt.
In this study, we report that Cdk5 regulates Akt activity through phosphorylation of the neuregulin receptors (ErbB2/ ErbB3) and regulation of PI3K/Akt kinase signaling pathways. We found that brain extracts and cortical neurons from Cdk5 knockout mice exhibited reduced PI3K/Akt activities and increased apoptosis. The Ser-1120 residue in the RSRSPR sequence in the ErbB3 receptor is a novel Cdk5 consensus sequence. Here we see that Cdk5 activity sustains neuronal survival by activating a survival-signaling pathway, upstream at the receptor itself.

EXPERIMENTAL PROCEDURES
Cell Culture-Cortical neurons from embryonic day 16.5 and 18 Cdk5 wild type and knockout mice (Cdk5Ϫ/Ϫ) were prepared as described by Li et al. (30). In brief, embryos were dissected and minced well with scissors. The dissociated cells were collected by centrifugation and resuspended in a serum-free neurobasal medium supplemented with B27 and 0.5 mM L-glutamine. Cells (25 ϫ 10 4 ) were plated in 35-mm plastic dishes precoated with polylysine (10 g/ml) (Gibco) for 7 days. HEK-293T and COS-7 cells were cultured in Dulbecco's modified Eagle's medium with 5% fetal calf serum.
Preparation of Brain Extracts-Brain of E18.5 from Cdk5Ϫ/Ϫ and wild type mice were rinsed with cold phosphate-buffered saline (PBS) and homogenized in a buffer containing 0.32 M sucrose, 5 mM HEPES, pH 7.4, and protease inhibitor mixture using poltroon. The homogenate was centrifuged at 10,000 ϫ g for 60 min. The supernatant was analyzed for in vitro phosphorylation and PI3K activity.
Phospholipid Extraction-Equal aliquots of 50 l (1 g/l) of supernatant from Cdk5Ϫ/Ϫ and wild type mice brain (E18) were phosphorylated as described by Li et al. (30). In brief, the samples were incubated in 15 Ci of [ 32 P]ATP in a buffer containing 50 mM Tris (pH 7.5), 50 mM NaCl, 5 mM MgCl 2 , 2 mM dithiothreitol for 2 h at 30°C with constant mixing. The reactions were stopped by the addition of chloroform/methanol (2:1, v/v). Following vigorous overtaxing, tubes were spun at 3000 rpm for 10 min. the organic phase containing phospholipids was collected, and a 0.8-ml mixture of chloroform/methanol/H 2 O (3:48:47) was added to the organic phase vortexes and spun for 10 min at 3000 rpm. The aqueous phase was discarded completely, and the organic phase was used to analyze the 32 P incorporation into the lipids. The extracted phospholipids were quantified by liquid scintillation.
PI3K Assay-Brain tissue from Cdk5Ϫ/Ϫ and wild type mice (E18) was homogenized in a buffer containing Nonidet P-40 (1%), 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM sodium orthovanadate, 2 mM sodium pyrophosphate, 50 mM NaF, 2 mM EDTA, 3 mM EGTA, and protease and phosphatase inhibitors. Cell lysates (200 g of total protein) were immunoprecipitated with anti-p85 subunit antibody. The precipitates were resuspended in 50 l of kinase assay buffer (10 mM Tris-HCl, pH 7.6, 100 mM NaCl, 1 mM EDTA, and 100 M Na 3 VO 4 ). The activity of PI3K was assayed essentially as described by Hii et al. (73). Briefly, 10 l of MgCl 2 (stock concentration of 100 mM), 10 l of PtdIns (stock concentration of 1 g), and 5 l of 400 mM Tris-HCl, pH 7.6, were added to the 50-l precipitates, and the reaction was started by the addition of 25 Ci of [␥-32 P]ATP. The reaction was incubated at room temperature for 10 min with constant mixing. The reaction was terminated by the addition of 40 l of 8 N HCl and 320 l of chloroform/ methanol (1:1). After vigorous mixing and centrifugation, the lipid phase was collected and washed with 500 ml of CH 3 OH/H 2 O (1:1). The inositol 3,4,5-trisphosphate and phosphatidylinositols were separated by TLC using CHCl 3 /CH 3 OH/H 2 O/30% NH 4 OH (90: 70:14.6:5.4, v/v/v/v) developing mixture. Phosphorylated phospholipids were detected by autoradiography.
Phosphorylation Studies-P7 rat brain extracts were prepared, and Cdk5 was immunoprecipitated using anti-Cdk5 antibody as described by Li et al. (30). The peptides were synthesized commercially (Peptide Technologies Inc.). For in vitro phosphorylation studies, we incubated peptides or histone H1 with the Cdk5 immunoprecipitate (IP). An Akt IP was used to phosphorylate H2B to determine Akt activity. Substrates (peptides or H2B) were incubated with [␥-32 P]ATP (0.1 mM) in a buffer containing 50 mM Tris-HCl (pH 7.4) with 1 mM EGTA, 1 mM dithiothreitol, 5 mM MgCl 2 , 0.5 M microcystin LR, and immunoprecipitated Cdk5 or Akt for 30 min at room temperature as described by Li et al. (30). To study the phosphorylation of ErbB-specific sites by Cdk5, the mutant and wild type C-terminal domain (residues 663-1339) of rat ErbB3 plasmids were transfected into COS-7 cells using Lipo-fectAMINE. HA-tagged ErbB3 wild type and mutant (T871A and S1120A) C-terminal domains (residues 663-1339) of the receptor were generated by standard cloning methods. The putative phosphorylation sites in ErbB3 were mutated using the QuikChange TM site-directed mutagenesis kit (Stratagene) according to the manufacturer's instructions. The mutations were verified by DNA sequencing.
Apoptosis Assays-DNA fragmentation associated with apoptosis was detected by TUNEL histochemistry staining. Cortical neurons with or without inhibitor treatment were directly mounted on cover slides and fixed with 4% paraformaldehyde in PBS and permeabilized with 0.2% Triton X-100 (20 min at room temperature and then incubated for nick end-labeling for 2 h at 37°C with TdT according to standard procedures (Roche Applied Science).
Immunofluorescence-Cortical neurons or brain sections were fixed in 4% paraformaldehyde in PBS for 30 min, washed in several changes of PBS for 30 min, and permeabilized in 0.2% Triton X-100 in PBS for 15 min. Monoclonal or polyclonal anti-Cdk5 antibody (1:200; Santa Cruz Biotechnology), polyclonal antibody ErbB3 (C-17; Santa Cruz), and phospho-Akt (Thr 308 ) (1:200; New England Biolabs) was incubated overnight at 4°C. After a wash in PBS (three times for 15 min each), Cells or sections were incubated with fluorescein isothiocyanate-conjugated goat anti-mouse IgG and rhodamine-labeled goat anti-rabbit IgG or rhodamine-labeled goat anti-mouse IgG secondary antibody for 1 h at room temperature. Fluorescent images were obtained using a Zeiss LSM-410 laser-scanning confocal microscope. Images were processed and merged using Adobe PhotoShop software.
Mass Spectrometry-Peptides were phosphorylated using nonradioactive ATP as described above. Phosphorylation of the peptides was determined by MALDI-TOF mass spectra on a Voyager-DE STR Biospectrometry Work station (Applied Biosystems) operating in the negative linear mode. ␣-Cyano-4-hydroxycinnamic acid matrix was utilized.

Reduced Brain Lipid Phosphorylation and PI3K Activity in
Cdk5Ϫ/Ϫ Mouse Brain-Previous studies have shown that neuronal Cdk5 activity is necessary for survival and nervous system development (29,30,46). Phospholipid kinases, the phosphoinositide kinases, are responsible for the phospholipid phosphorylation. Among three general families, PI3K selectively phosphorylates phosphatidylinositol 4,5-diphosphate (PtdIns(4,5)P 2 ) and produces phosphatidylinositol 3,4,5trisphosphate (PtdIns(3,4,5)P 3 ) upon stimulation by a variety of ligands in vivo as well as in vitro (47). PtdIns(3,4,5)P 3 is a key molecule involved in cell growth and survival signaling (48). To determine the identity of this fraction, phospholipids were extracted from wild type and Cdk5Ϫ/Ϫ mouse brain extracts after in vitro phosphorylation in the presence of [␥-32 P]ATP as described under "Experimental Procedures." The data presented in Fig. 1A show a 6-fold decrease in ␥-32 P incorporation into the phospholipids in brain extracts of Cdk5Ϫ/Ϫ as compared with wild type mice. In the absence of Cdk5, the synthesis of phospholipids is compromised. In order to explore the possibility of Cdk5-mediated interaction with the phospholipid-dependent PI3K signaling pathway, we compared PI3K activity in the cortical neurons derived from wild type and Cdk5Ϫ/Ϫ mice. The cortical neurons of Cdk5Ϫ/Ϫ mice showed a significantly decreased PI3K activity (Fig. 1B).
Akt Phosphorylation and Activity Is Decreased in Cdk5Ϫ/Ϫ Mouse Brain-Since Akt phosphorylation on both Thr-308 and Ser-473 by 3Ј-phosphoinositide-dependent kinase-1 (PDK1) is dependent on the levels of PtdIns(3,4,5)P 3 , the key product of PI3K activity and PtdIns(4,5)P 2 levels (49), we compared the Akt phosphorylation and activity levels in cortical extracts of E18-wild type and Cdk5Ϫ/Ϫ mice (Fig. 2, A and B). The total Akt and phosphorylated Akt expression levels were determined by Western blot analysis with specific anti-phospho-Akt (Thr-308 or Ser-473) and anti-total Akt antibodies. Akt kinase activity was assessed using IPs from brain extracts obtained with total Akt antibody and histone H2B as a substrate. We found that expression levels of Akt protein of wild type and Cdk5Ϫ/Ϫ mice were similar (Fig. 2C). However, the phosphorylation of Akt and its activity were significantly decreased in the Cdk5Ϫ/Ϫ mice compared with wild type mouse brain extracts (Fig. 2, C and D). Although the Akt activity was reduced to low levels in Cdk5Ϫ/Ϫ mice brain extracts, the corresponding Akt phosphorylation was not reduced proportionally (Fig. 2, C and   D). This may be due to some cross-reactively of phospho-Akt antibodies used in these experiments. In the absence of any Cdk5 activity in Cdk5Ϫ/Ϫ mice, the PI3K/Akt kinases are down-regulated, suggesting that their activities are dependent upon Cdk5 phosphorylation.
Neuregulin Receptor ErbB3 and ErbB2 Are Phosphorylated by Cdk5-The above observations suggest that Cdk5 activity is involved in the PI3K signaling pathway. To determine the target substrate for Cdk5, we based our analysis on the observation that the Cdk5-p35 complex is implicated in neuregulininduced acetylcholine receptor expression at the neuromuscular junction (13). This suggested that ErbB receptors might be a substrate for Cdk5 phosphorylation. Indeed, we found that the putative Cdk5 phosphorylation consensus sequence motifs are present in the ErbB2 and ErbB3 receptor molecules. Ser-1176 in the sequence SPGK of ErbB2, Thr-871 in the TPIK sequence, and Ser-1204 in the SPPR sequence of ErbB3 are putative phosphorylation sites in Cdk5 consensus motifs. To test whether these and other proline-directed serine residues in the peptides derived from ErbB2 and ErbB3 (see Fig. 3A) are the sites phosphorylated by Cdk5, we incubated these peptides and their mutant forms with Cdk5 IP from brain extracts and determined their phosphorylation by in vitro kinase assays. We found that Cdk5 significantly phosphorylated the Ser-1176 in the ErbB2 peptide RPKTLSPGKN and Thr-871 in the ErbB3 peptide AKTPIKWAL but weakly phosphorylated Ser-1204 in the ErbB3 proline-rich peptide RRGSPPRPPR (Fig. 3C). Interestingly, we found that the Ser-1120 in the basic amino acidrich sequence RSRSRSPRPR of ErbB3 was the most favorable substrate for Cdk5 (Fig. 3C). The peptide RSRSRSPRPR does not have the conventional consensus sequence ((K/R)(S/ T)PX(K/R), where X represents a basic residue) of cyclin-de- Cdk5 activity (B) in wild type and Cdk5Ϫ/Ϫ mice (E18) brain extracts. C and D, Western blot analysis of Akt phosphorylation using anti-phospho-Thr 308 and anti-phospho-Ser 473 antibodies in wild type and Cdk5Ϫ/Ϫ mice brain extracts (C) and Akt activity (D) by immunoprecipitating with anti-Akt antibody from wild type and Cdk5Ϫ/Ϫ mice brain extracts subjected to an in vitro kinase assay with histone H2B as a substrate. The low level of histone 2B phosphorylation is due to the lower exposure of the autoradiograph. Data in the histogram represent means Ϯ S.D. from four independent experiments. pendent kinase including Cdk5, but it is rich in basic amino acids and appears to be a novel Cdk5 phosphorylation consensus sequence. To confirm whether Cdk5 phosphorylates the Ser-1120 residue, we conducted in vitro kinase assays using various mutant peptides derived from RSRSRSPRPR in which alanines were substituted for serines (Fig. 3, B and D). Ser-1120 appeared to be the site for Cdk5 phosphorylation. The phosphorylation of Thr-871 of RPKTLSPGKN in ErbB2 and Ser-1120 of RSRSRSPRPR in ErbB3 were further investigated and confirmed by mass spectrometry (Fig. 3, E and F).
To investigate the Cdk5 phosphorylation of ErbB3 in vivo, the Thr-871 and Ser-1120 in the C-terminal domain of rat ErbB3 were mutated to Ala (T871A and S1120A). COS-7 cells were cotransfected with wild type HA-ErbB3 or mutant HA-ErbB3 (T871A and S1120A) C-terminal domains with Cdk5-p35. The levels of phosphorylated wild type HA-ErbB3 C terminus and HA-ErbB3 (T871A and S1120A) were measured by immunoprecipitation using anti-HA-tagged antibody, and their phosphorylation state was detected by Western blot using antiphosphoserine and anti-phosphothreonine antibody. We found that mutation of Thr-871 or Ser-1120 to Ala in ErbB3 showed no detectable Cdk5-dependent serine and threonine phosphorylation in HA-ErbB3 (T871A and S1120A) compared with the wild type HA-ErbB3 (Fig. 4, A and B). Similar results are obtained using HA-ErbB2 mutant (S1176A) and wild type (data not shown).
Cdk5 Associates with and Phosphorylates ErbB3 in Vivo-If Cdk5 phosphorylated the ErbB3 receptor, we would expect the kinase to co-localize with the receptor in cells. To examine this possibility in cortical neurons, we performed double-labeled immunofluorescence staining in rat cortical neurons and found that Cdk5 and ErbB3 are uniformly expressed throughout the cell (Fig. 5, A-C). To investigate whether Cdk5 is associated with ErbB3, we immunoprecipitated cell lysates from the rat cortical neurons with Cdk5 antibody by Western blot analysis and assayed for the co-elution of ErbB3. We found that ErbB3 is present in Cdk5 immunoprecipitate (Fig. 5D).
To examine whether Cdk5 specifically phosphorylated ErbB3, cell extracts from the wild type and Cdk5Ϫ/Ϫ brain were immunoprecipitated with anti-ErbB3 antibody and detected by Western blot using phosphotyrosine, phosphoserine, or phosphothreonine antibodies (Fig. 5E). Both phospho-Ser and phospho-Thr, but not phosphotyrosine, were significantly decreased in Cdk5Ϫ/Ϫ mice (Fig. 5, F and G), suggesting that ErbB3 is a putative substrate for Cdk5 phosphorylation in vivo.
Cdk5 Is Involved in Neuregulin-induced Akt Phosphorylation and Activity in Cortical Neurons-If Cdk5 acts as a modulator of the PI3K/Akt signaling pathway via its phosphorylation of A and B, the 10-mer peptide motif corresponding to Cdk5 phosphorylation consensus sequence and mutations in ErbB2 and ErbB3 were synthesized commercially. C and D, in vitro assays were performed by incubating peptide (50 g) with Cdk5 IP. E and F, identification of the Cdk5 phosphopeptides by MALDI-TOF spectra. MALDI-TOF spectra were acquired on a Voyager-DE STR Biospectrometry work station (Applied Biosystems) operating in the negative linear mode. ␣-Cyano-4-hydroxycinnamic acid was utilized as a matrix. E, MALDI-TOF linear negative mode spectrum of the phosphorylation reaction mixture of AKTPIKWMAL-amide. A major ion was observed at m/z 1237.75, corresponding to the monophosphopeptide. F, MALDI-TOF linear negative mode spectrum of the phosphorylation reaction mixture of RSRSRSPRPR-amide. A major ion was observed at m/z 1333.64, corresponding to the monophosphopeptide. Also observed was an ion at m/z 1412.91, corresponding to the diphosphopeptide. Other peptides were also analyzed (data not shown) .   FIG. 4. A and B, COS-7 cells were co-transfected HA-ErbB3 or mutant (T871A and S1120A) with Cdk5-p35. The wild type or mutant HA-tagged ErbB3 was immunoprecipitated using anti-HA antibody and subjected to Western blot using phospho-Thr or phospho-Ser antibody. Data in the histogram represent means Ϯ S.D. from four independent experiments. the ErbB3 receptor, then stimulation of the receptor by the addition of neuregulin should promote Cdk5 activity and ErbB3 phosphorylation. To test the prediction of this hypothesis, cortical neurons derived from rat E18 embryos were stimulated by neuregulin ␤ 1 and treated with either LY249002, a PI3K inhibitor, or roscovitine, a specific Cdk5 inhibitor. The cell lysates were subjected to Western blot analysis using phospho-Akt antibody. In addition, Akt kinase activity was assayed using an Akt IP with histone H2B as a substrate. We found that neuregulin markedly stimulated Akt phosphorylation and its activity (Fig. 6, A and B). Inhibition of PI3K activity by its specific inhibitor LY294002, even in the presence of neuregulin, resulted in a significant reduction of Akt phosphorylation.

FIG. 3. Phosphorylation of ErbB2 and ErbB3 in vitro and in intact cells by Cdk5.
The roscovitine effect on Akt phosphorylation and activity were less inhibitory, reaching control levels in the absence of neuregulin (Fig. 6, lanes 2 and 5). Roscovitine reduced neuregulinstimulated Akt phosphorylation and activity but had no significant effect on basal phosphorylation and activity. Akt activity and phosphorylation, however, were also reduced by half in the presence of roscovitine and neuregulin, suggesting that Cdk5 is involved in the neuregulin-PI3K/Akt signaling pathway (Fig. 6,  lanes 2 and 4).
Cdk5-mediated Neuroprotection Is Involved in the Neuregulin/PI3K Signaling Pathway-One of the key signaling pathways involved in the regulation of neuronal survival is the PI3K/Akt kinase intracellular signaling transduction pathway (50,51). The above results show that the phosphorylation of Akt induced by neuregulin was inhibited by a PI3K and a Cdk5 inhibitor, suggesting that Cdk5 may play an important role in neuronal survival through the regulation of Akt activity. Therefore, we investigated whether Cdk5 is involved in neuregulin-induced survival of cortical neurons after serum deprivation. Recent studies have indicated that the PI3K pathway is the primary signal transduction system that mediates the protective effect of neuregulin against serum deprivation in Schwann cells (52). We used cortical neurons derived from the rat embryonic brain (E18) as a model to measure cell apoptosis using TUNEL staining. The number of TUNEL-positive cells were quantified and compared. We found that the inhibitors, LY249002, a PI3K inhibitor, and roscovitine, a specific Cdk5 inhibitor, both increased the proportion of TUNEL-positive cells and reduced the extent of cell survival induced by neuregulin stimulation (Fig. 7). Roscovitine alone induced higher apoptosis compared in the presence of neuregulin and roscovitine. This suggests that Cdk5 activity is involved in neuron protection induced by neuregulin. DISCUSSION It has been demonstrated that a cell survival ligand, neuregulin, binds to ErbB receptors and recruits PI3K to the FIG. 5. Cdk5 co-localization and phosphorylation of ErbB3 in cortical neurons. A-C, rat cortical neurons were stained with monoclonal Cdk5 and polyclonal ErbB3 antibodies; Cdk5 and ErbB3 staining was visualized with a rhodamine-coupled secondary and fluorescein isothiocyanate-coupled secondary antibody, respectively. D, cortical neuronal extracts were prepared and immunoprecipitated with Cdk5 antibody and secondary IgG (control). The immunoprecipitates then were immunoblotted for ErbB3 antibody. E, cell lysates extracted from Cdk5Ϫ/Ϫ and wild type mice cortical neuron culture (7 days) was subjected to Western blot analysis using anti-Cdk5 antibody (C-8) (top) and anti-ErbB3 antibody (bottom). F, the immunoprecipitates with ErbB3 antibody was analyzed by Western blot using anti-phosphotyrosine, anti-phosphoserine, and anti-phosphothreonine. G, quantitative analysis of phosphotyrosine, phosphoserine, and phosphothreonine. Data in the histogram represent means Ϯ S.D. from four independent experiments.
FIG. 6. Cdk5 involved in neuregulin-induced phosphorylation of Akt. A, rat cultured cortical neurons were treated with human neuregulin ␤ 1 (100 ng/ml) for 10 min, and a PI3K inhibitor, LY294002 (50 M), and Cdk5 inhibitor, roscovintine (25 M), were added 1 h before treatment with NRG. The cell lysates were analyzed by Western blot using phospho-Akt (Thr-308 and Ser-473). B, the Akt kinase activity was assayed by in vitro kinase assay using histone H2B as a substrate. Phosphohistone H2B was quantified by liquid scintillation counting. Data in the histogram represent means Ϯ S.D. from four independent experiments. vicinity of the plasma membrane through its regulatory subunit p85 (53). The activation of PI3K generates the phosphoinositide phosphates inositol 1,4,5-diphosphate and inositol 1,4,5-trisphosphate at the inner surface of the plasma membrane, which, in turn, leads to the activation of PDK1. PDK1 phosphorylates Ser-473 and Thr-308 on Akt kinase. The activated Akt kinase targets many different apoptotic substrate molecules including Forkhead transcription factors, caspase-9 and Bad, inhibiting their activity and, thereby, promoting cell survival. This signaling pathway has been shown to play an important role in neuronal development and survival (51,54).
The work presented here shows that Cdk5 plays a role in modulating this cell survival pathway in neurons. The neuregulin receptor ErbB2 and ErbB3 were phosphorylated by Cdk5 at Cdk5 consensus motifs containing Ser-1176 of ErbB2 and Thr-871 of ErbB3, respectively. In addition, Ser-1120 of ErbB3 in the sequence RSRSRSPRPR was identified by in vitro phosphorylation as a preferred Cdk5 phosphorylation site, suggesting that RSRSRSPRPR is a novel Cdk5 phosphorylation consensus sequence. Transfection studies using COS-7 cells show that these residues are also phosphorylated in vivo. Al-though it has been shown that Cdk5 specifically phosphorylates peptides containing X(S/T)PXK motifs (where X represents a neutral/basic residue) (55,56), the ability of Cdk5 to phosphorylate this novel (SPRXR) consensus sequence motif in ErbB3 raises the possibility that Cdk5 targets other proteins in the nervous system than X(S/T)PX(K/R). In addition, this study suggests that Cdk5 is involved in the regulation of receptor protein-tyrosine kinases affecting downstream signaling transduction pathways in neuronal development and survival (Fig. 8).
Phosphorylation of ErbB receptors is probably essential to their function. ErbB3 has been shown to be well adapted to mediate PI3K signaling, because it contains in its C-terminal phosphorylation domain six such consensus p85 binding motifs (57). ErbB2 has also been found to associate with PI3K (58,59). Thus, the ErbB2/ErbB3 neuregulin co-receptor has been shown to couple to PI3K in a neuregulin-dependent manner.
A unique feature of our analysis is the utilization of the Cdk5 knockout mouse that lacks Cdk5 kinase activity. We demonstrated that brain extracts and/or cortical neurons of Cdk5Ϫ/Ϫ mice exhibit reduced PI3K and Akt kinase activities. The Akt activity was reduced significantly in the Cdk5Ϫ/Ϫ animals; however, PI3K activity was only reduced to 40% of wild type (Figs. 1B and 2D). These observations suggest that Cdk5 may regulate Akt activity independent of PI3K activation. In fact, there are several reports that the Akt activity can be regulated by PI3K-independent pathways; for example, Ca 2ϩ /calmodulindependent protein kinase kinase (68) and reagents that increase cAMP levels (69) as well as stress factors (70) can activate Akt. We suggest that some of these pathways may be compromised to reduce Akt activity in addition to PI3K activity in Cdk5Ϫ/Ϫ mice. In addition, the possibility exists that Cdk5 may regulate PDK1 activity by phosphorylating some of its proline-directed Ser/Thr residues, of which there are six. Two of them, QQTPPKL and VHTPNRT, are Cdk5 consensus motifs, whereas the Ser-244 residue in the KVLSPESK motif is essential for PDK1 activity. PDK1 exists in an active phosphorylated configuration, which can be membrane-localized due to its pleckstrin homology domain by PtdIns(4,5)P 2 . Accordingly, PDK1 does not require receptor activation; the presence of PtdIns(4,5)P 2 in unstimulated cells is sufficient to activate PDK1 and Akt phosphorylation (49).
ErbB3 immunoprecipitates from E18 cortical neurons of Cdk5-deficient mice exhibited a decreased serine and threonine phosphorylation but no significant reduction in tyrosine phosphorylation (Fig. 5G) compared with wild type. A conventional read-out for ErbB activity is usually associated with the levels of tyrosine phosphorylation. The data presented in Fig. 5G suggest that ErbB activity is modulated without affecting the tyrosine phosphorylation of some of the individual receptor subunits. Active ErbB receptors are heterodimers. It is important to note that ErbB3 is a unique in the sense that it has a kinase domain but shows no activity (71,72). ErbB2, which has both kinase domain and activity, is essential for receptor activity. In the present experiments, we used ErbB3 antibody to analyze the phosphorylation state of the receptor. The possibility exists that the ErbB3 tyrosine phosphorylation may have less effect on activation, and ErbB2 tyrosine phosphorylation may play a major role in ErbB2/ErbB3 activation. From these studies, we propose that proline-directed Ser/Thr phosphorylation of receptor in the cytoplasmic domain is required to fully activate the ErbBs. This study also suggests that neuregulindependent activation of Akt-enhanced survival of rat cortical neurons deprived of serum could be inhibited by roscovitine, a specific Cdk5 inhibitor. These results implicate that Cdk5 is involved in neuregulin-dependent activation of the PI3K/Akt neuronal survival pathway by regulating the phosphorylation of ErbB2/ErbB3.
Our results are consistent with studies showing that Cdk5 is involved in the neuregulin-mediated regulation of neuromuscular junction development (13). Cdk5 and its activator, p35, are highly concentrated at the neuromuscular junction, colocalize with acetylcholine receptors on the postsynaptic muscle membrane, and are physically associated with ErbBs, suggesting that the Cdk5-p35 complex is close to ErbB receptors in the signaling pathway. Moreover, neuregulin induced Cdk5 activation in myotubes that correlated with the expression of acetylcholine receptor. Cdk5 has been implicated in synaptic function in other systems, phosphorylating components of the presynaptic complex or the postsynaptic receptor (7,11,12). Our results suggest that the neuregulin/ErbB receptor signal cascade that regulates neuronal survival is another important site for Cdk5 modulation. Our data showed that specific inhibition of Cdk5 activity results in significantly reduced protection from induced apoptosis in cortical neurons by inactivating the neuregulin/ErbB survival pathway (see Fig. 8D).
Apoptosis is an active process occurring during both normal maturation of the nervous system and under stress-induced situations such as neurodegenerative diseases and stroke (60 -62). Activation of the PI3K/Akt pathway has been observed in promoting survival downstream of extracellular stimuli (63)(64)(65). What is most relevant to our observations is the recent evidence of neuregulin-induced Schwann cell survival via the PI3K/Akt pathway. In this system, both the Akt and mitogenactivated protein kinase pathways were activated in parallel downstream from PI3K, resulting in the phosphorylation and inactivation of BAD, a principal inducer of cell apoptosis. However, inactivation of the mitogen-activated protein kinase pathway by selective inhibition of mitogen-activated protein kinase/ extracellular signal-regulated kinase kinase did not affect neuregulin-mediated survival, leaving the Akt pathway as primarily involved. Our data on cortical neurons are in agreement with these observations but introduce Cdk5 as an important modulator of the pathway by virtue of its regulation of ErbB2/3 receptor phosphorylation.
The precise role of Cdk5 in induction and protection from apoptosis remains controversial. For example, in the developing mouse limb, regions of programmed cell death during digit formation correlate with elevated levels of Cdk5-p35, suggest-ing that Cdk5 activity is proapoptotic (14). Similar correlations between Cdk5 activity and apoptosis were reported in tissue remodeling of mammalian reproductive organs (15). Likewise, heat-shocked astrocytoma cells exhibit changes in morphology and increased apoptosis that correlate with Cdk5-p35 activity (16). Finally, in some neurodegenerative disorders, it has been suggested that deregulation of Cdk5 activity by cleavage of p35 to p25 correlates with neuronal pathology in Alzheimer's disease brains (18). On the other hand, a number of studies in addition to the results reported here show that Cdk5 is involved in cell survival. The absence of Cdk5 activity in the Cdk5 knockout mouse causes embryonic-lethal and induces cortical and cerebellar abnormalities and increased cortical apoptosis (24,30). The phenotype is rescued when Cdk5 (driven by a neuron-specific promoter, p35) is overexpressed in a transgenic Cdk5 knockout mouse host, suggesting a key role of neuronal Cdk5 activity in sustaining neuronal survival (29). Furthermore, we have also shown that Cdk5 can modulate neuronal survival by phosphorylating and inhibiting c-Jun Nterminal kinase 3 kinase activity, thereby blocking this apoptotic pathway (30). In this case, we see that Cdk5 supports neuronal survival by inhibiting a key site in an apoptotic pathway, whereas its phosphorylation of the ErbB receptors promotes neuronal survival by activating the PI3K/Akt survival pathway. The data presented in this study are summarized in Fig. 8. The ErbB receptors are activated by Cdk5 phosphorylation in their proline-directed Ser/Thr residues in the C-terminal tail domain; this in turn activates PI3K kinase, which induces activation of PDK1. PDK1 phosphorylates and activates Akt. Active Akt phosphorylates and down-regulates the molecules involved in cell death. The versatility of Cdk5 in development, function, and survival of the nervous system is probably due to a number of different factors, in addition to the wide range of target substrates, from cytoskeletal to synaptic proteins (66,67); its activity is also dependent upon the specific cell type, its regulatory proteins (p35, p39, p25), its localization within different cellular compartments, and the developmental and physiological state of the neuron.