1

Cyclin I and p35 determine the subcellular distribution of Cdk5 1 2 3

Henning Hagmann1;*, Yoshinori Taniguchi2;*, Jeffrey W. Pippin2, Hans-Michael Kauerz1, 4 Thomas Benzing1;3;4, Stuart J. Shankland2, Paul Thomas Brinkkoetter1 5

6 1 Department of Internal Medicine and Nephrology, Center for Molecular Medicine 7

University of Cologne, Cologne, Germany; 8 2 Division of Nephrology, Department of Medicine, University of Washington, Seattle, USA 9 3 Cologne Excellence Cluster on Cellular Stress Responses in Ageing-Associated Diseases, 10

University of Cologne, Germany 11 4 Systems Biology of Ageing Cologne, University of Cologne, 50931 Cologne, Germany, 12

13 14 To whom correspondence should be addressed: 15 16 Paul Brinkkoetter, 17 Department II of Internal Medicine and Center for Molecular Medicine Cologne, 18 University of Cologne, Germany. 19 Phone: +49-221-478 4480; Fax: +49-221-1423549; 20 Email: paul.brinkkoetter@uk-koeln.de 21 22

23 * These authors contributed equally. 24

25 26 Running title: Subcellular localization of Cdk5 depends on its activators. 27 28 29 Keywords: podocyte; apoptosis; axonal guidance; cyclin-dependent kinase 30 31 32 33 34 35 36 37

Articles in PresS. Am J Physiol Cell Physiol (December 10, 2014). doi:10.1152/ajpcell.00168.2014

Copyright © 2014 by the American Physiological Society.

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Abstract 38

The atypical Cyclin-dependent kinase Cdk5 serves an array of different functions in cell 39

biology. Amongst these are e.g. axonal guidance, regulation of intercellular contacts, cell 40

differentiation and pro-survival signaling. The variance of these functions suggests that Cdk5 41

activation comes to pass in different cellular compartments. The kinase activity, half-life and 42

substrate specificity of Cdk5 largely depend on specific activators, such as p25, p35, p39 and 43

Cyclin I. We hypothesized that the subcellular distribution of Cdk5 activators also determines 44

the localization of the Cdk5 protein and sets the stage for targeted kinase activity within distinct 45

cellular compartments to suit the varying roles of Cdk5. Cdk5 localization was analyzed in 46

murine kidney and brain slices of wildtype and Cyclin I- and/or p35-null mice by 47

immunohistochemistry and in cultured mouse podocytes using immunofluorescence labeling as 48

well as cell fractionation experiments. The predominance of Cyclin I mediates the nuclear 49

localization of Cdk5, whereas the predominance of p35 results in a membranous localization of 50

Cdk5. These findings were further substantiated by overexpression of Cyclin I and p35 with 51

altered targeting characteristics in HEK 293T cells. These studies reveal that the subcellular 52

localization of Cdk5 is determined by its specific activators. This results in the directed Cdk5 53

kinase activity in specific cellular compartments dependent on the activator present and allows 54

Cdk5 to serve multiple independent roles. 55

56

3

Introduction: 57

Unlike other Cyclin-dependent kinases (Cdk), Cyclin-dependent Kinase 5 (Cdk5) is not 58

involved in cell cycle progression but rather serves various functions in cell differentiation (14). 59

Cdk5 is most widely studied in post-mitotic cells, especially neurons. In neurons, Cdk5 controls 60

synaptic activity and plasticity, axonal guidance, migration and cytoskeletal remodeling (9, 20, 61

21, 36, 46). In addition, there is an increasing amount of literature on Cdk5 function in other 62

post-mitotic cells including cardiomyocytes and visceral epithelial cells of the mammalian 63

glomerulus, called podocytes (2, 6, 7, 11, 41). The activity of Cdk5 largely depends on the 64

distinct activators which likely vary in different cell types. For example, in neurons and 65

podocytes, Cdk5 is activated by the regulatory proteins p35. In neurons, Cdk5 is also activated 66

by p39, and the p35-cleavage product p25 (25, 51). These activators determine Cdk5 kinase 67

activity, half life, and subcellular localization, which play pivotal roles in neurodegenerative 68

disease including Amyotrophic Lateral Sclerosis, Alzheimer’s, Huntington’s and Parkinson’s 69

disease (13, 22, 28, 30, 31, 34, 35). 70

Our group identified Cyclin I as the only known cyclin activator of Cdk5, in both neurons and 71

podocytes (6). Interestingly, podocytes and neurons share several biological functions. 72

Podocytes are terminally differentiated cells which elaborate long, regularly spaced foot 73

processes that interdigitate with the processes of neighboring podocytes to form a specialized 74

intercellular junction, the slit diaphragm. At the slit diaphragm transmembranous signaling-75

proteins are orchestrated in cholesterol rich membrane domains to form synapse-like signaling 76

platforms allowing for highly active signal transduction between adjacent podocytes (23, 24, 77

40). This specialized cell-cell contact provides a selective filtration barrier within the 78

glomerulus. Previous studies have shown that mice lacking p35 or Cyclin I show increased 79

susceptibility to experimental glomerulonephritis. Of note, podocytes lack expression of p39 (1, 80

19). Similar to p35-Cdk5 and p39-Cdk5 in Neurons, p35-Cdk5 and Cyclin I-Cdk5 protect 81

4

podocytes from stress-induced apoptosis (6, 29, 49). Interestingly, Cyclin I influences mRNA 82

and protein levels of the pro-survival proteins bcl-2 and bcl-XL, whereas p35 exclusively 83

influences bcl-2 protein levels (6). 84

We hypothesized that this differential regulation may be due to a diverse subcellular localization 85

of Cdk5 depending on the localization of its activator. A number of studies have described the 86

subcellular localization of Cdk5-activators in neurons (4, 25, 36). A recent study shows 87

phosphorylation dependence of p35- and p39-distribution (3). Subcellular localization of the 88

Cyclin I- Cdk5 complex, however, is poorly understood. In this study we performed in vitro and 89

in vivo analysis of the subcellular distribution of Cdk5 in podocytes and cerebellar neurons. We 90

show that the Cdk5-activators p35, p25 and Cyclin I recruit Cdk5 to distinct cellular 91

compartments. p35-mediated targeting of Cdk5 to detergent resistant membrane domains 92

(DRM) is dependent on the myristoylation of p35. We further show that Cyclin I retains Cdk5 in 93

the nucleus rather than actively shuttling the kinase. Finally we suggest a model where the 94

subcellular localization of Cdk5 depends on the presence of its activators p25, p35 or Cyclin I. 95

96

5

Material and Methods: 97

Generation of mutant mice 98

We have previously reported on the generation of the cyclin I-/- mouse (19). The p35 mice were 99

obtained from Inez Vincent (21). Cyclin I-/-; p35-/- double mutant mice were generated by 100

intercrossing these two mouse lines. Mice were housed under standardized pathogen-free 101

conditions in the University of Washington animal facility. The experimental protocol was 102

approved by the Animal Care Committee of the University of Washington, Seattle. 103

Immunohistochemistry 104

Paraffin-embedded tissue of wildtype, Cyclin I -, p35 – or Cyclin I/p35 double knock-out mice 105

was cut to 4µm sections and stained with a Cdk5-specific polyclonal antibody (Cell Signaling). 106

Briefly, sections were deparaffinized in Xylene. After rehydration in graded ethanol and 107

blocking of endogenous peroxidases with 3% hydrogen peroxide sections were incubated 108

overnight at 4°C with primary antibody diluted in 1% BSA/PBS. The sections were washed 109

repeatedly in PBS before incubation with biotinylated anti-rabbit secondary antibody (Jackson 110

Immunoresearch, West Grove,PA) diluted in 1% BSA/PBS for 1 h at room temperature. The 111

ABCkit (Vector, Burlingame, CA) was used for signal amplification and 3,3=-112

diaminobenzamidine (Sigma-Aldrich) was used as a chromogen. Slides were counterstained 113

with hematoxylin (Sigma-Aldrich), dehydrated, and covered with Histomount (National 114

Diagnostics, Atlanta, GA). Images were acquired using a Leica DFC310 FX digital camera on a 115

Leica DMRB microscope operated by Leica Application Suite v4.0 software. Mice were housed 116

according to the standardized specific pathogen–free conditions in the University of Washington 117

animal facility. The Animal Care Committee of the University of Washington reviewed and 118

approved the experimental protocol. 119

Immunofluorescence 120

6

Immortalized podocyte cell lines from Cyclin I and p35 knock-out animals were grown on 121

collagen coated coverslips. Cells were fixed with 2% formaldehyde containing 4% sucrose for 122

10 minutes, then permeabilized with 0.3% Triton-X100 in PBS for 10 min and stained with 123

Cdk5-specific antibody (Cell Signaling, #2506) diluted in 1%BSA/PBS overnight at 4°C. Cells 124

were washed repeatedly with PBS and then incubated with fluorescence-labeled (Cy™3) 125

secondary antibody (Jackson ImmunoResearch, West Grove, PA) and mounted with Vectashield 126

containing 4’,6-Diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA, 127

USA). Images were acquired using a Leica DFC310 FX digital camera on a Leica DMIL 128

microscope operated by Leica Application Suite v4.0 software. 129

Assessment of Cdk5 positive podocyte number and Cdk5 localization 130

Quantification of positively stained cells was performed on individual animals using bright field 131

microscopy. The presences of brown color in the nucleus and/or cytoplasm by bright field 132

microscopy indicated positive staining for Cdk5. The number of cells with Cdk5 in the nucleus 133

only, cytosol only or both nucleus and cytosol were quantified in each glomerulus. Twenty 134

glomeruli were evaluated from each mouse in a blinded manner. The data is expressed as the 135

mean percentage ± SEM of Cdk5 positive cells with staining in nucleus only, cytosol only or 136

nucleus and cytosol. 137

Cell culture and transfection 138

HEK 293T cells were grown under standard conditions at 37°C in 5% CO2 in DMEM 139

(Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (Sigma-Aldrich). For 140

transfection experiments, cells were grown to 60–80% confluence, transfected with plasmid 141

DNA using the calcium phosphate method and grown for 24 hrs. 142

Conditionally immortalized podocytes were generated as previously described.(18, 44) 143

Quiescence and differentiation were induced by culturing the cells at 37°C for 10-12 days on 144

Primaria plastic plates (BD Biosciences) in the absence of IFN-γ. 145

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Cell Fractionation 146

Mouse podocytes were lysed in hypotonic buffer (10mM HEPES; 1.5mM MgCl2; 10mM KCl; 147

0.5mM DTT; 0.05% NP40; pH 7.9), homogenized in a dounce glass/glass homogenizer, and 148

then subjected to sequential centrifugation. Nuclei and mitochondria were segregated from the 149

sample by a 10.000 x g centrifugation step and membrane fractions (ER- and plasma membrane) 150

were collected as pellet after a final ultracentrifugation step at 100.000 x g in a Beckman TLA 151

55 rotor (16, 43). Samples were then analyzed on SDS-PAGE and analyzed by immunoblot. 152

Preparation of Detergent Resistant Membrane Domains (DRM) 153

DRMs were prepared from HEK 293T cells by ultracentrifugation in a sucrose density gradient 154

as described previously (8). Briefly, HEK 293T cells were lysed in buffer containing 1% Triton-155

X 100, loaded on a sucrose gradient from 45% to 5% and spun at 100.000 g for 16 hours in a 156

Beckman SW60Ti rotor. Fractions were separated on SDS-PAGE and analyzed by immunoblot. 157

Western blot analysis 158

Protein levels were measured by Western blot analysis as follows. Cells were washed three 159

times with cold phosphate-buffered saline (PBS) and cells were harvested by scraping on ice. 160

The cells were lysed in lysis buffer (50 mM Tris-HCl (pH 8.0), 5 mM EDTA, 150 nM NaCl, 1% 161

IP-40, 1% Triton X-100, 50 mM NaF, 1 mM Na-orthovanadate (all from Sigma-Aldrich) in the 162

presence of protease inhibitors (Roche, Indianapolis, IN, USA). Protein concentrations were 163

determined by BCA protein assay (Pierce, Rockford, IL, USA). The samples were separated on 164

a 10% sodium dodecyl sulfate-polyacrylamide electrophoresis gel and blotted on a 165

polyvinylidene difluoride membrane (Sigma-Aldrich). Membranes were incubated in 5% non-166

fat milk powder in TBS (10 mM Tris-HCl (pH 8.0), 150 mM NaCl) solution for 3 h at room 167

temperature. Thereafter, the blot was incubated overnight at 4°C with primary antibodies (Cdk5-168

specific antibody, Cell Signaling, #2506) followed by incubation with appropriate horseradish 169

peroxidase-conjugated secondary antibodies (Sigma-Aldrich). Proteins were visualized by 170

8

enhanced chemiluminescence according to the manufacturer’s instructions (GE Healthcare, 171

Piscataway, NJ, USA). In some cases, membranes were incubated in stripping buffer (100 mM 172

glycine, 1% SDS, pH 2.5) and re-stained as stated. Band densitometry was performed using 173

Image J (Wayne Rasband, U. S. National Institutes of Health, Bethesda, Maryland, USA, 174

http://imajej.nih.gov/ij.). Each experiment was performed 3 times and averaged. 175

Statistical analysis 176

All results are expressed as mean ± S.D. calculated with GraphPad Prism version 4.00c for 177

Macintosh (GraphPad Software, San Diego, CA, USA). Analysis of variance (ANOVA) with 178

Tukey-Kramer adjustment for multiple comparisons was applied. A p-value below 0.05 was 179

considered significant. 180

181

182

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Results: 183

The presence of Cyclin I or p35 determines the subcellular localization of Cdk5 in mouse 184

podocytes and Purkinje cells 185

To study the contribution of Cyclin I and p35 to the subcellular localization of Cdk5 in 186

podocytes, kidney sections of non-stressed wildtype mice and Cyclin I null, p35 null and p35 / 187

Cyclin I-double null mice were stained with a Cdk5 specific primary antibody (Fig. 1). Cdk5 188

immunoreactivity within podocytes was equally distributed in wildtype mice to the nucleus and 189

perinuclear regions (Fig. 1A+B). In sections from normal Cyclin I-null kidneys, there was a 5-190

fold increase in Cdk5 staining in the perinuclear region (49.4±13.4% Cyclin I-null vs. 9.5±8.1% 191

wildtype), whereas the intensity of nuclei staining was substantially reduced (Fig. 1 C+D). In 192

contrast, in p35 null mice kidneys, Cdk5 staining was increased approximately 5-fold in the 193

nucleus compared to the cytosol (60.4±15.1% p35 null vs 11.9±10.0% wildtype) (Fig. 1 E+F). 194

In mice lacking both Cyclin I and p35, the staining distribution of Cdk5 was evenly distributed 195

in both nuclei and cytosol, indistinguishable from wildtype mice (Fig. 1G+H). 196

To further validate this differential subcellular localization of Cdk5 in another cell type, 197

additional experiments were performed by staining mouse cerebellar sections of wildtype 198

animals and Cyclin I null, p35 null and p35 / Cyclin I-double null animals. Similar to podocytes, 199

in wildtype mice there was equal distribution of Cdk5 staining in the nucleus and cytosol (Fig. 1 200

I+J). In Cyclin I null animals nuclear staining was substantially reduced (Fig. 1 K+L). 201

Moreover, Cdk5 immunoreactivity was not only localized to the cytosol, but there was a clear 202

orientation towards the plasma membrane (Fig. 1 L). Similar to podocytes, p35 null brains had 203

predominant staining of Cdk5 in the nucleus (Fig. 1 M+N), whereas Cdk5-specific staining of 204

cerebellar slices of p35 / Cyclin I-double null animals resembled the wildtype situation (Fig. 1 205

O+P). Taken together, Cdk5 staining shifted from a nuclear and cytosolic pattern to a cytosolic 206

distribution in the absence of Cyclin I, and was predominantly in a nuclear distribution in the 207

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absence of p35. 208

Cyclin I and p35 specifically affect the subcellular localization of Cdk5 209

To substantiate the results showing Cyclin I- and p35- directed localization of Cdk5 in vivo, cell 210

culture studies were performed in immortalized podocyte cell lines derived from Cyclin I and 211

p35 null animals as previously reported (33). Immunofluorescence stainings for Cdk5 in these 212

cell lines recapitulated the findings from the immunohistochemical staining of in vivo mouse 213

tissue (Fig. 2 A – upper panels). In wildtype cells a balanced distribution of Cdk5 in the nucleus 214

and in the cytosol was found. In the absence of Cyclin I, Cdk5 accumulates in the cytosol and at 215

the plasma membrane, whereas in the absence of p35 all Cdk5 immunoreactivity is traced to the 216

nucleus. In cells lacking both Cyclin I and p35, the Cdk5 distribution resembles the wildtype 217

situation. 218

To demonstrate the specificity of the knock-out effects, we generated rescue cell lines of 219

Cyclin I null and p35 null podocytes, by retroviral re-expression of myc-tagged Cyclin I or V5-220

tagged p35, respectively. Immunofluorescence staining for Cdk5 was performed to analyze 221

subcellular distribution of the kinase in transduced null cells. The re-expression of Cyclin I.myc 222

in Cyclin-I deficient podocytes reversed the translocation of Cdk5 from the nucleus to the 223

cytoplasm, similar to that seen in wildtype cells (Fig. 2 A – middle panels). In podocytes lacking 224

p35, the almost exclusive nuclear signal of Cdk5 was redistributed to a nuclear and cytosolic 225

staining when p35.V5 was re-expressed (Fig. 2 A – right panels). 226

The cytosolic and nuclear localization of Cdk5 was next analyzed in cell fractionation 227

experiments. Hypotonic lysates of wildtype cells, Cyclin I and p35 null cells, as well as null 228

cells re-expressing Cyclin I or p35 were subjected to sequential centrifugation to segregate 229

nuclear and cytosolic fractions. These fractions were separated by SDS-PAGE, and analyzed by 230

Immunoblotting with a Cdk5-specific antibody. Α-Tubulin and TATA-box binding protein 231

served as controls for the purity of the preparation. In wildtype cells there was an equal protein 232

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distribution of Cdk5 in the cytosolic and nuclear fractions (Fig. 2 B). In Cyclin I null cells the 233

nuclear fraction of Cdk5 protein was reduced to about 25% of the total protein compared to 234

wildtype. Re-expression of Cyclin I.myc partially restored the distribution similar to that of 235

wildtype cells. In p35 null cells Cdk5 protein was predominantly localized to the nucleus (80% 236

nucleus/ 20% cytosol) (Fig. 2 C). However, when p35.V5 was re-expressed, the abundance of 237

Cdk5 protein was higher in the cytosol. 238

Taken together, these results show that in cultured podocytes, the subcellular localization of 239

cdk5 protein is governed by Cyclin I to a nuclear location, and by p35 to a cytosolic location, 240

which is consistent with the in vivo findings. 241

The myristoylation anchor of p35 mediates recruitment of Cdk5 into detergent resistant 242

membrane domains (DRM) 243

At the neuronal synapse, as well as at the podocyte slit-diaphragm, proteins and lipids are 244

orchestrated to form distinct signaling platforms. The high abundance of cholesterol in these 245

membrane domains is a requirement for efficient signal transduction, and makes them resistant 246

to detergents (10, 15, 32, 45, 54). Since p35-Cdk5 was recently shown to target signaling 247

receptors like ErbB4 and Dopamine D2 receptors which localize into DRMs, it was tempting to 248

ask whether p35 could recruit Cdk5 into these specialized membrane domains (26, 39). Since 249

endogenous levels of Cdk5 are not sufficient in podocytes to provide a signal in DRM-250

fractionation, HEK 293T cells were transiently transfected with FLAG-tagged Cdk5, together 251

with either p35.myc, p25.myc or Cyclin I.myc. Following cell lysis in a buffer containing 1% 252

Triton-X 100, lysates were subjected to density gradient ultracentrifugation on a sucrose 253

gradient. This allowed for purification of cholesterol rich membrane fractions. After 254

centrifugation 7 fractions were collected in each sample and separated on SDS-PAGE. 255

Immunoblotting with antibody specific for the myc-tag revealed that p35 localizes into 256

cholesterol rich membrane domains. p25 and Cyclin I, however, could not be detected in DRMs 257

12

(Fig. 3 A-C). Immunoblotting with anti-FLAG-antibody showed recruitment of Cdk5 into 258

DRMs only in the presence of p35, whereas Cdk5 was excluded from these membrane 259

compartments in the presence of p25 and Cyclin I. 260

Since p25 is a cleavage product of p35, lacking the N-terminal 97 amino acids which contain the 261

myristoylation anchor of p35, it was crucial to determine whether the functional myristoylation 262

of p35 was critical for Cdk5 recruitment into DRMs. As in most myristoylated proteins, in p35 263

the myristoylation occurs at the glycine residue at position 2. A prerequisite for myristoylation is 264

removal of the N-terminal methionine from the protein (5). This can be blocked by the addition 265

of a single amino acid N-terminally of the initial methionine. To this end a multi-cistronic 266

expression construct was generated containing the FLAG-tagged Cdk5 sequence, and the 267

p35.V5 sequence separated by a sequence encoding the viral 2A-peptide. The 2A-peptide 268

mediates co-translational cleavage to yield discrete protein products from a single open reading 269

frame (48). The specific amino acid sequence inhibits formation of a regular peptide bond 270

between a glycine and a proline residue, which results in ribosome skipping to the next codon. 271

The nascent peptide is therefore cleaved between a glycin and proline residue, leaving the 272

second peptide/protein with an N-terminal proline. 273

To test if myristoylation of p35 is a requirement for recruitment of p35-Cdk5 into DRMs, 274

Cdk5.FLAG.2A.p35.myc was transiently expressed in HEK 293T cells and lysates subjected to 275

ultracentrifugation on a sucrose gradient. In striking contrast to the earlier observation that Cdk5 276

is recruited to DRM fractions in the presence of p35, no Cdk5.FLAG was detected in DRM 277

fractions of cells expressing proline.p35 (Pro.p35) (Fig. 3 D). Together, these results show that 278

myristoylation of p35 is required for co-fractionation of Cdk5 with DRMs. Cyclin I and p25, in 279

contrast, fall short in recruitment of Cdk5 into DRMs. 280

Endogenous Cdk5 can be relocated to the plasma membrane by membrane targeted Cyclin I 281

The next aim was to investigate if endogenous Cdk5 could be artificially relocated in podocytes 282

13

lacking any of the known activators of Cdk5. To this end, a retroviral expression construct 283

coding for Cyclin I with an N-terminal CD16.7- and myc-tag (CD16.7.myc.Cyclin I) was 284

generated. Fusion of Cyclin I to the N-terminal CD16.7.-tag yields a chimeric integral 285

membrane protein (52). We hypothesized that membrane bound Cyclin I would also recruit 286

Cdk5 to the cell membrane. 287

To test this, CD16.7.myc.Cyclin I or control protein was stably re-expressed in Cyclin I / p35 288

null podocytes followed by cell fractionation experiments. Lysates of both cell lines were 289

subjected to sequential centrifugation steps to purify nuclear, membranous and cytosolic 290

fractions. The fractions were then separated on an SDS-PAGE and analyzed by immunoblot 291

with a Cdk5 specific antibody. In the absence of both p35 and Cyclin I only a faint signal for 292

Cdk5 was found in the membrane fraction (Fig. 4 A – left panel). When the membrane targeted 293

CD16.7.myc.Cyclin I was expressed, however, significantly more Cdk5 could be detected in the 294

membranous fraction (Cdk5 membrane/cytosol ratio: Control - mean 0.937; 295

CD16.7.myc.Cyclin I - 2.421; p= 0.001) (Fig. 4 A - right panel + quantification in B). Cdk5-296

specific immunofluorescence staining on CD16.7.myc.Cyclin I expressing Cyclin I / p35 null 297

podocytes and mock transfected control cells shows enrichment of Cdk5 at the plasma 298

membrane of CD16.7.myc.Cyclin I expressing cells and a cytoplasmic signal in control cell 299

(Fig. 4 C). This suggests that localization of Cdk5 depends on the subcellular localization of 300

Cyclin I more so than on its function. Expression levels of CD16.7.myc.Cyclin I in comparison 301

to endogenous Cyclin I are shown in Fig. 4 D. 302

Cyclin I does not affect nuclear import of Cdk5 303

Knowing that Cyclin I may recruit Cdk5 according to its own subcellular localization which is 304

typically localized to the nucleus, we addressed whether Cyclin I was involved in active nuclear 305

shuttling of Cdk5. To this end wildtype and Cyclin I null podocytes were treated with the 306

nuclear export inhibitor leptomycin B (LMB) and Cdk5 localization was analyzed by 307

14

immunofluorescence at different time points. In both wildtype and Cyclin I null podocytes 308

LMB-treatment of 4 hours resulted in a strong nuclear signal for Cdk5 (Fig. 5). This suggests 309

that nuclear import of Cdk5 is not affected in Cyclin I null cells. It is therefore unlikely that 310

Cyclin I serves as a shuttle for Cdk5 but rather retains Cdk5 in the nucleus by direct binding. 311

312

Discussion 313

The function and effect of the atypical cyclin-dependent kinase Cdk5 depends on its subcellular 314

localization. Nuclear Cdk5 activity was shown to play a role in the transcriptional regulation of 315

pro-survival genes (6), whereas perinuclear Cdk5 activity is associated with Tau-316

phosphorylation and neurodegenerative disease (37, 50, 55). Previous studies suggest that it is 317

the subcellular localization of the Cdk5-activators that determines the cellular distribution of 318

Cdk5. Asada et al. have shown that myristoylation and phosphorylation of p35 and p39 is 319

required for membrane association of these Cdk5-activators (3, 4). p35-Cdk5 was shown to be 320

protective in states of cellular stress (6, 12). Calpain-mediated cleavage of p35 to p25 in contrast 321

leads to perinuclear accumulation of p25 and degenerative phenotypes (17, 38, 47). Most studies 322

exploring subcellular localization of the Cdk5-activator complexes, however, have focused on 323

assessing the subcellular localization of Cdk5-activators assuming that the Cdk5 protein is 324

recruited to the respective localization by direct binding. The influence of the distinct activators 325

p35 and its cleavage product p25 in the presence of Cyclin I on the subcellular localization of 326

Cdk5 was the subject of this study. 327

The results from the experiments in mouse kidney and brain tissue as well as in cell culture 328

show that the subcellular localization of Cdk5 is determined by the allocation of either Cyclin I 329

or p35. In cells lacking p35, Cdk5 protein is predominantly localizing to the nucleus, whereas in 330

cells lacking Cyclin I, Cdk5 is predominantly found in the cytosol or even in the membrane. The 331

membrane localization of p35-Cdk5 has been described before (3, 4, 38). Besides confirming the 332

15

results of p35-mediated membrane association of Cdk5, this study adds additional knowledge on 333

the distribution of p35-Cdk5 in cholesterol rich and detergent-resistant membrane domains. 334

Those DRMs form the scaffolding for numerous signaling molecules. The myristoylation anchor 335

of p35 is required for co-fractionation of p35 and recruitment of Cdk5 into these signaling hubs. 336

It is well conceivable that from its membranous localization Cdk5 may target and influence 337

function and fate of signaling proteins such as cell surface receptors and ion channels. 338

If neither Cyclin I nor p35 are present (Cyclin I/p35-double null) Cdk5 takes on a subcellular 339

distribution as seen in wildtype cells. Notably, in Purkinje cells of Cyclin I/p35-double null mice 340

a faint signal for Cdk5 is seen in the plasma membrane. Unlike podocytes, Purkinje cells also 341

express the Cdk5 activator p39 which may target Cdk5 to the membrane in these cells (3, 4). 342

However, there is at least one other Cdk5 activator expressed in neurons called p67 for which 343

the subcellular localization remains elusive (53). 344

In additional experiments the mode of Cdk5 recruitment was addressed. In mouse podocytes 345

lacking any of the known Cdk5 activators Cdk5 could be relocated to the plasma membrane by 346

artificially membrane-targeted Cyclin I. 347

This study did not investigate additional docking proteins that may affect localization of Cdk5 or 348

its activators. However, p35 and Cyclin I bind Cdk5 directly (6, 27, 42). It is well conceivable 349

that p35, carrying the myristoylation anchor directly mediates Cdk5 localization to the plasma 350

membrane. The nuclear localization of Cdk5 in the presence of Cyclin I might be regulated by 351

proteins of the nuclear import machinery and by additional docking proteins within the nucleus. 352

In cells lacking Cyclin I, which show predominantly cytoplasmic localization of Cdk5, the 353

nuclear predominance of Cdk5 could be reconstituted after treatment with the inhibitor for 354

nuclear export leptomycin B. This means that nuclear import of Cdk5 is independent of Cyclin I. 355

These results suggest that Cdk5 activators play a passive role in recruitment of Cdk5 to the 356

specific subcellular location. The Cdk5 activators do not shuttle the kinase but rather retain the 357

16

Cdk5 protein at the specific subcellular locus of the activator (Fig. 6). These differences in 358

subcellular distribution of the Cdk5-activator complexes could at least partly explain the diverse 359

substrate specificity of Cdk5 dependent on its activators. Further studies will establish, how the 360

activity and the substrate specificity of Cdk5 differ in the different cellular compartments. 361

362

17

Acknowledgments: 363

We thank Angelika Köser for technical support and Petra Kleinwächter for graphical artwork. 364

365

Funding: 366

This study was funded by the Marga und Walter Boll-Stiftung to H.H. (HH 210-05-11), by FAU 367

Emerging Fields Initiative to S.S and by the Deutsche Forschungsgemeinschaft (BR2955/4-1) 368

and Köln Fortune Program of the University of Cologne to P.T.B.. T.B. received funding from 369

the Deutsche Forschungsgemeinschaft (SFB572, SFB635, and SFB829). 370

371

Disclosures: 372

The authors report no conflicts of interest. 373

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55. Zhang J, Cicero SA, Wang L, Romito-DiGiacomo RR, Yang Y, Herrup K. Nuclear 526 localization of Cdk5 is a key determinant in the postmitotic state of neurons. Proceedings 527 of the National Academy of Sciences 105: 8772–8777, 2008. 528

529

530

531

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Figure 1: The subcellular localization is altered in Cyclin I- and p35-null mice. 532

Immunohistochemical staining of kidney sections of wildtype mice and Cyclin I-null, p35-null, 533

and Cyclin I/ p35-double null mice with a Cdk5 specific primary antibody. In podocytes of 534

wildtype mice Cdk5 localizes to the nucleus and to the perinuclear regions (A/B). In Cyclin I-535

null mice significantly more Cdk5 is detectable in the perinuclear region (C/D). In p35-null mice 536

the immunoreactivity of Cdk5 is predominantly found in the nucleus (E/F). Knock out of both 537

Cyclin I and p35 restitutes the staining distribution of Cdk5 in nuclei and perinuclear region as it 538

is found in wildtype mice (G/H). Quantification data is provided below the panel. 539

540

Immunohistochemical analysis of cerebellar sections using Cdk5-specific antibody. In wildtype 541

sections Cdk5 staining is detected in the nuclei and in the perinuclear region of Purkinje cells 542

(I/J). In Cyclin I-null mice nuclear staining intensity of Cdk5 is lost and a stronger signal 543

originates from the perinuclear region (K/L). Knock out of p35 leads to a predominantly nuclear 544

signal of Cdk5 in neurons (M/N). Purkinje cells of p35/Cyclin I- double null mice show a 545

subcellular distribution of Cdk5 similar to wildtype (O/P). 546

547

548

Figure 2: Subcellular localization of Cdk5 in cultured mouse podocytes 549

Immunofluorescence staining with Cdk5 specific antibody of cultured mouse podocytes from 550

wildtype, Cyclin I-null, and p35-null mice show the same alterations in Cdk5-distribution as in 551

vivo (A, top panel). In Cyclin I-null podocytes Cdk5 localizes predominantly perinuclear and in 552

the plasma membrane, in p35-null cells Cdk5 is predominantly nuclear. Re-expression of either 553

Cyclin I.myc or p35.V5 restores the distribution to nucleus and cytoplasm as in wildtype cells 554

(A, lower panel). 555

24

Immunoblot analysis of cytosolic and nuclear fractions of cultured mouse podocytes from 556

wildtype and Cyclin I-null shows markedly reduced levels of Cdk5 in the nuclear fraction of 557

Cyclin I-null cells. This effect is partially resolved by re-expression of Cyclin I.myc (B). Cell 558

fractionation of p35-null podocytes show higher nuclear levels of Cdk5. Re-expression of 559

p35.V5 in p35-null podocytes redistributes Cdk5 to the cytosolic fraction (C). α-Tubulin and 560

Tata-box-binding protein served as control for the purity of the preparation. The experiments 561

were performed three times (n=3). 562

563

564

Figure 3: p35 recruits Cdk5 into detergent resistant membrane domains (DRMs) 565

DRM preparation and immunoblot of transiently transfected HEK 293T detects Cdk5.FLAG in 566

DRM fractions in the presence of p35.myc (A). In the presence of p25.myc or Cyclin I.myc 567

Cdk5.FLAG is only present in non-DRM fractions (B+C). The addition of an N-terminal proline 568

residue to p35 leads to the loss of Cdk5 from DRMs (D). Immunoblotting for Transferrin-569

Receptor (TFR) and Flotillin served as controls for non-DRM and DRM-fractions respectively. 570

571

Figure 4: Membrane targeted Cyclin I relocates Cdk5 to the plasma membrane 572

Cyclin I/p35-double null podocytes re-expressing membrane targeted Cyclin I 573

(CD16.7.myc.Cyclin I) show enhanced Cdk5-specific immunoreactivity in the membranous 574

fraction as compared to empty vector transfected cells (A). Transferrin-Receptor (TFR) and 575

Flotillin served as controls for the purity of the preparation. Quantification of the Cdk5 576

membrane/cytosol ratio shows significantly more membranous Cdk5 in the presence of 577

CD16.7.myc.Cyclin I (Control - mean 0.937; CD16.7.myc.Cyclin I - mean 2.421; p= 0.001) (B). 578

Immunofluorescence staining for Cdk5 reveals plasma membrane localization of Cdk5 in 579

CD16.7.myc.Cyclin I expressing cells, whereas Cdk5-staining is cytosolic and perinuclear in 580

25

control transfected cells (C). Immunoblot analysis with Cyclin I specific antibody of wildtype 581

cells expressing endogenous levels of Cyclin I and p35/Cyclin I null after re-expression of 582

CD16.7.myc.Cyclin I (D). 583

584

Figure 5: Leptomycin B antagonizes the effect of Cyclin I deficiency on Cdk5 localization. 585

Leptomycin B (LMB) treatment in wildtype cells does not affect the distribution of Cdk5 (top 586

panel). In Cyclin I-null cells where Cdk5 localizes in the cytoplasm predominantly LMB 587

treatment leads to a mostly nuclear distribution after 4 hours of treatment (lower panel). 588

589

Figure 6: The activators of Cdk5 determine subcellular localization of the kinase. 590

This schematic model depicts the effect of different activators on the subcellular distribution of 591

Cdk5. In the presence of both p35 and Cyclin I (CCNI) as in wildtype cells, Cdk5 is distributed 592

in the nucleus, in the cytoplasm, and at the plasma membrane (A). The lack of p35 and 593

subsequently p25 leads to a predominantly nuclear localization mediated by Cyclin I (B). Loss 594

of Cyclin I and predominance of p35 and p25 results in a membranous and cytosolic distribution 595

of Cdk5 (C). Deletion of both Cyclin I and p35 results in undirected distribution of Cdk5 similar 596

to the wildtype situation (D). 597