Background
CDK5 is a Serine/Threonine. protein kinase belonging to the CMGC subfamily. CDK5 is the catalytic subunit of an active heterodimeric complex consisting of CDK5 bound to either p35 or p39, two similar CDK5 cofactors encoded for by different genes (
CDK5R1 and
CDK5R2) [
1,
2]. These regulatory subunits have little primary sequence homology to cyclins but possess domains with three-dimensional structures similar to the Cdk-binding motif of cyclins [
3,
4] and are highly selective in their binding of CDK5 [
5]. The levels of p35 and p39 are not regulated through the cell cycle suggesting the function of CDK5 is not related to that of its cyclin binding relatives that are crucial regulators of cell cycle progression.
Mice lacking CDK5 die just before or after birth, with serious defects in neuronal layering of many brain structures [
6‐
8]. p35 null mice have a similar inverted cortical layering observed in the CDK5 null mouse but are viable with normal cerebellum, suggesting variable redundancy in p35 and p39 protein function across the brain [
9‐
11]. The p35 null mice exhibit increased susceptibility to seizures, while the p39 null mice have little apparent deficit, which may suggest that p35 is the more dominant regulator of CDK5 activity. Meanwhile, mice lacking both p35 and p39 have a very similar phenotype to that of the CDK5 null mouse providing evidence that p35 or p39 regulation of CDK5 is required for development of the brain [
12].
As such, CDK5 has predominantly been studied in post-mitotic neurons, the major site of expression of p39 and p35. The main mode of CDK5 regulation in neurons is currently thought to be modulation of the expression or stability of p35 and p39. The proteolytic clipping of these proteins by the calcium regulated protease calpain produces p25 and p29, respectively [
13,
14]. This alters the subcellular localization of the p25/p29 proteins, and the associated CDK5 catalytic subunit, since the N-terminal portion of p35/p39 that is lost contains a membrane localization domain. p25 is reported to be more stable than p35, and p25/CDK5 complexes are reported to contain intrinsically higher activity [
15], which would have obvious implications on CDK5 substrate phosphorylation in diseases with altered p35-p25 ratio. However the relevance of p35 to p25 ratio on steady state CDK5 substrate phosphorylation, and subsequent disease development remains to be fully appreciated.
There is a diverse array of proposed substrates of CDK5, although most have not been validated as true physiological substrates
in vivo or even in intact cells. Most substrates of CDK5 identified to date have key neuronal functions. These include tau [
16,
17], and CRMP2 [
18‐
20], with hyperphosphorylation of these proteins being associated with the generation of neurofibrillary tangles, one of the two hallmarks of Alzheimer’s disease. The phosphorylation of Pctaire 1, spinophilin, axin and neurabin 1 by CDK5 regulates the development of dendritic spines and axons [
21‐
23] while NMDA receptor activity is increased through the phosphorylation of its NR2A subunit by CDK5 [
24], and dopaminergic signalling is controlled by CDK5 through the phosphorylation of dopamine cAMP-regulated phosphoprotein of 32 kDa, DARPP32 [
25]. This substrate profile reflects the neuronal focus of CDK5 research and, combined with the lack of cell cycle regulation of its activity, means that CDK5 has generally not been associated with a key role in cancer initiation, progression or therapy. However, more ubiquitous cell regulatory actions of CDK5 outside of the brain are well described [
26,
27]. In addition there are many lines of evidence linking CDK5 to growth and cancer related actions. These include; i) the phosphorylation of oncogenic proteins such as Rb [
28], ATM [
29], Bcl-2 [
30], p53 [
31], STAT3 [
32], and talin [
33], ii) the observed dysregulation of CDK5 activity in leukaemia [
34] and pancreatic carcinoma cells [
35,
36], iii) a significant correlation between the expression of p35/CDK5 and the degree of differentiation and metastasis in non-small cell lung cancer [
37], as well as increased expression and activity of CDK5 in human hepatocellular carcinoma (HCC) [
38], iv) an association between polymorphisms in the CDK5 promoter and lung cancer risk in a specific Korean population [
39], v) the demonstration that CDK5 activation enhances medullary thyroid carcinoma (MTC) in a conditional mouse model [
40,
41], while inhibition of CDK5 activity reduces tumour growth, motility and metastasis in pancreatic cancer cells [
35] [
42,
43], and ablation/inhibition of CDK5 significantly decreased HCC cell proliferation [
38].
All of the above data suggests abnormal activation of CDK5 increases the risk of, or aggressiveness of, specific forms of cancer. However there are also reports that pharmacological (roscovitine) or siRNA inhibition of CDK5 enhances the proliferation of the breast cancer cell lines MCF-7 and MDA-MB321, while application of carboplatin, a chemotherapeutic used in the treatment of breast cancer, induces CDK5 activation [
44]. Similarly, CDK5 levels decrease in gastric cancer and its nuclear accumulation suppresses gastric tumorigenesis [
45].
Although this indicates a complex relationship between CDK5 activity and growth of different cancer types, the general theme is that tight regulation of CDK5 activity is important for normal cell physiology and that localised or temporal gain (or loss) of function is associated with abnormal cell proliferation. This complex relationship makes it vital to develop the means to accurately assess CDK5 activity in tissue to clarify the potential contribution that this kinase plays in tumourigenesis and whether it presents any novel opportunities for intervention.
The aims of our study were to identify high-confidence substrates as biomarkers of CDK5 activity in tissue and use these surrogate marker(s) of CDK5 activity to establish whether CDK5 activity was altered in human carcinoma.
Methods
Materials
Peptides (Additional file
1: Table S1) were synthesized by Pepceuticals Ltd, Enderby, Leicestershire UK. Active forms of the CMGC protein kinases were purchased from MRC Protein Phosphorylation Reagents, University of Dundee, except for p35/CDK5 and p25/CDK5 (Millipore UK Ltd, Herts, UK).
Antibodies: The pCRMP2 Ser522 and pCRMP4 Ser522 were generated in-house as previously described [
20] and are available from MRC Protein Phosphorylation Reagents, University of Dundee (mrcppureagents.dundee.ac.uk), while the pTau S202 (Cell Signalling, catalog. No.11834), pTau T205 (Invitrogen, catalog. No.44-738G), and pTau S235 (Bioworld, catalog. No.BS4193) antibodies were commercially available.
DNA Constructs: The generation of the expression constructs for human CRMP proteins have been described previously [
20], while human tau expression constructs were obtained from MRC Protein Phosphorylation Reagents, University of Dundee. Expression constructs for CDK5, p35 and p25 were generated by Dr Margereta Nikolic, Imperial College, London.
Cell culture
Embryonic primary cortical neurons were isolated from Sprague–Dawley rats at day 18 gestation. Briefly, following dissection, cortex was digested in 0.25 % trypsin in Hank’s balanced salt solution at 37 °C for 20 min. Cells were manually dissociated by trituration using a fire-polished Pasteur pipette and plated onto 0.01 % poly-l-lysine-coated coverslips at a density of 2–5 × 106 cells per 6 cm well, then incubated at 37 °C with 5 % CO2 in Neurobasal medium (Gibco) containing 2 % (vol/vol) B27 serum replacement (Invitrogen), penicillin (Sigma; 100 units/ml), streptomycin (Sigma; 100 μg/ml), and 1 % (vol/vol) L-glutamine (Sigma). HeLa and tumour cell lines were maintained in DMEM supplemented with 4.5 g/L glucose, 10 % (vol/vol) FCS, 1 % (vol/vol) penicillin (100 units/ml)/streptomycin (100 μg/ml) at 37 °C in 5 % CO2.
Plasmids were introduced into cells using Lipofectamine 2000 (Invitrogen) as per manufacturers instructions. Cells were incubated for 4 h at 37 °C before the transfection medium was removed and replaced with 5 ml growth medium. Cells were then incubated overnight at 37 °C, prior to lysis or fixation as below.
Cell lysis for protein isolation
Cells were lysed in ice-cold lysis buffer (1 % (v/v) Triton X-100, 50 mm Tris–HCl, pH 7.5, 0.27 M sucrose, 1 mM EDTA, 0.1 mM EGTA, 1 mM sodium orthovanadate, 50 mM sodium fluoride, 5 mM sodium pyrophosphate, 0.1 % (vol/vol) β-mercaptoethanol, and Complete protease inhibitor tablet (1 per 10 ml, Roche Applied Science, Basel, Switzerland)). Following centrifugation to remove insoluble material, supernatants were collected, and protein concentrations determined using the Bradford method.
Immunofluorescence
Neurons were fixed in 4 % (w/v) paraformaldehyde in PBS for 10 min at 4 °C, permeabilised with 0.1 % (v/v) Triton X-100 in TBS for 3 min at room temperature, blocked with 1 % (w/v) BSA in TBS containing 0.005 % (v/v) Tween-20 for 1 h at room temperature, and incubated with primary antibodies diluted 1:50 in PBS containing 5 % (w/v) BSA for 1 h at room temperature. Secondary antibodies conjugated to Cy-3 fluorophores were diluted 1:250 in PBS containing 5 % (w/v) BSA and incubated with neurons for 1 h at room temperature. Neurons were counterstained with 0.5 ug/mL DAPI solution (Invitrogen). Image acquisition was performed on a Leica SP-5 laser scanning confocal imaging system using 63× objectives.
Immunohistochemistry
Ethical approval was obtained by review through the Tissue Access Committee of Tayside Tissue Bank (approval # TR338) and the studies follow the Guidelines of the Declaration of Helsinki for the use of human tissues for research. Sections of formalin-fixed, paraffin embedded tissue were cut at a thickness of 4 μm, collected onto Polysine-coated microscope slides (VWR International) and dried overnight at 37 °C. Sections were dewaxed in Histoclear, rinsed in alcohol and endogenous peroxidase was quenched with 0.5 % hydrogen peroxide (100 volumes) in methanol at room temperature for 35 min. After washing in water, antigen retrieval was performed by boiling sections in 10 mM citrate buffer, pH 6.0 for 15 min in a microwave. After cooling, sections were rinsed in PBS and blocked with 5 % normal serum in PBS containing 5 % (v/v) avidin (Vector Laboratories, Peterborough, UK) for 30 min at room temperature. Sections were washed in PBS and incubated with primary antibody in 5 % normal serum in PBS containing 5 % (v/v) biotin at 4 °C overnight. After washing in PBS, sections were then incubated with biotinylated secondary antibody (1:250) (Vector Laboratories) for 30 min at room temperature, followed by streptavidin complexed with biotinylated peroxidase (Vectastain ABC kit; Vector Laboratories) at room temperature for 30 min. The peroxidase complexes were visualized using 0.25 mg/ml diaminobenzidine tetrahydrochloride (DAB) (Sigma) in PBS containing 5 mM imidazole (pH 7.0) and 0.075 % hydrogen peroxide for 10 min at room temperature. Cell nuclei were counterstained with haematoxylin (Sigma), dehydrated through graded alcohols, cleared in HistoClear and mounted in DPX. Images were taken using a Spot Insight QE digital camera or slides were digitally scanned (x40) using an Aperio ScanScope XT.
Cell fractionation
Adherent cells (1–10 × 106 cells) were harvested in 0.05 % (w/v) trypsin-EDTA and pelleted at 500× g for 5 min, washed 2x in PBS before subcellular fractionation which was preformed to the manufacturer’s specifications (Thermo Scientific- Cell fractionation Kit).
Assay of purified protein kinase activity
Specific activity (pmol/min) was determined for all protein kinases by incubating known amounts of kinase (0.01-1 μg) with the generic substrate myelin basic protein (MBP, 0.3 mg/ml final) in kinase buffer (25 mM MOPS pH 7.5, 0.05 % (v/v) Brij-35, 0.25 mM EDTA, 5 % (v/v) glycerol) plus 10 mM MgCl2, and 100 μM [γ-32P] ATP (approx 0.5 × 10
6 CPM/nmol) as previously described [
46]. Peptide kinase assays were performed with 2mUnits of each kinase as above, except MBP was replaced with the peptide at the concentration given in figure legends. One unit of activity of each protein kinase was calculated as 1 nmole of phosphate transferred/min.
Phosphorylation of protein substrates
Recombinant protein substrates were incubated with 2mUnits of each CMGC kinase as for MBP above for the times and at the concentrations given in figure legends. Reactions were terminated by the addition of SDS-PAGE loading buffer and heating to 70 °C for 15 mins. Aliquots were subjected to SDS-PAGE, stained with Coomassie Brilliant Blue (CBR-250), the gels were dried and radiolabeled bands visualized by autoradiography. Quantification of nmoles of phosphate incorporated was obtained by excising the stained protein band from the gel and counting in scintillation fluid.
Western blotting
SDS loading buffer was added to cell lysates and samples subjected to electrophoresis on 4-15 % polyacrylamide gels (Invitrogen) prior to transfer to nitrocellulose using the XCell II blot module (Invitrogen). Blots were blocked in 5 % (w/v) milk in TBST (50 mM Tris HCl pH7.4, 150 mM NaCl, and 0.1 % (v/v) Tween-20) and incubated overnight at 4 °C with the primary antibody diluted in 5 % (w/v) milk in TBST. Blots were washed in TBST and bound antibodies were detected using secondary antibodies linked to a fluorescent conjugate dye. Blots were visualized using a LICOR Odyssey® Infrared Imaging System (LICOR, Lincoln, NE).
Mass Spectroscopy
GST-tau (0.5 μM) was incubated with either p25/CDK5 or p35/CDK5 and MgATP for 5, 20 or 60 mins. Reactions were stopped by addition of 4× SDS-PAGE sample buffer prior to alkylation. GST-tau was isolated by SDS-PAGE, identified by coomassie staining and the destained protein band digested with 0.1 ml 2 g/ml trypsin in 50 mM TEAB overnight. Digests were extracted with 0.1 ml acetonitrile, supernatants dried, dissolved in 0.1 ml 5 % acetonitrile/0.25 % FA and 15 μl of sample from each time point separated on a 150 x 0.075 mm nanoC18 HPLC column prior to analysis on an Orbitrap-velos mass spectrometry system as described previously [
47]. LC-MS data was searched against Uniprot database using Mascot 2.4 and interrogated using Proteome Discoverer 1.4. Quantification of the identified phosphopeptides by generating extracted ion chromatograms was performed using Xcalibur 2.2 software.
Nuclear lysates were isolated as described above, and aliquots alkylated prior to separation by SDS-PAGE and either coomassie staining or western blot (with phospho specific antibodies to CRMP2 to identify CRMP2A). The protein band equivalent to the molecular mass of CRMP2A was excised and the destained protein band digested with 20 μl 12.5 μg/ml trypsin (Roche, Sequencing Grade) in 20 mM ammonium bicarbonate overnight at 30 °C. To each digest 20 μl of 100 % acetonitrile was added and incubated for 15 min then the supernatant removed. 30 μl of 5 % formic acid was then added to each gel piece and incubated for 15 min prior to the addition of an equal volume of 100 % acetonitrile (2.5 % formic acid final concentration). This extract was then removed and pooled with the original extract from the digest. A further 10 μl of 100 % acetonitrile was added to each and incubated for 10 min prior to pooling with the previous 2 extracts. The pooled extracts were then dried down, resuspended in 10 μl of 5 % formic acid then diluted to 1 % prior to injection. 15 μl of sample from each time point was separated on a PepMap RSLC C18, 2 μM column (75 μM × 50 cm nanoViper) (Thermo Scientific) connected to an Ultimate3000 RSLCnano System (Thermo Scientific) coupled to a LTQ Orbitrap Velos Pro (Thermo Scientific) via a EasySpray source Thermo Scientific). Orbitrap Velos Pro .RAW data files analysed with Proteome Discoverer (Ver. 1.4.1) using Mascot (Ver. 2.4.1) as the search engine against the IPI Human Database and sequence of CRMP2A.
Statistical analysis
All statistical analysis was performed using Prism 6.0 software (GraphPad software, CA, USA). Calculation of the mean was used to determine central tendency and standard error of the mean was calculated to quantify the precision of the mean. For comparison of substrate phosphorylation following transfection of p35/CDK5 and p25/CDK5 with untransfected control, statistical analysis was performed by one-way analysis of variance (ANOVA) with Tukey’s post hoc test as comparisons between each group. For comparisons between squamous cell carcinoma and adenocarcinoma, a student’s t-test was performed. A p value of <0.05 was considered significant and p values are expressed in relevant figures using asterisks where * represents <0.05, ** represents <0.001, and *** represents <0.0001.
Conclusions
We demonstrate that an antibody that selectively detects a validated CDK5 phosphorylation site on the substrate CRMP2 robustly stains NSCLC, B-cell lymphoma and to a lesser extent breast carcinoma. Furthermore we show for the first time that it is a specific splice variant of CRMP2 that localises to the nucleus of cancer cells. We propose that CDK5 regulation of CRMP2A could contribute to cancer initiation and progression, and this is supported by recent evidence implicating CDK5 activity in taxol-induced cancer metastasis [
61]. Phosphorylation of CRMP2 by CDK5 is associated with altered function in neurons [
62], however the role of phosphorylation of CRMPs by CDK5 in cancer has not yet been studied. We demonstrate that there are no inherent differences in the activity of p35/CDK5 and p25/CDK5 towards any substrates tested. Whilst the CRMP4 isoform is proposed as a metastasis suppressor in prostate cancer the role of CRMP4 phosphorylation in this action has not been investigated [
63]. However our data questions whether CRMP4 is a substrate for CDK5 in healthy cells, or when we increase CDK5 expression. Therefore we propose the CDK5 upregulation would influence CRMP2 but not CRMP4, and furthermore propose that it is the CRMP2A isoform that is a novel oncogenic target for CDK5. This work provides the opportunity for development of additional tools aimed at this CDK5-CRMP2A axis to combat cancer initiation, progression and metastasis.
Competing interests
The authors’ declare that they have no competing interests.
Author’s contributions
NJG carried out all of the molecular studies, PJC supervised the tumour collection and staining and initial analysis, YLW and FAC performed the quantitative assessment of tumour samples, SEB helped collect, store and prepare the tumour samples, NAM performed the phosphosite mapping, DJL performed the Mass Fingerprinting, CJH generated phosphospecific antibodies and recombinant proteins, while CDS conceived and supervised the project. NJG, PJC and CDS drafted the manuscript, while all authors read, modified and approved the final manuscript.