Báo cáo Y học: ik3-1/Cables is a substrate for cyclin-dependent kinase 3 (cdk 3) pdf

Here, we report that Ser274 of p70ik3-1is phosphorylated by cdk2 or cdk3 bound to cyclin A and to cyclin E in vitro.. We also found that in COS7 cells in which cyclin E and cdk3 were ect

ik3-1/Cables is a substrate for cyclin-dependent kinase 3 (cdk 3)

Tadanori Yamochi1, Kentaro Semba2, Keitaro Tsuji1,3, Kiyohisa Mizumoto3, Hiroko Sato1,

Yoshiharu Matsuura4, Ikuo Nishimoto1and Masaaki Matsuoka1

1 Department of Pharmacology, KEIO University School of Medicine, Tokyo, Japan;2Department of Cellular and Molecular Biology, The Institute of Medical Science, University of Tokyo, Japan;3Department of Biochemistry, School of Pharmaceutical Sciences, Kitasato University, Tokyo, Japan;4Research Center for Emerging Infectious Diseases, Research Institute for Microbial Diseases, Osaka University, Japan

p70ik3-1(a 70-kDa protein) contains a cyclin box, and binds

to p35cdk3in vivo and in vitro [Matsuoka, M., Matsuura, Y.,

Semba, K & Nishimoto, I (2000) Biochem Biophys Res

Commun 273, 442 – 447] In spite of its structural similarity

to cyclins, p70ik3-1 does not activate cyclin-dependent

kinase 3 (cdk3)-mediated phosphorylation of pRb, histone

H1, or the C-terminal domain of RNA polymerase II Here,

we report that Ser274 of p70ik3-1is phosphorylated by cdk2

or cdk3 bound to cyclin A and to cyclin E in vitro We also found that in COS7 cells in which cyclin E and cdk3 were ectopically overexpressed, the phosphorylation level of Ser274 in coexpressed p70ik3-1is upregulated We therefore conclude that p70ik3-1 is a substrate for cdk3-mediated phosphorylation

Keywords: cdk3; ik3-1; phosphorylation

Mammalian G1 phase progression is regulated by G1 cyclin

and cyclin-dependent kinases (cdks) Cdk4 or cdk6 is

associated with D-type cyclins while cdk2 binds to cyclin E

or cyclin A to become an independent and essential kinase

[1,2] Cdk3 is another putative G1 cdk, whose cyclin

partners have not been identified [3] In vitro, cdk3 is an

active kinase in association with either cyclin E or cyclin A

[4,5] In eukaryotes, overexpression of a dominant-negative

cdk3 induces G1 arrest, which is not rescued by

upregu-lation of wild-type cdk2, suggesting that the function of

cdk3 is distinct from that of cdk2 and independently

essen-tial for the mammalian G1 – S transition [6] Cdk3

partici-pates in the G1 – S progression at least partially by binding to

E2F-1, E2F-2, or E2F-3 through DP-1 and by enhancing

their transcriptional activities [7]

To further understand the role of cdk3 in mammalian

G1 – S transition, we searched for new molecules interacting

with p35cdk3and cloned ik3-1 (designated ik3-1 from an

interaction with cdk3) [8] p70ik3-1seems to belong to the

cyclin family, as its C-terminal domain, composed of 124

amino acids, resembles the highly conserved cyclin box

p70ik3-1also binds to p35cdk3in vivo and in vitro The ik3-1

gene may belong to a multigene family and is highly

conserved during evolution The expression pattern of ik3-1

suggests that it may work mainly in the G1 phase [8]

Parrallel with our findings, ik3-1 was also cloned

independently by Zukerberg et al [9] as a putative adaptor

molecule connecting cdk5, a neuron-specific kinase, with

c-abl in neuronal cells, and hence named Cables by this

group Cables enhances neurite growth in association with cdk5 and c-abl It should be noted that while cdk5 activity is detected restrictedly in postmitotic neurons [10], ik3-1 (Cables) is nevertheless expressed ubiquitouly [8,9] It is thus tempting to investigate whether and how ik3-1 functions in non-neuronal cells

In search of the functional relationship between ik3-1 and cdk3 in self-replicating cells, we examined how ik3-1 could modify cdk3 activity and whether ik3-1 could be a substrate for cdk3-mediated phosphorylation Here, we report that ik3-1 is a novel substrate for cdk3/cyclin E and for cdk3/ cyclin A

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

Cell culture and transfection Transient transfection to COS7 cells was performed with lipofectAMINE PLUSTMreagents according to the manu-facturer’s instructions (GibcoBRL) COS7 cells (80 – 100% confluency) in 100-mm dishes were incubated for 3 h with precomplexed DNAs and the lipofectAMINE PLUSTM reagents Unless specified, 7 mg of each DNA, 20 – 30 mL of PLUS, and 15 – 25 mL of lipofectAMINE reagents were used for each dish At 40 – 48 h after the start of transfection, cells were harvested

Plasmids and point mutations pCMV – cdk3, pCMV – cdk3dn (dominant-negative cdk3), pCMV – cdk2, pCMV – cdk2dn (dominant-negative cdk2), pCMV–cyclin E, pCMV–cyclin A, and the backbone vector (pCMV–neo-Bam) were as described previously [6–8] pMF–ik3-1 and pMF–ik3-1DN were as described previously [8] ik3-1DN is the ik3-1 partial cDNA in which the N-terminal 139 amino-acid region of ik3-1 is deleted The ik3-1 cDNA and the ik3-1DN cDNA were inserted into pGEX (Pharmacia, UK) vectors for expression of GST-tagged proteins in bacteria (GST – ik3-1 and GST – ik3-1DN) A His-tagged p27Kip1plasmid, pET21a (1)mp27

Correspondence to M Matsuoka: Department of Pharmacology, KEIO

University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo

160-8582 Japan, Fax: 1 81 3 3359 8889, Tel.: 1 81 3 5363 3751,

E-mail: sakimatu@mc.med.keio.ac.jp

(Received 17 April 2001, revised 2 July 2001, accepted 24 September

2001)

Abbreviations: cdk 3, cyclin-dependent kinase 3; DMEM, Dulbecco’s

modified Eagle’s medium; Tn5, Trichoplusia ni 5 cells; TLC, thin-layer

cellulose.

was the gift of A Koff (Memorial Sloan Kettering Cancer

Center, NY, USA) GST – pRb and GST – cdk2 plasmids

were as described previously [11]

To replace Ser274 in ik3-1 cDNA with Thr or Ala,

mutagenic primers, 50-TCTCCGGAGATGTCGAACACT

(corre-sponding to amino-acid residues 264 – 277), were used for

PCR amplification

Immunoprecipitation, immunoblotting, and metabolic

labeling

Rabbit polyclonal antibodies to cdk3 (Y-20) and cdk2 (M2)

were purchased from Santa Cruz Biotech (Santa Cruz, CA,

USA) An anti-FLAG mouse monoclonal antibody (M2)

was from Eastman Kodak

Immunoprecipitation and immunoblotting procedures were

as described previously [8] Briefly, cells were suspended at

5  106mL21in a NP40 lysis buffer (50 mMHepes, 150 mM

NaCl, 1 mMEDTA, 1 mMdithiothreitol, 0.2% Nonidet P-40)

containing 2.5 mg:mL21of leupeptin, 5 mg:mL21of

apro-tinin, 20 mM b-glycerophosphate, 0.2 mM

phenymethane-sulfonyl fluoride and 0.1 mMorthovanadate and sonicated at

4 8C The cleared supernatants were then incubated for 2 h

with the indicated antibodies and precipitated for 1 h with

20 mL of 1 : 1 slurry of protein G – Sepharose FF per ml

lysate at 4 8C The washed immunoprecipitates were used

for further experiments Immunoblotted signals were

visualized with an ECL detection kit (Amersham, UK)

For metabolic labeling, COS7 cells transfected with

indicated plasmids were washed twice and preincubated

with phosphate-free Dulbecco’s modified Eagle’s medium

(DMEM) supplemented with 10% dialyzed fetal bovine

serum for 1 h Then cells were incubated in the same fresh

medium containing 0.3 – 0.5 mCi:mL21 of [32

P]orthophos-phate (Amersham) for 5 h

Baculovirus system

Cdk2, cyclin A, and cyclin E baculoviruses were as

described previously [11,12] The recombinant baculovirus

encoding cdk3 was generated by homologous

recombina-tion as described previously, using the pAcYM-1

baculo-viral vector with the cdk3 cDNA [13] Trichoplusia ni (Tn)5

insect cells (1  106) grown in serum-free EX-CELL medium

(JRH Biosciences, Lenexa, KA, USA) were infected or

coinfected with indicated baculoviruses at a multiplicity of

infection of two At 48 h after infection, cells were lysed

in 200 mL of the kinase buffer (50 mM Hepes, 1 mM

dithiothreitol, 20 mM b-glycerophosphate, 10 mM MgCl2)

containing protease and phosphatase inhibitors for 1 h on

ice The cleared supernatants were used for kinase assays

Kinase assays

Immunoprecipitates were washed three times with the

NP40 lysis buffer and three times with the kinase buffer,

and then incubated in 15 mL of the kinase buffer

contain-ing 1 mg of GST-pRb, 1 mg of GST – ik3-1, 1 mg of GST –

ik3-1DN, or 5 mg of GST – cdk2 as substrates in the

presence of 25 mM ATP and 0.5 mCi of [g-32P]ATP

(6000 Ci:mmol21) (Amersham) at 30 8C for 1 h [14] For

kinase assays with insect-cell derived cyclin/cdks, 0.5 mL

of cleared lysates from Tn5 insect cells were used for each lane Phosphorylated substrates were visualized with

FLA-2000 (Fuji Film, Japan) Bacterially expressed proteins were purified as described previously [14]

Two-dimensinal radioactive peptide mapping Radioactive bands excised from dried gels were eluted in

50 mM ammonium bicarbonate (pH 7.3) at 37 8C for 3 h The proteins in the supernatants were precipitated with 18% trichloroacetic acid Dried precipitates were dissolved in

50 mM ammonium bicarbonate (pH 8.0) and incubated at

37 8C for 8 h in the presence of 20 mg (Tos-Phe-CH2 -Cl)-trypsin (Worthington Biochem.) After lyophilization, digested phosphopeptides were electrophoresed with the

pH 1.9 system in the first dimension and then fractionated

by the ascending chromatography with the phospho-chromatography buffer system in the second dimension using a thin-layer cellulose (TLC) plate, as described pre-viously [15] For two-dimensional phosphoamino-acid analysis, acid hydrolysis was performed by incubation of purified phosphopeptides at 110 8C for 60 min with 6M HCl, followed by two-dimensional TLC electrophoresis as described previously [15]

R E S U L T S

P70ik3-1is phosphorylated by cyclin A/cdk2, cyclin E/cdk2, cyclin A/cdk3 or cyclin E/cdk3 which is produced in the baculoviral system

We initially tested a hypothesis that p70ik3-1 might be a regulatory cyclin for p35cdk3 For this purpose, we co-transfected COS7 cells with expression vectors for ik3-1 and cdk3 A kinase assay with p35cdk3immunoprecipitated from COS7 cells indicated that p70ik3-1 did not activate the cdk3-mediated phosphorylation of pRb, histone H1, or the C-terminal domain of RNA polymerase II (data not shown)

To examine another possibility, namely that p70ik3-1is a substrate for cdk3, we generated GST-tagged proteins of ik3-1 in E coli, and tested them as substrates for cdk3-containing kinases produced in the baculoviral system We also tested whether p70ik3-1 is phosphorylated by cdk2-containing kinases In order to reconstitute active kinases,

we coexpressed either cyclin A or cyclin E in association with either cdk2 or cdk3 baculovirally in insect cells (Fig 1A, lanes 6 – 9 of each panel) In parallel, we expressed each cyclin or each cdk separately as negative controls (lanes 2 – 5) As described earlier [4], either cyclin A or E was able to become a partner cyclin for cdk2 as well as cdk3

to phosphorylate pRb if reconstituted in the baculovirus system (upper panel) Likewise, any of cyclin A/cdk3, cyclin A/cdk2, cyclin E/cdk3, or cyclin E/cdk2, reconsti-tuted in insect cells, phosphorylated GST – ik3-1 (middle panel) but not GST – cdk2 (lower panel), indicating that p70ik3-1is a potential substrate for these kinases

P70ik3-1is phosphorylated by anti-cdk2 immunoprecipitates from COS7 cells For further analysis, the endogenous cdk2 was immuno-precipitated with anti-cdk2 Ig from COS7 cells and used for

kinase assays (Fig 1B) On the grounds that both GST – pRb

and GST – ik3-1DN were phosphorylated by anti-cdk2

immunoprecipitates (lanes 2 and 6) and their

phosphoryl-ation was inhibited by coincubphosphoryl-ation with a cdk inhibitor,

p27Kip1, which was generated in E coli (lanes 4 and 8),

p70ik3-1could again be considered as a potential substrate

for cdk2 in this system If we used GST – ik3-1 instead of

GST – ik3-1DN, we obtained similar results (data not

shown) However, we could not assess whether p70ik3-1is

a substrate for cdk3 immunoprecipitated from COS7 cells

because expression of cdk3 is too low [6] and the

endogenous cdk3-mediated kinase activity could not be

detected even in the usual immunoprecipitation kinase assay

using pRB or histone H1 as substrates (Fig 2A, lane 2)

P70ik3-1is phosphorylated by either cyclin A/cdk3 or

cyclin E/cdk3 reconstituted in COS7 cells

To obtain a sufficient amount of cdk3-mediated kinase

activity from COS7 cells, we ectopically expressed cdk3 in

association with either cyclin E or cyclin A by transient transfection (Fig 2A) Kinase assays indicated that pRb-phosphorylating activity was prominently upregulated in the anti-cdk3 immunoprecipitates from COS7 cells transfected with pCMV – cdk3 in association with either the pCMV – cyclin A or pCMV – cyclin E (lanes 3 and 4) In parallel, GST – ik3-1-phosphorylating activity was also upregulated (Fig 2A, lanes 7 and 8) although the degree of upregulation was relatively low In order to exclude that this phosphorylating activity originated from asocociated other kinases, we performed a similar experiment using cdk3 dominant-negative form instead of wild-type cdk3 (Fig 2B) If we compare lanes 3 and 4, we can recognize that phosphorylating activity of ik3-1 increased only in lysates from cells where cyclin A and wild-type cdk3, not cdk3 dn, were expressed, supporting the theory that cyclin/ cdk3 actually phosphorylates ik3-1

P70ik3-1is phosphorylated by either cyclin A/cdk3 or cyclin E/cdk3in vivo

Furthermore, to examine whether p70ik3-1 is also phos-phorylated by cdk3 in vivo, we transfected COS7 cells with both pCMV – cyclin E and pCMV – cdk3 in association with pMF – ik3-1, which were then metabolically labeled with [32P]orthophosphate By immunoprecipitation with the

Fig 1 p70ik3-1is phosphorylated by both p35cdk3and p33cdk2in

vitro (A) Lysates from Tn5 insect cells not infected (lane 1), infected,

or coinfected with indicated baculoviruses were utilized for kinase

assays with GST – pRb (upper panel), GST – ik3-1 (middle panel), or

GST – cdk2 (lower panel) as substrates A, E, k2, and k3 correspond to

the baculoviruses encoding cyclin A, cyclin E, cdk2, and cdk3.

Approximately 0.5 mg lysates were used for each reaction (B) Lysates

from COS7 cells (1  106) were immunoprecipitated with the

nonimmune rabbit serum (N) and the anti-cdk2 Ig (k2) in the presence

or absence of 5 mg of BSA or the bacterially generated His-tagged

p27Kip1 Immunoprecipitates were then used for kinase assays with

GST – pRb (lanes 1 – 4) and GST – ik3-1DN (lanes 5 – 8) as substrates.

Because phosphorylation of GST – ik3-1DN and GST – ik3-1 by the

baculovirally generated cyclin/cdk kinases occurs in a similar fashion

(data not shown), we used GST – ik3-1DN as substrates in this

experiment.

Fig 2 p70ik3-1is phosphorylated by cyclin/cdk3 reconstituted in COS7 cells (A) Lysates from cells (1  10 6 ) tranfected with pCMV – cyclin A (lanes 1, 3, 5, and 7), pCMV – cyclin E (lanes 4 and 8), or the backbone vector (lane 2 and 6) in association with either pCMV – cdk3 (indicated as 1) or the backbone vector ( – ), were immunoprecipitated with the nonimmune rabbit serum (N) and the anti-cdk3 Ig (k3) Approximately 250 mg of lysates were contained in each immunopre-cipitation Immunoprecipitates were then used for kinase assays with GST – pRb (lanes 1 – 4) and GST – ik3-1 (lanes 5 – 8) as substrates (B) Lysates from cells (1  106) tranfected with pCMV – cyclin A (lanes

2 – 5), or the backbone vector (lane 1) in association with either pCMV – cdk3, pCMV – cdk3 dominant-negative form (cdk3 dn) or the backbone vector ( – ), was immunoprecipitated with the nonimmune rabbit serum (N) and the anti-cdk3 Ig (k3) Approximately 250 mg of lysates was contained in each immunoprecipitation Immunoprecipitates were then used for kinase assays with GST – ik3-1 as substrates.

anti-FLAG Ig, we obtained a larger amount of32P-labeled

FLAG – p70ik3-1 from these cells (Fig 3A, lane 3 of the

upper panel) than that from cells in which neither cdk3 nor

cyclin E was overexpressed (lane 2), or that from cells in

which both dominant-negative cdk3 and cyclin E were

overexpressed (lane 4) The lower panel of Fig 3A

demonstrates that similar amounts of FLAG – p70ik3-1were

expressed in each transfection If cyclin E was replaced with

cyclin A in the system, a similar result was obtained

(Fig 3B) We could therefore conclude that p70ik3-1 is a

substrate for cdk3-mediated phosphorylation On the contrary, however, the amount of labeled FLAG – p70ik3-1 was not apparently increased in COS7 cells in which cyclin E/cdk2 activity was potentiated by the cotransfection

of pCMV – cyclin E and pCMV – cdk2 (Fig 3C) Currently,

we cannot therefore conclude that p70ik3-1 is a substrate for cdk2-mediated phosphorylation As an answer to the question of why the dominant-negative cdk3 did not reduce phosphorylation of FLAG – p70ik3-1 below the normal state (compare lanes 2 and 4 of the upper panel in Fig 3A, and lanes 1 and 3 in Fig 3B), we assume that other types of kinases phosphorylate FLAG – p70ik3-1at different sites (see Fig 4) and render obscure the effect of the dominant-negative cdk3 on its total phosphorylation

Ser274 of ik3-1 is phosphorylated by cdk3in vitro Although there are no classical consensus sites (S/T-P-X-R/ K) for cdk-mediated phosphorylation in ik3-1, Ser274 followed by a P-R-P-K sequence resembles the site in p53 which is phosphorylated by cyclin A/cdk2 [16] We therefore asked whether this position is the phosphorylated

Fig 3 p70ik3-1 is phosphorylated by cyclin/cdk3 in vivo (A – C)

COS7 cells (2  10 6 ) transfected with indicated vectors or the

backbone vectors, were labeled with [32P]orthophosphate Cleared

lyasates were immunoprecipitated with the anti-FLAG Ig (F) or the

nonimmune rabbit serum (N) The same set of unlabeled transfected

cells (5  105) was harvested in parallel to estimate FLAG – p70ik3-1

expression with sequential immunoprecipitation-immunoblotting with

anti-FLAG Ig in the lower panel of (A) E, A and dn indicate cyclin E,

cyclin A and a dominant-negative form of cdk.

Fig 4 Ser274 of p70ik3-1 is phosphorylated by cyclin E/cdk3 in vitro Lysates from Tn5 insect cells not infected (lanes 1 – 3), or coinfected with baculoviruses producing cyclin E and cdk3 (lanes 4 – 6) were used for kinase assays with GST – ik3-1 (lanes 1 and 4), GST – (S274T)ik3-1 (lanes 2 and 5), or GST – (S274A)ik3-1 (lanes 3 and 6) as substrates GST – ik3-1 and its mutant proteins eluted from lanes 4 – 6 were subject to digestion with trypsin and two-dimensional peptide mapping Spots A and B of peptide mapping for lane 4, and spots A 0 and

B 0 of peptide mapping for lane 5, were subject to two-dimensional phosphoamino-acid analysis, as shown in the bottom panels Radioactive phosphopeptides were visualized with FLA 2000 Bioimage Analyzer after five-day exposure (4 and 5), 10-day exposure (6) Radioactive phosphoamino acids were visualized after 4-day exposure.

O indicates the origin of electrophoresis W, S-T, and S-A indicate GST – ik3-1, GST – (S274T)ik3-1, and GST – (S274A)ik3-1 S, T and Y indicate phosphoserine, phosphothreonine and phosphotyrosine.

site in ik3-1 To this end, we produced mutant GST – ik3-1

proteins as substrates for in vitro kinase assays by replacing

Ser274 with threonine [(S274T)ik3-1] or alanine

[(S274A)ik3-1] using the site-directed mutagenesis

tech-nique As expected, baculovirally generated cyclin E/cdk3

phosphorylated both wild-type GST – ik3-1 and GST –

(S274T)ik3-1 efficiently (Fig 3, lanes 4 and 5) while it

phosphorylated GST – (S274A)ik3-1 to a much smaller

degree (lane 6), indicating that Ser274 is the main target for

cdk3-dependent phosphorylation This was also true if the

baculovirally produced cyclin E/cdk2 or cyclin A/cdk3 as

well as anti-cdk2 immunoprecipitates from COS7 cells were

used as kinase source in place of the baculovirally produced

cyclin E/cdk3 (data not shown)

Furthermore, to analyze in detail, we eluted radioactive

GST – ik3-1 proteins from dried gels and digested them with

(Tos-Phe-CH2Cl)-trypsin, and then conducted

two-dimen-sional peptide mapping analysis (Fig 4, middle panels) We

recognized two major phosphopeptide spots, A and B, with a

few spots with weaker radioactivity in the wild-type GST –

ik3-1 panel (middle left panel) Spot B in the wild-type

p70ik3-1panel was not observed in the S274A mutant panel

(middle right panel) At a glance, spot A seemed to exist in

the same mutant panel indicated as C (middle right panel)

However, if we estimate the relative radioactivity of spot A

or C against other spots, we could speculate that the

phos-phopeptide corresponding to spot C in the S274A mutant

seemed to represent one of the background phosphopeptides

that was normally hidden behind the spot corresponding to

A This interpretation was also supported by the observation

that the migration pattern of spot C was similar to but

apparently not the same as that of phosphopeptide A (middle

left and right panels) Here we came to notice that both spot

A and spot B disappeared if Ser274 was replaced with

alanine Regarding the relationship between spot A and spot

B, we have speculated that the phosphopeptides

correspond-ing to spot A and B arised by incomplete tryptic cleavage of

the Ser274-containing region In fact, phosphoamino-acid

analysis of spots A and B indicated that both spots A and B

contained phosphoserine (Fig 4, bottom left two panels)

Furthermore, we observed that re-digestion with trypsin of

the phosphopeptide purified from spot A gave rise to both

spot A and spot B by another two-dimensional peptide

mapping (data not shown), indicating that the assumption is

true Spots A0 and B0 in the S274T mutant panel may

correspond to mutated phosphopeptides, in which threonine

substituted for serine was phosphorylated by cyclin E/cdk3,

and which therefore migrated a little differently from

wild-type ones (middle central panel) Phosphoamino-acid

analysis of spot A0and B0 indicated that both spots A0and

B0 contained phosphothreonine (Fig 4, bottom right two

panels), strongly supporting that Ser274 is phosphorylated

by cyclin/cdk3

Ser274 of ik3-1 also is phosphorylated by cdk3in vivo

Next, we asked whether Ser274 of p70ik3-1is phosphorylated

by cyclin E/cdk3 intracellularly in mammalian cells Another

in vivo labeling experiment for this purpose indicated that

in COS7 cells cotransfected with both pCMV – cyclin E and

pCMV – cdk3, the phosphorylation level of the wild-type

FLAG – p70ik3-1, but not that of FLAG – (S274A)p70ik3-1, is

upregulated as compared with control cells (Fig 5, upper

panel, lanes 1 vs 2, lanes 3 vs 4) Purified FLAG – p70ik3ÿ1 was then digested with trypsin and subjected to two-dimensional peptide mapping Here we observed that there were several phosphopeptide spots including two spots (indicated as A and B) that seemed to migrate similarly to spots A and B in the wild-type GST – ik3-1 panel (compare Fig 4, lower panel 1 with Fig 3, lower left panel) To confirm that these spots A and B in the in vivo labeled FLAG – p70ik3-1panel were the same as spots A and B in the wild-type GST – ik3-1 panel, we mixed whole phosphopep-tides generated by digestion with trypsin from the wild-type FLAG – p70ik3-1 labeled in vivo, and phosphopeptides purified from spots A and B of in vitro labeled wild-type GST – ik3-1 on a TLC plate (Fig 3, the lowest panel) We then conducted another two-dimensional peptide mapping (Fig 4, panel ‘mix’) We were able to see that the

Fig 5 Ser274 of p70 ik3-1 is phosphorylated in vivo COS7 cells (2  106) transfected with indicated vectors were labeled with [32P]orthophosphate Cleared lysates were immunoprecipitated with the anti-FLAG Ig WT and S-A indicate pMF – ik3-1 for expression of wild-type FLAG – p70ik3-1(lanes 1 and 2) and pMF – (S274A)ik3-1 for expression of FLAG – (S274A)p70 ik3-1 (lanes 3 and 4), respectively They were cotranfected with pCMV – cyclin E and pCMV – cdk3 (lanes

2 and 4) or the backbone vectors (lanes 1 and 3) Wild-type FLAG – p70ik3-1(lanes 1 and 2) and S274A mutant FLAG – p70ik3-1(lanes 4) were eluted, digested with trypsin, and subjected to two-dimensional peptide mapping (three middle panels) In vivo labeled wild-type FLAG – p70ik3-1was purified from another gel, digested with trypsin, mixed with one-fifth amount of phosphopeptides purified from spots A and B of the GST – ik3-1 in the lower left panel of Fig 4, and then subjected to two-dimensional peptide mapping (panel ‘mix’) Radioactive phosphopeptides were visualized by FLA 2000 Bioimage Analyzer after 14-day exposure (1 and 2) and 21-day exposure (4) O indicates the origin of electrophoresis.

phosphopeptides, corresponding to spots A and B derived

from the in vivo labeled FLAG – p70ik3-1, comigrated in a

similar fashion with those from in vitro labeled GST – ik3-1,

indicating that the above assumption is true

Moreover, if we estimate the radioactivity of

phospho-peptide spots A and B against those of the other spots, we can

recognize that the radioactivity of peptides corresponding to

spots A and B from FLAG – p70ik3-1in lane 2, are apparently

upregulated as compared with that from FLAG – p70ik3-1in

the lane 1 (Fig 5, lower panels 1 vs 2), suggesting that

cotransfected cyclin E/cdk3 increased the overall

phos-phorylation level of FLAG – p70ik3-1 by increasing

phos-phorylation of Ser274 This fact confirms that Ser274 is the

major target residue phosphorylated by cdk3-mediated

kinase activity, not only in vitro but also in vivo

Furthermore, to examine whether spots corresponding to

phosphopeptides A and B derived from FLAG – p70ik3ÿ1

disappear if Ser274 is disrupted by site-directed

mutagen-esis, we eluted S274A mutant FLAG – (S274A)p70ik3-1from

the lane 4 gel of Fig 5 and conducted two-dimensional

peptide mapping (Fig 5, middle right panel 4) The A- and

B-corresponding spots disappeared while the

D-correspond-ing spot still existed Unexpectedly, a weak spot

corre-sponding to spot E also seemed to disappear We do not

know why this happened However, we speculate that

phosphorylation giving rise to the spot E may occur only

when Ser274 is phosphorylated, or alternatively the S274A

mutation may induce a conformational change of the protein

blocking phosphorylation of the spot E-corresponding

peptide We obtained similar results when we analyzed

S274A mutant FLAG – (S274A)p70ik3-1from the lane 3 gel

of Fig 5 by two-dimensional peptide mapping (data not

shown) Thus, although we cannot completely exclude the

possibility that the S274A mutation induces a

confor-mational change blocking phosphorylation of non-Ser274

sites by cyclin/cdk3, we could assume that Ser274 is

phosphorylated by cyclin/cdk3 in vivo as it is in vitro

Based on these data, we can consider that p70ik3-1is one

of the substrates for cdk3, while it is still possible that cdk3

indirectly increases phosphorylation of p70ik3-1 through

some other unknown mechanism

D I S C U S S I O N

ik3-1 cDNA was originally cloned by its protein – protein

interaction with p35cdk3 [8] Both in vitro and in vivo,

p70ik3-1is considered to be a substrate for cdk3-dependent

phosphorylation Furthermore, p70ik3-1could also be a

sub-strate for cyclin/cdk2 in vitro (Fig 1) However, its

inter-action with p33cdk2 is relatively weak [8], and in vivo

phosphorylation of p70ik3-1was not apparently enhanced by

overexpression of cyclin/cdk2 (Fig 3C), suggesting that the

ik3-1-mediated pathway is mainly regulated by cdk3 This

result reminds us of the foregoing observation that the

dominant-negative cdk3-mediated G1 arrest is not

com-pletely rescued by wild type cdk2 in human osteosarcoma

cells [6], indicating that the function of cdk3 is at least

partially distinct from that of cdk2 in G1 progression

Intriguingly, ik3-1 has no classical cdk sites (S/T-P-X-R/K)

that are phosphorylated by cyclin/cdk2 or cyclin B/cdc2

Instead, it contains a S-P-R-P-K sequence at residues 274–278

that resembles the S-P-Q-P-K-K sequence of human p53,

which is phosphorylated by cyclin A/cdk2 [16] Site-directed

mutagenesis procedures and the two-dimensional peptide mapping analysis have established that Ser274 in p70ik3-1is phosphorylated by both cdk3/cyclin A and cdk3/cyclin E in vitro (Fig 4) The same residue is also phosphorylated by both cdk3/cyclin A and cdk3/E in vivo (Fig 5) Analysis of ik3-1 amino-acid sequence indicates that ik3-1 has a putative ZRXL (Z and X are typically basic) motif that would allow it to be a substrate by cyclin/cdk3 as well as cyclin/cdk2 through binding

to cyclin, leading to speculation that binding of the cyclin subunit, but not cdk3, to ik3-1 might be required for phosphorylation of ik3-1 The fact that cyclin/cdk2 could phosphorylate ik3-1 in vitro supports this assumption

In postmitotic neurons, ik3-1 or Cables [9] may enhance neurite growth by potentiating c-abl-mediated tyrosine phosphorylation of cdk5 Tyr14-phosphorylated cdk5 is more active in vitro, and ik3-1 is phosphorylated by p35/ cdk5 in vitro [9] In spite of this observation, it still remains

to be clarified how ik3-1 functions in non-neuronal cells because ik3-1 is basically expressed ubiquitously and cdk5

is inactive in non-neuronal cells [10] In COS7 cells, cdk5 activity is also undetectable even after ik3-1 is over-expressed (M Matsuoka, unpublished observation) Accordingly, we can conclude that in COS7 cells, Ser274

in p70ik3-1is phosphorylated by endogenous kinases other than cdk5 (Fig 4), at least one of which is cdk3 as shown in this work Currently, however, the question of how ik3-1 function is modified by the cdk3-mediated phosphorylation

of Ser274 remains to be addressed

One of the major issues in the cell-cycle field is how G1 cyclin/cdks accelerates the mammalian G1 – S progression and commits to DNA replication To address this question, the substrates and target molecules need to be clarified So far, it has been shown that cyclin E/cdk2 phosphorylates pRb, which is also the sole known substrate for cyclin D/cdk4 or cyclin D/cdk6 at this time Hyperphosphorylating pRb and upregulating free E2F, both cyclin E/cdk2 and cyclin D/cdk4 or cyclin D/cdk6 co-operatively promote the transcription of various genes, including the cyclin E gene necessary for G1 – S transition [1,2] Other candidate sub-strates for cyclin E/cdk2 include NPAT [17], components of the premRNA splicing machinery [18] and Id2 [19], and more are emerging In this respect, the functional analysis of ik3-1, a candidate target for cdk3, will contribute to the further understanding of cdk3 function in the mammalian G1 – S transition which is distinct from the cdk2 function in self-replicating cells

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

We are indebted to Tomo Yoshida, Kazumi Nishihara, Kouichi Tsuchiya, Fusano Igarashi and Dovie Wylie for expert technical assistance; Drs Jiyong Zhao, S van den Heuvel, Ed Harlow, Andrew Koff, Charles J Sherr, Hiroshi Hirai, Makoto Nakanishi and Hitoshi Matsushime for providing us with plasmids and baculoviruses This work is supported in part by grant from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, the Organization for Pharmaceutical Safety and Research and KEIO University Special Grant-in-Aid for Innovative Collaborative Research Projects.

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