Báo cáo khoa học: A peptide derived from cyclin-dependent kinase activator (p35) specifically inhibits Cdk5 activity and phosphorylation of tau protein in transfected cells pdf

A peptide derived from cyclin-dependent kinase activator p35specifically inhibits Cdk5 activity and phosphorylation of tau protein in transfected cells Ya-li Zheng, Bing-Sheng Li, Niranj

A peptide derived from cyclin-dependent kinase activator (p35)

specifically inhibits Cdk5 activity and phosphorylation of tau protein

in transfected cells

Ya-li Zheng, Bing-Sheng Li, Niranjana D Amin, Wayne Albers and Harish C Pant

Laboratory of Neurochemistry, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, USA

Cyclin-dependent kinase-5 (Cdk5) is a serine/threonine

kinase activated by its neuron-specific activator, p35, or its

truncated form, p25 It has been proposed that the

deregu-lation of Cdk5 activity by association with p25 in human

brain tissue disrupts the neuronal cytoskeleton and may be

involved in neurodegenerative diseases such as Alzheimer’s

disease In this study, we demonstrate that a short peptide

(amino acid residues 154–279; Cdk5 inhibitory peptide;

CIP), derived from p35, specifically inhibits Cdk5 activity

in vitro and in HEK293 cells cotransfected with the

peptide and Cdk5/p25, but had no effect on endogenous

cdc2 kinase activity Moreover, we demonstrate that the

phosphorylation of tau in HEK293 cells, cotransfected with Cdk5/p25 and CIP, is effectively reduced These results suggest that CIP specifically inhibits both Cdk5/p25 complex activity and the tau hyperphosphorylation induced by Cdk5/ p25 The elucidation of the molecular basis of p25 activation and CIP inhibition of Cdk5 activity may provide insight into mechanisms underlying the pathology of Alzheimer’s dis-ease and contribute to therapeutic strategies

Keywords: Cdk5, p35, Cdk5 inhibitory peptide (CIP), Tau phosphorylation, Alzheimer’s disease

Cdk5 is a serine/threonine kinase with close homology to

the mitotic Cdks [1,2] It plays a critical role in brain

development and neuronal migration [3–5] In contrast to

other members of the Cdk family, Cdk5 is activated by

binding the neuron-specific noncyclin molecules, p35 or p39

[6–9] Mice lacking p35 are viable and fertile but show

lamination defects in the cerebral cortex and mild disruption

in the hippocampus and cerebellum [10], whereas mice

deficient in Cdk5 die perinatally and show severe and

widespread defects in neuronal migration [3–5,11] p35/

Cdk5 kinase activity promotes neurite growth and

phos-phorylates a wide variety of substrates [12] Deregulation of

Cdk5 activity by proteolytic conversion of p35 to p25 has

been implicated in neurodegenerative diseases [13,14]

Computer modeling and mutagenesis studies have

pre-dicted that p35 adopts a cyclin-like tertiary structure

[15–17] Although, to produce full activity, in addition to

cyclin binding most members of the Cdk family require

phosphorylation of an intramolecular domain called the

T-loop by another kinase [18] Cdk5 differs in that full

activity can be achieved by binding to p35 in the absence of

T-loop phosphorylation [17,19]

The p35 activation domain was mapped to the region of

amino acid residues 150–291 [16,17] More recently Amin

et al found that residues 138–291 constitute the smallest

fragment (p16) of p35 that fully activates Cdk5 [20] (Fig 1A) That study found that further truncation of p16, removing either the N-terminal 11 residues (part of the p35 aNT helix, Fig 1A) or the C-terminal four residues of p16 (the p35 a7 helix, Fig 1A), produces peptides that bind

to Cdk5 with moderate affinity and do not activate it in vitro, but instead competitively inhibit Remarkably, the peptide that remains after both C- and N-terminal truncations (p35 residues 154–279) has a much higher affinity for Cdk5 This Cdk5 inhibitory peptide (CIP) markedly inhibits the activity

of Cdk5 in vitro [20] The high affinity of CIP suggested that

it might act as a specific Cdk5 inhibitor in a cellular environment as well We explored this possibility by examining the specificity of its inhibition in HEK293 cells transfected with Cdk5/p25 We find that CIP specifically inhibits the activity of Cdk5/p25 but does not affect the activity of cdc2 kinase in transfected HEK293 cells We also observed that CIP reduces the phosphorylation of tau in HEK293 cells cotransfected with tau, CIP and Cdk5/p25 These results indicate that transfection of CIP efficiently and specifically inhibits Cdk5/p25 complex activity and, in p25-transfected cells, reduces tau phosphorylation Finally, we discuss the molecular basis of CIP inhibition of Cdk5/p25 activity in relation to the recently published Cdk5/p25 crystal structure [21]

M A T E R I A L S A N D M E T H O D S

Materials p35 (N-20), p35 (C-19) polyclonal antibody, Cdk5 (C-8) polyclonal antibody, Cdk5 (J-3) monoclonal antibody, cdc2 P34 (H-297) polyclonal antibody, and cdc2 p34 (17) monoclonal antibody were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA) Anti-tau (AT8) Ig

Correspondence to H C Pant, Laboratory of Neurochemistry,

NINDS, NIH, Bldg 36, Rm 4D04, 9000 Rockville Pike,

Bethesda, MD 20892-4130, USA.

Fax: + 1 301 496 1339, Tel.: + 1 301 402 2124,

E-mail: panth@ninds.nih.gov

Abbreviations: Cdk5, cyclin-dependent kinase-5; CIP, Cdk5 inhibitory

peptide; HEK293, human embryonic kidney 293.

(Received 2 May 2002, revised 2 July 2002, accepted 23 July 2002)

was obtained from Innogenetics (Gent, Belgium), PS202

polyclonal antibody (phosphorylated tau epitopes at

Ser202) and TAU-5 monoclonal antibody (reacts with the

nonphosphorylated as well as the phosphorylated forms of

tau) were purchased from Biosource International,

Inc (Camarillo, CA, USA) Other antibodies include

anti-Xpress–HRP and anti-Xpress–FITC (which detect fusion proteins containing the eight amino acid Xpress epitope: Asp-Leu-Tyr-Asp-Asp-Asp-Asp-Lys; Invitrogen, Carlsbad, CA, USA) and anti-T7 Tag monoclonal antibody (Novagen, San Diego, CA, USA) A pcDNA/Amp euk-aryotic expression vector and LipofectAMINE Reagent were purchased from Invitrogen (Carlsbad, CA) pGEM-T vector was obtained from Promega Corporation (Madison,

WI, USA) The Cdk5 inhibitor, roscovitine, was obtained from Biomol Research Laboratories, Inc (Plymouth Meeting, PA)

Plasmids and constructs Construction of CMV expression vectors for Cdk5, p35, and p25 were made according to a procedure described by Nikolic et al [22] A 126-residue peptide (CIP) correspond-ing to peptide fragment Cys154 to Pro279 of p35 was

(5¢-TGCCTGGGTGAGTTTCTC-3¢) and a reverse primer (5¢-TGGGTCGGCATTTATCTG-3¢) derived from p35 (Fig 1A) A CMV-tau fragment (amino acids 181–242) was generated by PCR from a rat brain cDNA library The primer was designed according to the rat tau sequence, as follows: a forward primer (5¢-ACACCACC CAGCTCTGGT-3¢) and a reverse primer (5¢-GCG GCTCTTGGCGGAAGA-3¢) The PCR amplified frag-ments were gel purified with a GenecleanII kit (Bio101, Inc from BCH Medical Supplies Co.) After cutting with Not1 and EcoRI, the fragments were inserted into Not1/EcoRI site of a linearized CMV (pcDNAC3) vector The constructs

of CIP and tau peptides were verified by sequencing Cell culture and transfection

Human embryonic kidney (HEK293) cells were obtained from the American Type Culture Collection, cultured in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum, supplemented with 100 UÆmL)1 penicillin and 100 lgÆmL)1 streptomycin at 37C in a humidified atmosphere of 5% CO2 The cells were transiently transfected using LipofectAMINE (Life Technologies) according to the manufacturer’s instructions The above described constructs of CIP, p25, p35, wild-type Cdk5 and tau were transfected independently or cotransfected Twenty-four hours post-transfection, the cells were starved

in the presence of 0.2% fetal bovine serum overnight (to reduce any background stimulation by serum factors) The cells were fixed for immunocytochemistry analysis, or lysed with lysis buffer for immunoprecipitation and Western blot analysis

Western blot analysis Cells were harvested by scraping from dishes and lysed in ice-cold lysis buffer (20 mM Tris, pH 7.5, 150 mM NaCl,

1 mMEDTA, 1 mMEGTA, 1% Triton X-100, 0.1% SDS, 2.5 mM sodium pyrophosphate, 1 mM 2-glycerol phos-phate, and 1 mMNa3VO4, supplemented with a mixture of protease inhibitors and 1 mMphenylmethanesulfonyl fluo-ride) by passing through a 21 gauge needle several times and incubation for 30 min on ice After centrifugation for

20 min at 13 000 g at 4C, the protein concentrations of

Fig 1 Identification of the Cdk5/p25 inhibitory peptide (CIP) derived

fromp35 (A) Mapping of p25, p16 and CIP to p35 (human sequence).

Red segments are the alpha helices in p25 as determined by Tarricone

et al [21] The sequences comprising p25, p16 and CIP are indicated by

the labelled arrows (B) A combination of N-terminal and C-terminal

truncations of p35 produces a nonactivating fragment (154–279, CIP)

that inhibits Cdk5 activity and binds with high affinity (C) Inhibition

of Cdk5 activity by CIP Cdk5 kinase activity was determined by

preincubating various amount of CIP with Cdk5/p25 for 2 h at 30 C

followed by incubation in the kinase reaction for an additional hour in

the presence of [c- 32 P]ATP and histone H1 as described in the

mate-rials and methods section.

the supernatants were determined using BCA protein

reagent An equal amount of total protein (20 lg of

protein per lane) was resolved on a 4–20%

SDS-polyacryl-amide gel and blotted onto a poly(vinylidene difluoride)

membrane This membrane was blocked by incubating in

blocking buffer containing 20 mM Tris/HCI(pH 7.4),

150 mM NaCI, and 0.1% (v/v) Tween 20 (Tris/NaCl/

Tween) plus 5% dry milk (w/v) for 1 h at room temperature

This was followed by incubation overnight at 4C in

primary antibodies: anti-Cdk5 (C-8, 1 : 200), p35 (N-20 and

C-19, 1 : 200), anti-Xpress–HRP (1 : 3000), anti-cdc2 P34

(H-297 and 17, 1 : 200), anti-PS202, and TAU-5 mAb

(1 : 1000 and 1 : 500, respectively) diluted in blocking

buffer The membranes were then washed in Tris/NaCl/

Tween (4· 5 min) This was followed by incubation in

secondary antibody (goat- anti-mouse or goat anti-(rabbit

IgG H + L)–HRP conjugate at a dilution of 1 : 3000) for 2

h at room temperature and washing four times in Tris/

NaCl/Tween Western blots were analyzed using the

Amersham Enhanced Chemiluminescence (ECL) kit

fol-lowing the manufacturer’s instructions

Immunoprecipitation and kinase assays

Cells were lysed in ice-cold lysis buffer without SDS,

described as above, and immunoprecipitated with

anti-Cdk5 (C-8), anti-cdc2 P34 (17) or anti-Xpress The

immunoprecipitates were washed twice with lysis buffer

and twice with kinase buffer Kinase activity assays were

performed as described previously [23] In brief, a total

volume of 50 lL of kinase assay mixture was used,

containing 50 mM Tris/HCl (pH 7.4) with 1 mM EGTA,

1 mM dithiothreitol, 5 mM MgCI2, 0.5 mM

micro-cystinLR, 10 lg of histone H1, and 10 lL of cdk5 or

Xpress immunoprecipitates The phosphorylation reaction

was initiated by the addition of 0.1 mM [c-32P]ATP and

incubated at 30C for 30 min The reaction was

termin-ated by spotting 25 lL of the reaction mixture on P81

phosphocellulose pads that were washed five times in

75 mM phosphoric acid followed by rinsing with 95%

ethanol The radioactivity was measured in a liquid

scintillation counter SDS/PAGE and autoradiography

assessed the phosphorylated histone H1

Immunocytochemistry

After HEK293 cells were cultured and transfected on

glass coverslips coated with poly L-lysine, cells were

washed twice in NaCl/Pi and fixed for 30 min at room

temperature in 4% paraformaldehyde, NaCl/Pi, and

10 mM EGTA washed and permeabilized (with 25 mM

Tris, pH 7.4, 150 mM NaCI, and 0.2% Triton X-100) for

15 min The coverslips were incubated overnight at 4C

with primary antibodies: polyclonal anti-Cdk5 (C-8,

1 : 50), p35 (N-20 and C-19, 1 : 50), anti-cdc2 P34

(H-297 and 17, 1 : 50) antibodies; anti-tau, AT8

(1 : 500), anti-PS202 (1 : 250) and monoclonal TAU-5

mAb (1 : 100); monoclonal anti-T7.Tag antibody

(1 : 500) and Anti-Xpress–FITC (1 : 500) All antibodies

were diluted in NaCl/Pi with 1% Triton X-100 After a

wash in NaCl/Pi(3· 15 min), the cells or coverslips were

incubated with 1 : 50 fluorescein isothiocyanate

(FITC)-conjugated goat anti-(mouse IgG) and rhodamine-labeled

goat anti-(rabbit IgG) secondary antibody for 1 h at room temperature This was followed by three washes with NaCl/Pi, and then the cells were embedded in aqueous medium Fluorescent images were observed with

a Zeiss LSM-410 laser-scanning confocal microscope Images were processed and merged by AdobePHOTOSHOP software

R E S U L T S

Identification of the Cdk5/p25 inhibitory peptide (CIP) derived from p35

As discussed in the introduction, p35 residues 138–291 (p16) are essential for effective Cdk5 activation and we found that the peptide corresponding to p35 residues 154–279 (CIP) (Fig 1A,B) is a highly effective in vitro inhibitor of Cdk5 [20] A dose–response relationship for CIP on Cdk5 activity

in vitro is shown in Fig 1C Cdk5 kinase activity was determined by incubating various amounts of CIP with Cdk5/p25 for 2 h at 30C followed by incubation in the kinase reaction mixture for an additional hour in the presence of [c-32P]ATP and histone H1 Figure 1C shows that the activity of Cdk5 is markedly inhibited in vitro by less than 1 lMCIP

CIP inhibits the activity of Cdk5 kinase

in transfected HEK293 cells

To explore the inhibitory effects of CIP on the Cdk5/p25 complex activity in vivo, we cotransfected HEK293 cells with CIP and appropriate control expression constructs One set of transfections was carried out with the vector alone (control), a second set was cotransfected with p25 and Cdk5, and a third was cotransfected with p25, Cdk5 and CIP Cell lysates were subjected to Western blot analysis using anti-Cdk5 (C-8) and anti-p35 (C-19) to detect the expression of Cdk5 and p25 proteins, respectively Anti-Xpress–HRP that recognizes specifically constructed plas-mids [24] was employed to detect the expression of CIP (Fig 2A) We found that there is no endogenous p25 or CIP, but endogenous Cdk5 is present in HEK293 cells (Fig 2A, left lane) There were clear bands of expression of transfected Cdk5, p25 (Fig 2A, middle and right lanes), and CIP (Fig 2A, right lane, bottom) These results were confirmed by immunocytochemical analysis in transfected cells (Fig 2B) The antibodies used were anti-Xpress–FITC (CIP), C-19-rhodamine (p35) and C-8-rhodamine (Cdk5) There was clear expression of transfected CIP (Fig 2B, a and b), Cdk5 and p25 (Fig 2B, c and d, respectively) in the transfected cells

To determine whether CIP inhibits the protein phospho-rylation activity of Cdk5/p25 in transfected cells, kinase activity assays were performed on the extracts The activity

of Cdk5/p25 in cells transfected with the Cdk5/p25 constructs (Fig 2C, middle lane) was markedly higher than that in control cells In comparison, the cells transfected with Cdk5/p25 plus CIP was much lower (Fig 2C, com-pared middle and right lanes) Transfection of p25 alone produced a threefold increase in Cdk5 activity compared to control cells (no transfection), and was inhibited by CIP transfection (data not shown) These results were confirmed by kinase activity assays of anti-Xpress Ig

immunoprecipitates of the Cdk5/p25 and Cdk5/p25/CIP

complexes from the same cell lysates (data not shown)

These results demonstrate that CIP can substantially inhibit

Cdk5/p25 activity in transfected cells

P25 phosphorylates tau more effectively than

p35 when Co-transfected with Cdk5

Cdk5 has been implicated, along with other kinases (e.g

MAPK, GSK3, MARK), in the phosphorylation of tau

in transfected cells and mouse models of

neurodegenera-tive diseases [14,25–27] If CIP inhibits the activity of

Cdk5/p25 as suggested by the above data, tau

phospho-rylation by Cdk5 might also be inhibited in

CIP-trans-fected cells To compare CIP inhibitory effects on tau

phosphorylation, we first studied the effect of

Cdk5/p25-induced tau phosphorylation in cotransfected HEK293

cells (Fig 3) We detected tau phosphorylation using

phospho-S202, a phospho-epitope-specific antibody [28]

p35 and p25 were expressed at similar levels in the

transfected cells (Fig 3A), but the tau phosphorylation in

the cells transfected with tau/p25/Cdk5 was markedly

higher than that in cells transfected with tau/p35/Cdk5 (Fig 3B) The occurrence of more extensive phosphory-lation of tau by Cdk5/p25 than by Cdk5/p35 is confirmed

by immunocytochemistry staining with the antitau antibody, AT8 Again significantly increased tau phosphorylation is shown to occur in cells transfected with tau/p25/Cdk5 (Fig 3C, c) compared with cells transfected with tau/Cdk5/p35 (Fig 3C, d) These results agree with previous studies indicating that Cdk5/p25 causes tau hyperphosphorylation, whereas Cdk5/p35 does not effectively phosphorylate tau in vivo [14,29]

CIP inhibits tau phosphorylation in cotransfected HEK293 cells

Experiments by Patrick et al showed by both Western blot analysis and immunohistochemistry that p25 accumulates

in brains of patients with Alzheimer’s disease They also demonstrated that the Cdk5/p25 complex hyperphosphory-lates tau in cultured neurons and is accompanied by cytoskeletal disruption, morphological degeneration and apoptosis [14]

Fig 2 Analysis of Cdk5, p25, and CIP expression, and Cdk5 activity in transfected HEK293 cells HEK293 cells were transiently transfected with the following expression constructs: vector only; p25 with Cdk5; p25, Cdk5, CIP (A) Western blot analysis of Cdk5, p25 and CIP expression Forty-eight hours after cotransfection of p25 and Cdk5 with or without CIP the cell lysates were prepared and subjected to Western blot analysis using (from top to bottom) anti-Cdk5 (C-8), anti-p35 (C-19) (detecting p25) and anti-Xpress (detecting CIP) Ig Equal amounts of protein were used in each case The left lane, control (vector only); the middle lane, Cdk5 and p25; and right lane, Cdk5, p25 and CIP transfected cells (B) Immunocytochemical analysis of CIP, Cdk5, and p25 Confocal micrographs illustrate the cells cotransfected with CIP (a and b), Cdk5 (c), and p25 (d) Cells were fixed and double-stained with anti-Xpress-FITC and polyclonal anti-Cdk5 (C-8) antibodies (a, c, and e) and with anti-Xpress-FITC and polyclonal antip35 (C-19) antibodies (b, d, and f) Images were obtained using a Zeiss LSM 410 laser scanning confocal microscope (C) Analysis of Cdk5 kinase activity using in vitro kinase assays After HEK293 cells were cotransfected with vector alone, p25 and Cdk5 with or without CIP for 48 h, the cell lysates were immunoprecipitated with anti-Cdk5 (C-8) Ig and subjected to a kinase activity assay using histone H1 as a substrate The transfections of expression constructs were the same as shown in Fig 2A The left lane, control (vector only); the middle lane, cotransfection of Cdk5/p25; the right lane, cotransfection of Cdk5/p25/CIP Data represent mean ± SD of three experiments.

To determine whether CIP can inhibit the

hyperphos-phorylation of tau, we cotransfected tau, p25, Cdk5, and

CIP constructs into HEK293 cells in the following four sets:

tau only, tau with Cdk5/p25, tau with p25/Cdk5/CIP, and

tau with p25/Cdk5 treated with roscovitine (10 lm), a Cdk5

inhibitor [30] After 48 h transfection, the cells were lysed

and subjected to Western blot analysis First, we used

anti-Cdk5 (C-8), anti-p35 (C-19), anti-Xpress-HRP, and anti-tau

(TAU-5) Ig to detect the levels of expression of transfected

Cdk5, p25, CIP, and tau, respectively Transfected Cdk5

p25, CIP, and tau are clearly shown at 35, 25, 12.5, and

6.7 kDa., respectively (Fig 4A) That Cdk5/p25 expression

significantly increases tau phosphorylation is evident by

both Western blot (Fig 4B, lane 3) and

immunofluores-cence staining (Fig 4C, b) with anti-(phospho-S202) To

observe whether tau phosphorylation decreases in

trans-fected cells in the presence of CIP, we detected tau

phosphorylation using tau phospho-S202 in Western blots

of these cell lysates Cotransfection of CIP significantly

decreases tau phosphorylation in the cells (Fig 4B, lane 2)

An inhibitor of Cdk5 (roscovitine) also decreases tau

phosphorylation (Fig 4B, lane 4) These results are

con-firmed by immunocytochemical staining with

anti-(phos-pho-S202) The level of phosphorylated tau in the cells

cotransfected with CIP was significantly lower than that in

the cells cotransfected without CIP (Fig 4C, compare a

with b) Thus, CIP can effectively decrease tau phosphory-lation in Cdk5/p25 transfected cells

CIP transfection does not inhibit Cdc2 activity

in HEK293 cells Cdk5 is a member of cdc2-related kinase family [2] and cdc2 kinase is a nuclear protein that plays an essential function in the normal cell cycle progression in eukaryotes To assess the specificity of CIP inhibition, we examined the effect of CIP and roscovitine on cdc2 kinase activation in transfected HEK293 cells (Fig 5) The expressions of transfected CIP and endogenous cdc2 protein in HEK293 cells are showed

by Western blots using anti-Xpress–HRP and anti-cdc2 p34 (17) Ig (Fig 5A, upper and lower, respectively) cdc2 kinase activity assays are performed using the same cell lysates The activities of cdc2 kinase were similar in nontransfected cells (control, Fig 5B, lane 1) and in cells transfected with CIP (Fig 5B, lane 3), but cells treated with roscovitine (10 lM) had distinctly lower activity (Fig 5B, lane 2, compared with other lanes) These results indicate that CIP does not affect cdc2 kinase activation, whereas roscovitine inhibits both Cdk5 and cdc2 activity To evaluate the specifity of anti-cdc2 p34 (17) and anti-Cdk5 (C-8), we immunoprecipitated the cell lysates of nontransfected (vector only) or cotrans-fected with Cdk5/p25 using anti-Cdk5 or anti-cdc2 p34 (17)

Fig 3 Phosphorylation of tau by Cdk5/p25 or Cdk5/p35 in transfected HEK293 cells HEK293 cells were transiently cotransfected with rat tau (181–242) and Cdk5 plus p25 or p35 (A) Analysis of p25 and p35 expression by Western blot using anti-p35 (C-19) Left lane, cotransfected with Cdk5, p35 and tau; Right lane, cotransfected with Cdk5, p25 and tau (B) Total tau and phospho-tau were analyzed by Western blot using anti-tau (bottom) and phospho-tau S202 (top) Ig in aliquots of the same cell lysates Left lane, cotransfected with Cdk5, p35 and tau; right lane, cotransfected with Cdk5, p25 and tau (C) Immunocytochemical analysis of p25, p35, and phospho-tau After HEK293 cells were cotransfected with Cdk5/p25/tau (left column) and Cdk5/p35/tau (right column) for 48 h, they were fixed and double-stained with polyclonal antip35 (C-19) and anti-AT8 Ig (a, c, and e) and with polyclonal antip35 (N-20) and anti-AT8 Ig (b, d, and f) a and b expressed transfected p25 and p35, respectively;

c and d expresses phosphorylated tau Images were obtained using a Zeiss (Thornwood, NY) LSM 410 laser scanning confocal microscope.

antibodies, and then performed the kinase activity assays using histone H1 as a substrate (Fig 5C,D) We found that the activity of Cdk5 was significantly higher than the activity

of cdc2 in cotransfected cells (Fig 5D), while the cdc2 activity was markedly higher than Cdk5 activity in nontransfected cell (Fig 5C) These results suggest that

Fig 4 CIP reduced tau phosphorylation in cotransfected HEK293 cells.

HEK293 cells were transiently cotransfected with rat tau protein (181–

242), Cdk5, and p25 with or without CIP (A) Analysis of Cdk5, p25,

CIP, and tau expression by Western blot using Cdk5 (C-8),

anti-p35 (C-19), anti-Xpress-HRP, and antitau (TAU-5) Ig, respectively.

Lane 1, tau only; lane 2, Cdk5/p25/CIP/tau; lane 3, Cdk5/p25/tau; lane

4, Cdk5/p25/roscovitine There are clear bands at 36, 25, 12.5, and

6.7 kDa of expressed Cdk5, p25, CIP, and tau, respectively (B) The

phosphorylated tau was analyzed by Western blot using

anti-(phos-pho-tau) (S202) Samples were as described for Fig 4A,C

Immuno-cytochemical analysis of total tau and phospho-tau HEK293 cells

were cotransfected with Cdk5, p25 and tau with CIP (left column) or

without CIP (right column) After 48 h the cells were fixed and stained

with polyclonal phospho-tau (S202, a and b) and monoclonal antitau

(total tau, 1 : 100; c and d) antibodies FITC-conjugated (total tau)

goat anti-(mouse IgG) and rhodamine-labeled (phospho-tau) goat

anti-(rabbit IgG) secondary antibodies (Sigma, 1 : 100) were used.

Images were obtained using a Zeiss LSM 410 laser scanning confocal

microscope.

Fig 5 CIP specifically inhibits Cdk5 HEK293 cells were transiently cotransfected with CIP only or treated with roscovitine (10 l M ) (A) CIP and cdc2 expressions were analyzed by Western blot Cells were transfected with or without CIP (vector only) for 48 h, the cell lysates were prepared and subjected to Western blot analysis using anti-cdc2 p34 (17) (lower), anti-Xpress–HRP for CIP identification (upper) antibodies Lane 1, vector only cells; lane 2, roscovitine treated cells; lane 3, transfected with CIP (B) Analysis of cdc2 kinase activity using

in vitro kinase assay After HEK cells were cotransfection with CIP for

48 h, the cell lysates were immunoprecipitated with anti-cdc2 p34 (17)

Ig and subjected to kinase activity assay using histone H1 as a sub-strate Lane 1, nontransfected cells; lane 2, roscovitine treated cells; lane 3, transfected with CIP Data represent mean ± SD of three experiments (C and D) The evaluation of kinase activities using anti-Cdk5 (C-8) and anti-cdc2 (p34, 17) Ig in transfected and nontrans-fected HEK293 cells After HEK cells were cotransfection with P25/ Cdk5 for 48 h, the cell lysates were immunoprecipitated with anti-Cdk5 (C-8) and anti-cdc2 p34 (17) Ig, respectively, and subjected to kinase activity assay using histone H1 as a substrate (C) shows non-transfected cells; (D) shows cells non-transfected with p25/Cdk5.

there is no significant cross-reactivity between the anti-Cdk5

(C-8) and anti-cdc2 p34 (17)

D I S C U S S I O N

In this study we demonstrate that CIP, a peptide derived

from p35 that markedly inhibits the activity of Cdk5/p25

in vitro(Fig 1) [20], also inhibits Cdk5 activity in transfected

cells (Fig 2) We find that tau phosphorylation induced by

Cdk5/p25 transfection of HEK293 cells is greater than that

induced by Cdk5/p35 transfection (Fig 3) and that CIP

cotransfection can substantially decrease tau

phosphoryla-tion in Cdk5/p25 transfected cells (Fig 4) CIP inhibiphosphoryla-tion of

Cdk5 is relatively specific, in that it does not affect the

activity of the closely related cdc2 (Fig 5)

Some insight into the mechanism of CIP inhibition of

Cdk5 activity may be derived from our previous

observa-tions that CIP, comprised of 126 residues [p35(154–279)]

exhibits higher affinity for Cdk5 than do the related

inhibitory peptides, p35(143–279), p35(154–283) or even

the activators, p35, p25, and p16 [20] From the structure of

Cdk5/p25 [21] one can see that the smallest effective

p35-derived activator, p16, encompasses the eight a-helices,

p35(153–280) (Fig 1A) The helices a1–a5 have the tertiary

structure of a cyclin box, even though there is little sequence

homology between p35 and the conventional cyclins

The cyclin box structures of p35 and cyclin A bind to

analogous aspects of their respective kinases, but the

properties of their binding sites are quite different The

cyclin A repositioning of the cdc2 T-loop is largely achieved

through polar and electrostatic interactions including the

Thr160 phosphorylation site In contrast, activation of Cdk5

is postulated to result from a repositioning of its T-loop by

interactions between the T-loop (particularly Ile153 of Cdk5)

and a hydrophobic ridge and pocket formed by the

C-terminal portions of the a3 and a6 helices of p25 [21],

whereas it does not directly involve T-loop phosphorylation

Repositioning of the T-loop in both cases appears to be a

consequence of the rigidity of the cyclin box structure of the

activator compared to the relatively more flexible T-loop

Judging from the crystal structure, the two critical

segments that make p16 an effective activator and, when

removed, transform CIP into a high affinity inhibitor are

also the segments that normally link together the N- and

C-terminal ends of the activating domain of p35 and confer

the required rigidity This suggests an explanation for the

increased affinity of CIP relative to the activating peptides

As noted above, a major interaction site of p25 with the

Cdk5 T-loop includes hydrophobic residues that remain

intact in CIP Thus the increased flexibility of CIP relative to

the p16 structure may allow these residues to conform more

closely to the contours of the Cdk5 interaction site without

inducing the T-loop displacement necessary for kinase

activation

Cdk5 phosphorylates the KSPXK sequence motif in a

large number of proteins including NF-H and NF-M,

neurofilament high and middle molecular-mass proteins [31],

and the tau protein [25] Cdk5 hyperactivity is toxic to

cultured neurons and may underlie the

neurodegenera-tion associated with diseases like Alzheimer’s disease [14] It

has been suggested that conversion of p35 to p25 is

responsible for Cdk5 kinase hyperactivity and the induction

of pathological alterations in neurons in vivo [13,14,29] p25

overexpression decreases the substrate selectivity of Cdk5, causing, tau hyperphosphorylation, and triggering neuritic dystrophy in cultured neurons [14] Tau becomes hyper-phosphorylated in p25-overexpressing transgenic mice that show Alzheimer’s-like lesions [13] In this study, our results confirm previous studies [14,27] that the expression of p25/ Cdk5 induces tau phosphorylation in transfected HEK293 cells, while p35/Cdk5 does not We found that tau phospho-rylation was inhibited in Cdk5/p25 transfected cells in the presence of CIP and was also decreased when transfected cells were exposed to roscovitine, a Cdk5 inhibitor Inhibitors

of Cdk5, such as roscovitine, are nonspecific in that they inhibit the activities of other Cdks with similar efficiency (Fig 5B) Therefore, we compared the effect of CIP with roscovitine on cdc2 kinase activation in CIP transfected HEK293 cells As shown in Fig 5A,B), CIP does not inhibit the activity of cdc2 kinase

In summary, we provide evidence that (a) CIP is a specific inhibitor of Cdk5 kinase; (b) CIP effectively and specifically inhibits the activity of Cdk5/p25 complex in vivo as well as

in vitro; (c) CIP inhibits the phosphorylation of tau induced

by Cdk5/p25 expression in transfected cells CIP, as a specific inhibitor of Cdk5/p25 complex, may prove useful in exploring the role of this kinase complex in the tau pathology of Alzheimer’s disease and other neurodegener-ative disorders, and may contribute to the development of therapeutic strategies

A C K N O W L E D G E M E N T S

The authors thank Drs Philip Grant and Harold Gainer for their excellent suggestions and for critically reading this manuscript.

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