Degradation of the cyclin-dependent kinase inhibitor …labs.biology.ucsd.edu/estelle/publications/Ren.et.al...Degradation of the cyclin-dependent kinase inhibitor KRP1 is regulated

Degradation of the cyclin-dependent kinase inhibitor KRP1is regulated by two different ubiquitin E3 ligases

Hong Ren1, Aaron Santner1, Juan Carlos del Pozo2, James A. H. Murray3 and Mark Estelle1,*

1Department of Biology, Indiana University, Bloomington, IN 47405, USA,2Dpt. Biotecnologıa, Instituto Nacional de Investigacion y Tecnologıa Agraria y Alimentaria (INIA), 28040 Madrid, Spain, and3Institute of Biotechnology, University of Cambridge, Cambridge CB2 1QT, UK

Received 25 May 2007; revised 21 September 2007; accepted 17 October 2007.*For correspondence (fax 1 812 855 6082; e-mail [email protected]).

Summary

In animals and fungi, a group of proteins called the cyclin-dependent kinase inhibitors play a key role in cell

cycle regulation. However, comparatively little is known about the role of these proteins in plant cell cycle

regulation. To gain insight into the mechanisms by which the plant cell cycle is regulated, we studied the

cyclin-dependent kinase inhibitor KRP1 in Arabidopsis. KRP1 interacts with the CDKA;1/CYCD2;1 complex

in planta and functions in the G1–S transition of the cell cycle. Furthermore, we show that KRP1 is a likely

target of the ubiquitin/proteasome pathway. Two different ubiquitin protein ligases, SCFSKP2 and the RING

protein RKP, contribute to its degradation. These results suggest that SCFSKP2b and RPK play an important role

in the cell cycle through regulating KRP1 protein turnover.

Keywords: Arabidopsis, cell cycle, KRP ubiquitin.

Introduction

The cell cycle consists of a series of events that ultimately

lead to the formation of two daughter cells. In eukaryotes,

the fundamental mechanisms of cell cycle regulation are

highly conserved. The cycle is divided into four phases, G1,

S, G2 and M, and has two major checkpoints to control cell

cycle progression: the G1–S transition and the G2–M tran-

sition. Cell cycle progression is controlled by the activities of

cyclin-dependent kinase (CDK)/cyclin complexes. Different

combinations of CDKs and cyclins regulate passage from

one phase of the cycle to the next (De Veylder et al., 2003;

Dewitte and Murray, 2003). The activities of CDK/cyclin

complexes can be regulated by CDK inhibitors (CKIs), which

function as negative regulators of CDK activity (Sherr and

Roberts, 1999). CKIs have been identified in yeast, mammals

and plants (De Clercq and Inze, 2006). In mammals, there are

seven CKIs, which are classified into two families: the INK4

family and the Cip/Kip family (Nakayama and Nakayama,

1998; Vidal and Koff, 2000). Plants do not appear to have

INK4-type CKIs, but proteins related to the Cip/Kip family

have been identified in Arabidopsis, tobacco, maize and

tomato (Bisbis et al., 2006; Coelho et al., 2005; De Veylder

et al., 2001; Jasinski et al., 2002; Wang et al., 1997). The

Arabidopsis genome encodes seven proteins related to the

mammalian CKI p27Kip1, known as Kip-related proteins

(KRPs) or interactors/inhibitors of Cdc2 kinase (ICKs; De

Veylder et al., 2001; Vandepoele et al., 2002; Zhou et al.,

2002a,b). The only sequence similarity between KRPs and

p27Kip1 is in the conserved CDK-binding/inhibitory domain

(De Veylder et al., 2001; Wang et al., 1997). Recently, an

additional CKI was identified in Arabidopsis called SIAMESE

(SIM; Churchman et al., 2006). The SIM protein includes a

cyclin binding domain and a domain also found in the KRP

proteins. Genetic studies indicate that SIM has a role in the

control of endoreduplication.

Recent studies have begun to shed light on the function of

plant CKIs in cell cycle regulation and growth. Ectopic

expression of KRP1, KRP2, KRP4 and KRP6 confirm that

these proteins function as inhibitors of the cell cycle,

resulting in dwarfed plants with reduced cell number and

organ size (Bemis and Torii, 2007; De Veylder et al., 2001;

Wang et al., 2000; Zhou et al., 2003). Studies of transcrip-

tional regulation have shown that KRP1 transcription is

increased by low temperature and abscisic acid (ABA),

whereas KRP2 transcription is downregulated by auxin

during lateral root initiation (Himanen et al., 2002; Wang

et al., 2000). The expression patterns of KRPs during the cell

ª 2008 The Authors 705Journal compilation ª 2008 Blackwell Publishing Ltd

The Plant Journal (2008) 53, 705–716 doi: 10.1111/j.1365-313X.2007.03370.x

cycle were characterized using synchronized Arabidopsis

cultured cells (Menges and Murray, 2002; Menges et al.,

2005). These data show that there are three main patterns of

transcriptional regulation of KRP genes. KRP1 is highly

expressed in non-dividing cells and is strongly downregu-

lated during the G1 phase in cell cycle re-entry. KRP1 shows

a further clear peak of expression at the G2–M transition,

although this is threefold lower than the expression in non-

dividing cells. KRP2 is highly expressed in non-dividing

cells, and is unique in showing a peak of expression only

during the G1 phase as cells re-enter the cell cycle. In

contrast, KRP3, KRP4, KRP5, KRP6 and KRP7 are not highly

expressed in non-dividing cells, but are upregulated or peak

during the S and early G2 phases. These results implicate

KRP1 and KRP2 as primary candidates for controlling

activation of division by non-dividing cells. Among the plant

CKIs, KRP1 and KRP2 are known to be degraded by the 26S

proteasome (Jakoby et al., 2006; Verkest et al., 2005a).

Interestingly, degradation of KRP2 requires its phosphory-

lation by the CDKB1;1 complex (Verkest et al., 2005b).

However, the detailed mechanisms of KRP1 and KRP2

degradation remain to be elucidated.

In eukaryotes, ubiquitin-mediated protein degradation

plays a critical role in the cell cycle by destroying many

important cell cycle regulators (Hershko, 2005). Conjugation

of ubiquitin to a substrate requires the sequential action of

three enzymes: ubiquitin-activating enzyme (E1), ubiquitin-

conjugating enzyme (E2) and ubiquitin-protein ligase (E3).

The E3 enzymes are responsible for the specificity of the

pathway, and several classes of E3s have been implicated in

cell cycle regulation including the SCF (SKP1-cullin-F-box

protein) and RING domain ubiquitin ligases. An SCF com-

plex is composed of four subunits: RBX1, CUL1, SKP1 and an

F-box protein. The CUL1 subunit functions as a scaffold to

bind RBX1 and the SKP1-F-box protein subcomplex. The

F-box protein subunit provides specificity for SCFs and

binds substrates (Moon et al., 2004; Petroski and Deshaies,

2005). In yeast and mammals, the role of SCFs in cell cycle

regulation has been extensively investigated. SCFs are

responsible for the degradation of cyclins, CKIs, transcrip-

tion factor E2F-1 and many other cell cycle regulators

(Hershko, 2005). A well-known example is the regulation of

p27Kip1 degradation by SCFSKP2 in mammals. SCFSKP2

targets p27Kip1 for degradation to trigger the G1–S transition

of the cell cycle (Sutterluty et al., 1999; Tsvetkov et al., 1999).

The Arabidopsis genome encodes two F-box proteins

(called SKP2a and SKP2b) that are related to mammalian

SKP2 (del Pozo et al., 2002). SKP2a appears to recruit the

phosphorylated form of the transcription factor E2Fc for

degradation. Whether SKP2b also regulates E2Fc degrada-

tion is unknown. In addition to E2Fc, another cell cycle

regulator that may be an SCF substrate is CYCD3;1. This

cyclin is unstable and its degradation depends on the 26S

proteasome (Planchais et al., 2004). In transgenic plants with

reduced levels of RBX1, CYCD3;1 accumulates indicating

that SCF is involved in its degradation. However, the F-box

protein component of this SCF has not been identified

(Lechner et al., 2002; Liu et al., 2004).

To investigate the post-translational regulation of plant

CKIs, we focused on KRP1. Our data demonstrate that KRP1

interacts with the CDKA;1/CYCD2;1 complex in planta. Fur-

thermore, we show that KRP1 degradation is dependent on

SCFSKP2b and the RING protein RKP. These results provide

new insight into the mechanisms by which the plant cell

cycle is regulated by protein degradation.

Results

KRP1 expression

Previous studies by RNA blot and RT-PCR have shown that

KRP1 is expressed in roots, stems, leaves, flowers, inflo-

rescences and actively dividing cultured cells (De Veylder

et al., 2001; Lui et al., 2000; Wang et al., 1998). In addition,

KRP1 expression was examined in leaves and in the shoot

apex by in situ hybridization (Ormenese et al., 2004). KRP1

RNA was detected in endoreduplicating tissues of leaves,

but not in dividing cells of the shoot apical meristem. To

further characterize KRP1 expression, we generated Ara-

bidopsis transgenic lines in which the bacterial b-glucuron-

idase reporter gene (GUS) was placed adjacent to the KRP1

promoter. Over 10 independent transgenic lines were ana-

lyzed, and all lines exhibited similar GUS expression pat-

terns. In young seedlings, GUS staining was first observed in

cotyledons (Figure 1a). With longer incubation times, stain-

ing became apparent in roots, hypocotyls and emerging

leaves (Figure 1b). In older seedlings, GUS staining was

equivalent in cotyledons and rosette leaves (Figure 1c). In

the flower, GUS staining was observed in the sepals, anthers

and mature pollen (Figure 1d,e). GUS staining was also

detected in siliques, with peak expression at the base (Figure

1f,g). These results show that KRP1 is expressed in various

tissues and organs throughout plant development. An

examination of publicly available expression data confirms

this view (http://jsp.weigelworld.org/expviz/expviz.jsp; Sch-

mid et al., 2005). KRP1 transcript is detected in all tissues

examined, but is most abundant in cotyledons and leaves

(Table S1).

KRP1 overexpression inhibits auxin-mediated

pericycle cell division during lateral root initiation

To understand the role of KRP1 in plant growth and devel-

opment, we investigated the effects of KRP1 loss of function

and gain of function on plant growth and development. A

krp1 line with a T-DNA insertion in the third intron was

identified in the SALK collection, and RT-PCR results indicate

that the mutation prevents formation of a functional KRP1

706 Hong Ren et al.

ª 2008 The AuthorsJournal compilation ª 2008 Blackwell Publishing Ltd, The Plant Journal, (2008), 53, 705–716

protein (data not shown; Alonso et al., 2003). Despite the fact

that the krp1 mutant was grown in a wide variety of growth

conditions, we were not able to detect any changes in phe-

notype compared with the wild-type line (data not shown).

To determine the effects of KRP1 overexpression, we gen-

erated Arabidopsis transgenic plants that express a c-Myc

epitope tagged KRP1 under the control of the Cauliflower

mosaic virus (CaMV) 35S promoter. More than 30 indepen-

dent lines exhibited similar phenotypes. Plants had serrated

rosette leaves, reduced apical dominance, reduced fertility

and a reduced numbers of lateral roots. This phenotype is

very similar to that conferred by overexpression of KRP1

(Wang et al., 2000), indicating that Myc-KRP1 is a functional

protein in planta. A 35S:Myc-KRP1 line that has a weak

phenotype and carries a single T-DNA insertion was cho-

sen for further analysis. Interestingly, KRP1 overexpressors

were temperature sensitive. If grown at 18�C, plants were

more robust and exhibited increased fertility (data not

shown).

An interesting and previously uncharacterized aspect of

the KRP1-overexpression phenotype is a severe defect in

lateral root formation. The 35S:Myc-KRP1 line exhibited only

a slight decrease in primary root growth, but lateral root

formation was dramatically inhibited (Figure 2a,b). The

density of emerged lateral roots was reduced by 41% and

96% in the hemizygous and homozygous 35S:Myc-KRP1

plants, respectively, suggesting that the effect of KRP1 on

lateral root formation is dose dependent. This was con-

firmed by determining the level of Myc-KRP1 in these lines

by protein blot (Figure 2e).

To learn more about how KRP1 functions in lateral root

formation, we introduced a CYCB1;1:GUS transgene into

35S:Myc-KRP1 plants by crossing. CYCB1;1, a mitotic cyclin,

is expressed in late G2 and M phase, and is therefore a

marker for cell cycle progression from the G2 to the M phase.

Two experiments were performed with these plants. In the

first we used the activity of the CYCB1:1:GUS gene as a

marker for initiation of lateral root primordia. When both

lateral root primordial and emerged lateral roots were

counted, overexpression of Myc-KRP1 resulted in a decrease

in the total number of lateral roots (Figure 2c). However, a

much larger fraction of these lateral roots did not emerge in

the overexpression lines compared with the wild type

(Figure 2d). These results indicate that increased levels of

KRP1 inhibit both formation of lateral root primordia and

continued growth of primordia once they are formed.

We then used the CYCB1:1:GUS transgene to examine the

effect of KRP1 overexpression on auxin-mediated pericycle

cell division. Seedlings were grown using lateral root

induction conditions developed by Himanen et al. (2002).

Seedlings were first treated with the auxin transport inhib-

itor N-1-naphthylphthalamic acid (NPA). This compound

prevents pericycle cell division, and all pericycle cells remain

in the G1 phase. Subsequently, seedlings were treated with

the auxin 1-naphthaleneacetic acid (NAA) to activate pericy-

cle cells, causing them to pass the G1–S and G2–M transi-

tions and to undergo cell division. After NPA treatment, wild-

type and 35S:Myc-KRP1 seedlings did not exhibit GUS

staining in the pericycle (Figure 2f). As expected, treatment

of wild-type seedlings with NAA produced significant GUS

staining in the pericycle, showing that these cells have

passed through the G2–M transition. In contrast, no staining

was observed in the 35S:Myc-KRP1 plants (Figure 2f),

indicating that KRP1 overexpression inhibits auxin-medi-

ated pericycle cell division during lateral root initiation.

KRP1 interacts with CDKA;1 and CYCD2;1 in planta

A crucial step towards understanding the role of KRP1 in cell

cycle regulation is to identify its CDK/cyclin complex targets.

Analyses using the yeast two-hybrid system have shown

that KRP1 interacts with CDKA;1 and D-type cyclins

(CYCD1;1, CYCD2;1 and CYCD3;1), but that KRP1 does not

interact with CDKB1;1, CYCA2;2 and B-type mitotic cyclins

(CYCB1;1 and CYCB2;1; De Veylder et al., 2001; Wang et al.,

1998). However, these interactions have not been confirmed

in the plant. Because we have transgenic plants over-

expressing Myc-KRP1 as well as antibodies to CDKA;1,

CDKB1;1 and CYCD2;1, we tested for these interactions by

immunoprecipitation. Protein extracts prepared from wild-

type and 35S:Myc-KRP1 seedlings were immunoprecipitated

with an a-c-myc antibody. Immunoblot analyses were per-

formed with a-CDKA;1, a-CDKB1;1 and a-CYCD2;1 antibod-

ies. As shown in Figure 2g, CDKA;1 and CYCD2;1, but not

(a) (b) (c)

(d) (e) (f) (g)

Figure 1. KRP1 is broadly expressed.

A 2062-bp KRP1 promoter was fused to GUS, and GUS expression was

examined by GUS staining of transgenic plants carrying a KRP1:GUS

transgene.

(a) and (b) 7-day-old light-grown seedling stained for 4 (a) or 24 h (b).

(c) Rosette leaves of a 10-day-old light-grown seedling stained for 4 h.

(d) Mature flower.

(e) A closer look at the anthers and mature pollens shown in (d).

(f) Siliques.

(g) A closer look at the base of siliques shown in (f).

KRP1 degradation by E3 ligases 707


CDKB1;1, co-immunoprecipitates with Myc-KRP1. These

results indicate that KRP1 interacts with CDKA;1 and

CYCD2;1 in planta.

KRP1 is an unstable protein and its degradation

depends on the 26S proteasome

KRP1 is related to mammalian CKI p27Kip1, a protein that

is degraded through the action of two ubiquitin ligases

called SCFSKP2 and KPC1 (Carrano et al., 1999; Kamura

et al., 2004b; Sutterluty et al., 1999; Tsvetkov et al., 1999).

Although KRP1 has been shown to be degraded by the 26S

proteasome (Jakoby et al., 2006), the detailed mechanism of

KRP1 degradation has not yet been described. To study

KRP1 protein stability in planta, we generated Arabidopsis

transgenic plants that express KRP1–GUS fusion protein

under the control of the KRP1 promoter. In 10-day-old

KRP1:GUS plants stained for 4 h, GUS staining was

observed in the cotyledons and leaves (Figure 1c). To assist

in comparison, this image is also shown in Figure 3. In

contrast, no GUS staining was observed in KRP1:KRP1–GUS

seedlings that were incubated in staining solution for 24 h

(Figure 3b). As GUS is a stable protein, these results indicate

that KRP1 destabilizes GUS, suggesting that KRP1 is an

unstable protein that is quickly degraded in planta (Jeffer-

son et al., 1987).

To determine whether the 26S proteasome is involved in

KRP1 degradation, we examined the effect of MG132, a

proteasome inhibitor, on KRP1–GUS and Myc-KRP1 protein

stability. Unlike the DMSO-treated seedlings, GUS staining

was observed in the cotyledons of MG132-treated

KRP1:KRP1–GUS seedlings (Figure 3c,d). Similarly, MG132

treatment resulted in increased Myc-KRP1 levels in 35S:Myc-

KRP1 seedlings (Figure 3e). CDKA;1 was used as a loading

(a) (b)

(c)

(e) (f) (g) (h) (i) (j)

(d)

Figure 2. KRP1 overexpression inhibits auxin-mediated pericycle cell division.

(a) Primary root length of 2-week-old light-grown plants. He, hemizygous; Ho, homozygous.

(b) Number of emerged lateral roots in 2-week-old light-grown plants.

(c) Total lateral roots/seedling (primordia + emerged) visualized using the CYCB1;1:GUS marker.

(d) Percentage lateral root primordia from (c).

(e) Immunoblot analysis of Myc-KRP1 levels with an a-c-myc antibody. Protein extracts were prepared from 2-week-old light-grown plants. An unknown protein

recognized by the a-c-myc antibody was used as a loading control.

(f) Root of a CYCB1;1:GUS seedling grown on an ATS + 10 lM N-1-naphthylphthalamic acid (NPA) plate for 72 h.

(g) CYCB1;1:GUS seedling transferred to an ATS + 10 lM 1-naphthaleneacetic acid (NAA) plate for 12 h after initial growth on NPA.

(h) Root of a 35S:Myc-KRP1 (homozygous) seedling carrying a CYCB1;1:GUS transgene grown on an ATS + 10 lM NPA plate for 72 h.

(i) GUS expression in the root of a 35S:Myc-KRP1 (homozygous) seedling transferred to an ATS + 10 lM NAA plate for 12 h after initial growth on NPA.

(f–i) All seedlings were stained for GUS.

(j) KRP1 interacts with CDKA;1 and CYCD2;1 in planta. Protein extracts from wild-type (Col) and 35S:Myc-KRP1 seedlings were immunoprecipitated with an a-c-myc

antibody. Immunoblot analyses of protein extracts from wild-type (Col) and a-c-myc immunoprecipitates were performed with a-CDKA;1, a-CDKB1;1 and a-CYCD2;1

antibodies. Asterisk indicates an unknown protein recognized by the a-CYCD2;1 antibody.

708 Hong Ren et al.


control. MG132 did not affect CDKA;1 protein levels

(because this protein is stable). The stabilization of both

KRP1–GUS and Myc-KRP1 by MG132 indicates that KRP1

degradation depends on the 26S proteasome.

The AXR1-dependent RUB conjugation

pathway regulates KRP1 degradation

Like ubiquitin, RUB1 (related to ubiquitin-1, also called

NEDD8) is a post-translational modifier that regulates

diverse cellular processes. At present the best-characterized

RUB1 targets are the cullin proteins, including the CUL1

subunit of SCFs (Parry and Estelle, 2004; Petroski and

Deshaies, 2005). In Arabidopsis, RUB1 conjugation to CUL1

requires the AXR1-ECR1 heterodimer (RUB-activating

enzyme E1), RCE1 (RUB-conjugating enzyme E2) and RBX1

(RUB-protein ligase E3). RUB conjugation modulates the

activity of many, perhaps all, CUL1-based SCFs (Parry and

Estelle, 2004). Our previous data demonstrated that the

ubiquitin-proteasome pathway regulates KRP1 degradation.

To determine if the RUB conjugation pathway is required for

this degradation, we examined KRP1–GUS and Myc-KRP1

protein stability in the axr1-3 mutant, which has an impaired

RUB conjugation pathway (del Pozo et al., 1998).

We introduced the KRP1:KRP1–GUS transgene into axr1-3

plants by crossing. The results shown in Figure 4a,b indicate

that the axr1-3 mutation acts to stabilize KRP1–GUS in both

the cotyledon and hypocotyl. Homozygous KRP1:KRP1–GUS

plants are very similar to wild type in appearance, whereas

axr1-3 plants exhibit a pleiotropic phenotype that includes

reduced stature and decreased apical dominance (Figure

4c,d). Interestingly, the introduction of the KRP1:KRP1–GUS

transgene into the axr1-3 background enhances this mutant

phenotype. The effects of the transgene were variable,

but 34% (29/85) had a severe phenotype as illustrated in

Figure 4c,d.

Myc-KRP1

CDKA;1

DMSO MG132

(a) (b) (c)

(d) (e)

Figure 3. KRP1 degradation depends on the 26S proteasome.

(a) GUS staining of a 10-day-old light-grown KRP1:GUS seedling stained for

4 h.

(b) GUS staining of a 10-day-old light-grown KRP1:KRP1–GUS seedling

stained for 24 h.

(c) GUS staining of 4-day-old light-grown KRP1: KRP1–GUS seedlings treated

with DMSO for 8 h.

(d) GUS staining of 4-day-old light-grown KRP1:KRP1–GUS seedlings treated

with 50 lM MG132 for 8 h. MG132 is a 26S proteasome inhibitor. DMSO was

used as a control. Arrows indicate GUS staining.

(e) Immunoblot analysis of protein extracts from 35S:Myc-KRP1 (homozy-

gous) seedlings treated with DMSO and 50 lM MG132 for 12 h, respectively,

with a-c-myc and a-CDKA;1 antibodies. CDKA;1, a stable protein, was used as

a loading control.

1

(a)

(e)

(b)

2 3 4

Myc-KRP1

(c)

(d)

Figure 4. The AXR1-dependent RUB conjugation pathway regulates KRP1

degradation.

(a) GUS staining of a 4-day-old light-grown KRP1:KRP1–GUS seedling.

(b) GUS staining of a 4-day-old light-grown axr1-3 seedling carrying a

KRP1:KRP1–GUS transgene.

(c) Seven-week-old plants. From left to right, wild-type (Col-0), axr1-3, axr1-3

KRP1:KRP1–GUS, and KRP1:KRP1–GUS. Bar = 2 cm.

(d) Inflorescences of 7-week-old mature plants shown in (c). Bar = 1 cm.

(e) Immunoblot analysis of Myc-KRP1 with an a-c-myc antibody. Protein

extracts were prepared from 2-week-old light-grown plants. An unknown

protein recognized by the a-c-myc antibody was used as a loading control

(bottom). Lane 1, Col-0; lane 2, 35S:Myc-KRP1 (hemizygous); lane 3, axr1-3

35S:Myc-KRP1 (hemizygous); lane 4, axr1-3.



We also introduced the 35S:Myc-KRP1 transgene into

axr1-3 plants by crossing. As shown in Figure 4e, the axr1-3

mutation stabilized Myc-KRP1. These data indicate that the

AXR1-dependent RUB conjugation pathway regulates KRP1

degradation.

KRP1 degradation is dependent on SCFSKP2b

The RUB conjugation pathway is probably required for

the function of all cullin-based E3 ubiquitin ligases,

including SCF, CUL3-BTB and CUL4-DDB E3s (Bernhardt

et al., 2006; Chen et al., 2006; Parry and Estelle, 2004). To

determine if an SCF might be involved in KRP1 degrada-

tion, we introduced the KRP1:KRP1–GUS and 35S:Myc-

KRP1 transgenes into the axr6-3 mutant by crossing. The

axr6-3 mutant contains a recessive and temperature-sen-

sitive mutation of CUL1 that has been shown to stabilize

SCF substrates (Quint et al., 2005). As shown in Figure

5a,b,d, the axr6-3 mutant stabilized both KRP1–GUS and

Myc-KRP1, exhibiting GUS staining in the cotyledon and

an increased Myc-KRP1 protein level. Interestingly, over-

expression of either KRP1–GUS or Myc-KRP1 enhances

the axr6-3 growth defects. Plants exhibited a dramatically

decreased height and were sterile (Figures 5c and 6e).

(a) (b)

(f) (g)

(h)

(i)

(c)

(d)

(e)

Figure 5. KRP1 degradation is dependent on SCFSKP2b.

(a) GUS staining of a 6-day-old light-grown KRP1:KRP1–GUS seedling.

(b) GUS staining of a 6-day-old light-grown axr6-3 seedling carrying a KRP1:KRP1–GUS transgene.

(c) Eleven-week-old mature plants grown at 18�C. From left to right, wild-type (Col-0), axr6-3, axr6-3 KRP1:KRP1–GUS and KRP1:KRP1–GUS. Scale bar = 2 cm.

(d) Immunoblot analysis of Myc-KRP1 with an a-c-myc antibody. Protein extracts were prepared from 40-day-old plants grown at 18�C. An unknown protein

recognized by the a-c-myc antibody was used as a loading control (lower panel). Lane 1, wild-type (Col-0); lane 2, axr6-3; lane 3, axr6-3 35S:Myc-KRP1 (hemizygous);

lane 4, 35S:Myc-KRP1 (hemizygous).

(e) Eleven-week-old mature plants grown at 18�C. From left to right, wild-type (Col-0), axr6-3, axr6-3 35S:Myc-KRP1 (homozygous) and 35S:Myc-KRP1 (homozygous).

Scale bar = 2 cm.

(f) RT-PCR analysis of SKP2a, SKP2b and ACTIN2 expression in wild-type (Col-0) and SKP2-RNAi line (RNAi-29). Total RNAs were extracted from 7-day-old light-grown

seedlings. PCRs were performed for 25 cycles (ACTIN2) and 40 cycles (SKP2a and SKP2b).

(g) Left panel: GUS staining of 5-day-old light-grown KRP1:KRP1–GUS seedlings in the wild-type (Col-0) and SKP2-RNAi line (RNAi-29) (arrow). Right panel: close-up

view of the SKP2-RNAi KRP1:KRP1–GUS seedling.

(h) Three-week-old plants. From left to right: Col-0, 35S:SKP2b-TAP, 35S:SKP2b-TAP 35S:Myc-KRP1 (hemizygous), 35S:Myc-KRP1 (hemizygous). Scale bar = 0.5 cm.

(i) Myc-KRP1 and Myc-KRP1 levels in plants shown in (h). Top two panels show Myc-KRP levels measured with an a-c-myc antibody. Protein extracts were prepared

from 3-week-old plants. An unknown protein recognized by the a-c-myc antibody was used as a loading control. Bottom three panels show Myc-KRP1, SKP2b-TAP

and ACTIN2 levels measured by RT-PCR. Total RNA was extracted from 3-week-old plants. The c-myc epitope and tandem affinity purification (TAP) tag transcripts

were amplified to show Myc-KRP1 and SKP2b-TAP expression, respectively. PCRs were performed for 25 cycles.

710 Hong Ren et al.


These results indicate that CUL1 is required for KRP1

degradation.

The involvement of CUL1 in KRP1 degradation reveals

that an SCF mediates KRP1 protein turnover. Among the SCF

subunits, the F-box protein recognizes and binds substrates.

In mammals, the F-box protein SKP2 binds CKI p27Kip1

(Sutterluty et al., 1999; Tsvetkov et al., 1999) and the

transcription factor E2F-1 (Marti et al., 1999), as well as

other cell cycle regulators (Nakayama and Nakayama, 2005),

targeting them for degradation. Arabidopsis has two SKP2-

related F-box proteins SKP2a and SKP2b that are 83%

identical at the amino acid sequence level. It has been

shown that SKP2a binds the transcription factor E2Fc and

appears to mediate its degradation (del Pozo et al., 2002,

2006). Because of the sequence and functional relationship

between KRP1 and p27Kip1, we decided to investigate the

possibility that Arabidopsis SKP2 is involved in KRP1

degradation.

To test this possibility, we examined KRP1–GUS protein

stability in the SKP2a and SKP2b T-DNA insertion mutants.

We identified SKP2a and SKP2b T-DNA insertion mutants in

the GABI-KAT and SALK collection (Alonso et al., 2003;

Rosso et al., 2003; see Figure S1a). Neither skp2a-1 and

skp2b-1 nor a skp2a-1 skp2b-1 double mutant exhibited an

obvious phenotype. In addition, none of these mutants

stabilized KRP1–GUS (data not shown). To understand the

molecular nature of the skp2a-1 and skp2b-1 mutants, we

examined SKP2a and SKP2b expression by RT-PCR in the

skp2a-1 skp2b-1 double mutant. The full-length transcripts

of SKP2a and SKP2b could not be detected. However,

truncated transcripts were detected at a high level for SKP2a

and at a low level for SKP2b (see Figure S1c). Therefore,

truncated SKP2a and SKP2b proteins could be produced. If

these truncated proteins exist, SKP2a and SKP2b will have

the F-box domain, as well as four and two leucine-

rich repeats (LRRs), respectively (see Figure S1b). Thus,

it is possible that truncated SKP2a and SKP2b could

form a functional SCF complex that targets substrates for

degradation.

As an alternative, we examined KRP1–GUS protein sta-

bility in SKP2 RNA interference (RNAi) transgenic plants with

reduced levels of both SKP2a and SKP2b. We worked with

two independent lines and obtained similar results. SKP2-

RNAi transgenic lines did not exhibit any obvious defects in

growth and development (data not shown). Here, we show

results for the line RNAi-29. Compared with wild type, the

expression of both SKP2a and SKP2b was strongly de-

creased in this line (Figure 5f). The KRP1:KRP1–GUS trans-

gene was introduced into SKP2-RNAi transgenic plants by

crossing, and F1 plants were examined for GUS expression.

GUS staining was observed in the cotyledon of RNAi-29

seedlings (Figure 5g). Therefore, SKP2-RNAi transgenic

plants stabilize KRP1–GUS, indicating that SKP2a and/or

SKP2b are involved in KRP1 degradation.

To gain further evidence for a role for Arabidopsis SKP2 in

KRP1 degradation, we examined the effect of SKP2 overex-

pression on KRP1 degradation in planta. We generated

Arabidopsis transgenic plants that express a TAP (tandem

affinity purification) tagged SKP2a or SKP2b under the

control of the CaMV 35S promoter. We introduced the

35S:SKP2a-TAP and 35S:SKP2b-TAP transgenes into

35S:Myc-KRP1 plants by crossing. A 35S:SKP2a-TAP line

did not appear to alter the effects of Myc-KRP1 overexpres-

sion (data not shown). Interestingly, two independent

35S:SKP2b-TAP lines suppressed the effects of Myc-KRP1

overexpression. Here, we show results for line 5. An obvious

phenotype of KRP1 overexpressers is serrated rosette

leaves. Plants that overexpress both SKP2b-TAP and Myc-

KRP1 did not exhibit serrated rosette leaves (Figure 5h). The

loss of serrated leaf phenotype was associated with a

decreased Myc-KRP1 protein level. Three-week-old

35S:SKP2b-TAP 35S:Myc-KRP1 plants had much less Myc-

KRP1 than 35S:Myc-KRP1 plants (Figure 5i). We also exam-

ined Myc-KRP1 transcript levels in both lines and found

them to be similar (Figure 5i), confirming that decreased

Myc-KRP1 protein levels are caused by increased degrada-

tion. Taken together, our data indicate that KRP1 degrada-

tion is dependent on an SCF complex that consists of CUL1

and SKP2b.

The RING protein RKP also contributes to KRP1 degradation

Recent studies in mammalian cells indicate that at least two

different E3s contribute to p27Kip1 degradation: SCFSkp2 and

a RING-type E3 called KPC1 (Kamura et al., 2004b; Kotoshiba

et al., 2005). Because KRP1 is related to p27Kip1, a similar

KPC-dependent protein degradation mechanism might exist

in Arabidopsis to regulate KRP1 protein turnover. Interest-

ingly, there is a KPC1-related RING finger protein called

At2g22010 in Arabidopsis. Like mammalian KPC1,

At2g22010 has a RING finger domain in the C-terminus and a

SPRY domain with an unknown function near the N-termi-

nus (Stone et al., 2005; Figure 6a). The two proteins are

approximately 25% identical, and this conservation extends

along the entire length of the proteins. Based on this rela-

tionship we have named this protein RKP (related to KPC1).

Microarray experiments indicate that RKP RNA is present

throughout plant development, with particularly high levels

of expression in the embryo and senescing leaves (Schmid

et al., 2005).

To determine if RKP functions in KRP1 degradation, we

first examined the effect of loss of RKP on KRP1–GUS levels.

A RKP T-DNA insertion mutant was identified in the SAIL

collection (Sessions et al., 2002). The rkp-1 mutant contains

a T-DNA insertion in exon 2 of RKP that prevents the

formation of full-length RKP RNA. As any protein encoded

by the truncated transcript would not include the RING

domain, rkp-1 is likely to be a null mutant. When the



KRP1:KRP1–GUS transgene was crossed into the rkp-1 line

we found higher levels of GUS staining in mutant cotyle-

dons and flowers, indicating that RKP participates in KRP1

degradation (Figure 6b). To further investigate the role of

RKP in KRP1 degradation, we examined the effect of RKP

overexpression on KRP1 protein turnover in planta. We

introduced a 35S:HA-RKP construct into the 35S:Myc-KRP1

line exhibiting the serrated leaf phenotype typical of high

KRP1 levels (Figure 6c). In the T1 generation, 92% (34/37) of

kanamycin- and hygromycin-resistant 35S:HA-RKP

35S:Myc-KRP1 plants did not exhibit serrated rosette leaves.

We examined Myc-KRP1 protein levels in six independent

lines that lost the serrated rosette leaf phenotype by

immunoblot analysis using an a-c-myc antibody. All six

lines had much lower Myc-KRP1 protein levels than the

35S:Myc-KRP1 plants (data not shown). Here we show

results for line 6. Figure 6c shows that RKP overexpression

suppressed the serrated rosette leaf phenotype of 35S:Myc-

KRP1 plants. In addition, this suppression was associated

with reduced levels of Myc-KRP1 (Figure 6d).

Despite the clear role of RKP in KRP1 degradation, the rkp1

mutant did not exhibit any clear defects in growth and

development. As our results indicate that both RKP and

SKP2 participate in KRP1 degradation, we crossed the

SKP2:RNAi transgene into rpk-1 plants. The results in Figure

6b show that KRP1–GUS levels are higher in this line than

either parental line, confirming that both SKP2 and RPK

contribute to KRP1 degradation, probably independently.

However, again no defects in plant growth and development

were observed.

Discussion

In recent years, the ubiquitin-proteasome pathway has

been implicated in many aspects of cellular regulation and

development in plants, particularly hormone signaling.

Here, we provide clear evidence for an important role for

two different ubiquitin protein ligases in cell cycle regula-

tion. We show that both SCFSKP2b and the RING protein

RKP mediate the degradation of KRP1. In addition, the

absence of a clear growth defect in mutant lines deficient

in both E3s suggests that additional mechanisms regulate

KRP1 levels.

KRP1 interacts with the CDKA;1/CYCD2;1 complex

In eukaryotes, cell cycle progression is controlled by the

activities of CDK/cyclin complexes. In Arabidopsis, there are

five CDKs with known direct roles in the cell cycle, and at

least 31 cyclins of the three main classes of A, B and D types

(Menges et al., 2005; Vandepoele et al., 2002). In previous

studies, KRP1 was shown to bind CDKA;1 and three D-type

cyclins (CYCD1;1, CYCD2;1 and CYCD3;1) in yeast, but not

with CDKB1;1 (De Veylder et al., 2003; Jakoby et al., 2006;

Wang et al., 1998; Zhou et al., 2003). Furthermore, overex-

pression of CDKA;1 and CYCD2;1 in Arabidopsis whole

plants and trichomes, respectively, suppress the effects of

KRP1 overexpression (Schnittger et al., 2003; Zhou et al.,

2003).We have extended these results by showing that KRP1

forms a complex with CDKA;1 and CYCD2;1 in vivo, but not

with CDKB1;1.

A number of lines of evidence suggest that CDKA;1/

CYCD2;1 has a critical role in the G1–S transition (Healy

et al., 2001; Planchais et al., 2004). The in vivo interactions

between KRP1 and CDKA;1/CYCD2;1 strongly suggest that

KRP1 functions to regulate the G1–S transition, consistent

with genetic studies in which KRP1 overexpression was

shown to inhibit cell division and endoreduplication ( Wang

et al., 2000; Zhou et al., 2002a,b).

HsKPC1

(a)

(b)

(c)

(d)

RKP

SPRYdomain

RING fingerdomain

Myc-KRP1

KRP1:KRP1-

GUSKRP1

:KRP1

-GUS

rkp-

1 KRP1:KRP1-

GUS

rkp-

1,SKP2-

RNAi

Col-0 35S:HA-R

KP

35S:Myc-K

RP1

35S:H

A-RKP

35S:M

yc-K

RP1

Figure 6. KRP1 degradation is dependent on the RING protein RKP.

(a) Structure of human KPC1 (HsKPC1) and Arabidopsis RKP showing the

SPRY and RING domains (not drawn to scale).

(b) Seven-day-old seedlings or mature flowers stained for 24 h.

(c) Three-week-old plants.

(d) Immunoblot analysis of Myc-KRP1 levels. Protein extracts prepared from

4-week-old plants.

712 Hong Ren et al.


In addition to the CDKA;1/CYCD2;1 complex, KRP1 may

have other targets. A recent study reported that besides its

role in the G1–S transition, KRP1 also functions in the G2–M

transition to regulate mitosis entry (Weinl et al., 2005).

However, the CDK/cyclin complex target of KRP1 at the

G2–M transition is unknown. It will be important to identify

other CDK/cyclin complex targets of KRP1 in the future to

further define the role of KRP1 in cell cycle regulation.

Ectopic expression of KRP1 inhibits pericycle

activation during lateral root initiation

Lateral root formation is an example of post-embryonic

de novo organogenesis. In Arabidopsis, lateral roots are

derived from pericycle cells adjacent to the xylem poles

(Casimiro et al., 2003; Himanen et al., 2002). Pericycle acti-

vation appears to involve changes in the level of a protein

related to KRP1, called KRP2 (Himanen et al., 2002). The

KRP2 gene is expressed in pericycle cells and downregu-

lated in conditions that induce lateral root formation. Based

on these results it was proposed that KRP2 controls the G1–S

transition of pericycle cells during pericycle activation.

The KRP1 gene is also expressed in roots and is down-

regulated during lateral root formation (Himanen et al.,

2002). We demonstrate that KRP1 overexpression inhibits

auxin-mediated pericycle cell division, resulting in a dra-

matic decrease in the number of lateral roots. These results

suggest that KRP1 may also have a role in regulating

pericycle cell activation. However, the lack of a clear effect

of the krp1 mutation on lateral root development precludes a

definitive conclusion. In any case, it is clear that KRP1 is not

the only factor regulating this process.

SCFSKP2b mediates KRP1 degradation

Previous studies demonstrated that KRP1 is an unstable

protein that is degraded by the 26S proteasome (Jakoby

et al., 2006; Zhou et al., 2003). We have extended this work

using a KRP1–GUS fusion protein. Our results demonstrate

that the AXR1-RUB conjugation pathway is required for

KRP1 degradation. Because the only known substrates for

RUB conjugation are the cullin proteins, these results imply

that KRP1 degradation requires a cullin-based E3, such as an

SCF. Indeed we show that CUL1 is required for KRP1 deg-

radation, indicating that an SCF regulates KRP1 degradation.

In mammals p27Kip1 is degraded by SCFSkp2. In Arabid-

opsis, there are two proteins related to Skp2: SKP2a and

SKP2b. Despite the fact that KRP1 is only related to p27Kip1

through the cyclin- and CDK-binding/inhibitory domain, our

results indicate that SKP2b is involved in KRP1 degradation.

SKP2-RNAi lines with strongly decreased levels of both

SKP2a and SKP2b stabilize KRP1–GUS. Furthermore, we

have shown that SKP2b overexpression promotes KRP1

degradation in planta. Surprisingly, overexpression of the

closely related SKP2a protein did not suppress the effects of

Myc-KRP1 overexpression.Whether this is caused by a

difference in the function of SKP2a and SKP2b, or by an

artifact related to the specific 35S:SKP2a line, is unknown.

Thus, our results suggest that SKP2b targets KRP1 for

degradation, but do not exclude the possibility that SKP2a

has a similar function. It will be important to examine the

biochemical interactions between KRP1 and SKP2b in the

future. Taken together, our data suggest that an SCF

complex that is composed of CUL1 and SKP2b mediates

KRP1 degradation.

KRP1 degradation is also regulated by the RING protein RKP

Previous studies have identified two domains on KRP1 that

contribute to its instability, suggesting that there may be two

independent mechanisms of KRP degradation (Jakoby et al.,

2006). In mammalian cells, p27Kip1 degradation is mediated

by both SCFSkp2 and a RING protein called KPC1 (Kamura

et al., 2004b). Our genetic studies suggest that RKP, an

Arabidopsis protein related to KPC1, also contributes to

KRP1 degradation. The rkp-1 mutant stabilizes KRP1–GUS.

In addition, RKP overexpression promotes KRP1 degrada-

tion in planta. The observation that KRP1–GUS is signifi-

cantly more stable in rkp 35S:SKP2-RNAi plants than

either the rkp or 35S:SKP2-RNAi lines suggest that the

two E3s function independently. Furthermore, both path-

ways appear to be active in the same tissue. In mammals,

SCFSkp2-dependent degradation of p27Kip1 occurs in the

nucleus, whereas KPC functions in the cytoplasm (Kamura

et al., 2004a). Jakoby et al. (2006) have shown that KRP1

localizes primarily to the nucleus, but is found in different

subnuclear domains. It will be interesting to determine if

RKP and SCFSKP2b mediate KRP1 degradation in distinct

cellular compartments.

Despite the clear involvement of SCFSKP2 and RKP in KRP1

degradation, plants deficient in either or both of these E3s do

not display any growth defects. This is surprising, as

relatively modest changes in KRP1 levels exert an effect on

development. For example, the KRP1:KRP1–GUS transgene

acts to enhance the phenotype of the axr1-3 and axr6-3

mutants. Our failure to detect a phenotype in SKP2-RNAi and

rkp lines may be caused by residual SCFSKP2 activity in the

RNAi lines and redundancy between SCFSKP2 and RPK.

Alternatively, it is possible that Arabidopsis has an addi-

tional mechanism of KRP1 degradation, perhaps via another

E3 enzyme.

Experimental procedures

Plant materials and growth conditions

Arabidopsis thaliana plants were grown under 24-h light conditionsat 22�C, or 18�C when necessary. All mutants and transgenic lines



were in the Columbia ecotype. The skp2a-1 mutant (GABI-Kat293D12), a T-DNA insertion in At1g21410, was acquired fromGABI-KAT; Rosso et al., 2003). The skp2b-1 mutant (SALK_028396),a T-DNA insertion in At1g77000, was acquired from the ArabidopsisBiological Research Center (ABRC, http://www.arabidopsis.org;Alonso et al., 2003). The rkp mutant (SAIL_3_E3), a T-DNA insertionin At2g22010, was acquired from the ABRC (Sessions et al., 2002).All T-DNA mutants were confirmed by PCR and sequencing. Seedswere surface sterilized in a 30% bleach and 0.04% Triton X-100solution, and were cold treated for 2–3 days at 4�C to synchronizegermination. Seeds were grown on ATS medium [1% sucrose,5 mM KNO3, 2.5 mM KH2PO4 (pH 5.6), 2 mM MgSO4, 2 mM Ca(NO3)2,50 lm CuSO4, 1 lM ZnSO4, 0.2 lm NaMoO4, 10 lm NaCl and0.01 lM CoCl2) with or without 0.8% agar. N-1-Naphthylphthalamicacid (NPA), 1-naphthaleneacetic acid (NAA), herbicide basta(AgroEvo) and antibiotics were added to autoclaved ATS medium,when necessary.

Transgenic lines

A 2062-bp KRP1 (At2g23430) promoter was amplified from genomicDNA with primers KRP1-PF (5¢-GTTCAAGCGAGTGACACATCTC-3¢)and KRP1-PR (5¢-CTTCGATTTAGGTTACGTGTGCG-3¢). TheKRP1:GUS plasmid was constructed by cloning the KRP1 promoterto pCB308 containing the Escherichia coli GUS (Xiang et al., 1999). A602-bp KRP1 full-length cDNA was amplified from a cDNA librarywith primers KRP1-F (5¢-ACGCACACGTCACCTAAATC-3¢) and KRP1-R (5¢-CTTCACTCTAACTTTACCCATTCG-3¢). The KRP1:KRP1–GUSplasmid was constructed by cloning both the KRP1 promoter and theKRP1 full-length cDNA without the stop codon TGA to pCB308. Tomake a 35S:Myc-KRP1 construct, the KRP1 full-length cDNA withoutthe start codon ATG was fused to the C-terminus of six c-mycepitopes in pGEM7Z. The Myc-KRP1 insert was then cloned topROK2.

To make a 35S:SKP2-RNAi construct, a 433-bp cDNA fromnucleotides 256–688 of SKP2a (At1g21410) was cloned in anopposite orientation to pHANNIBAL (Wesley et al., 2001). TheSKP2-RNAi insert was then cloned to pBIN19. To make a35S:SKP2b-TAP construct, an SKP2b (At1g77000) full-length cDNAwithout the stop codon TGA was fused to the N-terminus of a TAP(tandem affinity purification) tag (Rigaut et al., 1999). The SKP2b-TAP insert was then cloned to pPILY (Ferrando et al., 2000).

A 3861-bp RKP full-length cDNA without the start codon ATG wasamplified with primers RKP-F (5¢-CACCTTGGCTGAAGACAGCCT-ACGG-3¢) and RKP-R (5¢-GCAACTAACCCGAGCTTCATGTGC-3¢), andwas cloned to pENTR/D-TOPO using the pENTR directional TOPOcloning kit (Invitrogen, http://www.invitrogen.com). The RKP insertin pENTR/D-TOPO was then cloned to pGWB15 (http://bio2.ipc.shimane-u.ac.jp/pgwbs/index.htm) containing the CaMV 35S pro-moter and three hemagglutinin epitopes to make a 35S::HA-RKPconstruct.

All the above constructs in the binary vectors were introducedinto Agrobacterium tumefaciens strain GV3101. Plants were trans-formed by the vacuum infiltration method (Bechtold and Pelletier,1998). Transgenic plants were selected on ATS plates supplementedwith the necessary antibiotics or herbicide. The KRP1:GUS andKRP1:KRP1–GUS transgenic plants are basta resistant. The35S:Myc-KRP1 and 35S::HA-RKP transgenic plants are kanamycin-and hygromycin-resistant, respectively.

GUS assays

To examine GUS expression, seedlings were incubated in a GUSstaining solution at 37�C as described by Oono et al. (1998). GUS-

stained seedlings were incubated in 70% ethanol to remove chlo-rophyll. GUS staining patterns were examined under a NikonSMZ1500 dissecting microscope (http://www.nikonusa.com).

RT-PCR analysis

Total RNAs were extracted using the TRI reagent (Sigma, http://www.sigmaaldrich.com). The first-strand cDNAs were synthesizedfrom 5 lg total RNAs using oligo(dT)20 primer and SuperScript IIRNase H) reverse transcriptase (Invitrogen). PCRs were performedwith the following gene-specific primers: SKP2a-F, 5¢-CCGCTTC-ATTTTAGTCATTAAAC-3¢; SKP2a-R1, 5¢-GGCCGTTTATATATACAA-CATAAC-3¢; SKP2a-R2, 5¢-TGATTGCAGTTATTCCCAATAG-3¢;SKP2b-F, 5¢-CATATTTACTTTTGATCTCGTGG-3¢; SKP2b-R1,5¢-CATACTAGAGAGTAGTAGACC-3¢; SKP2a-R2, 5¢-CGAGTTTAGT-CAGGTTAGTA-3¢; Myc-F, 5¢-GACTCTAGAGGATCCCCAAAGC-3¢;Myc-R, 5¢-AGCCGAATTCGATGGGGTACCG-3¢; TAP-F, 5¢-TAG-CCGTCTCAGCAGCCAACC-3¢; TAP-R, 5¢-CTTCCCCGCGGAATTCGC-GTC-3¢; ACTIN2-F, 5¢-GGCTGAGGCTGATGATATTC-3¢; ACTIN2-R,5¢-TCTGTGAACGATTCCTGGAC-3¢.

Immunoblot analysis and immunoprecipitation

Protein extracts were prepared as described by Gray et al. (1999).For immunoblot analysis, 50 lg protein extracts were mixed withSDS-PAGE sample buffer and were boiled for 5 min. Denaturedproteins were separated on a 10% acrylamide SDS gel and weretransferred to a nitrocellulose membrane. The membrane wasimmersed in Tris-buffered saline (pH 7.6) containing 5% non-fat drymilk and 0.1% Tween 20 to block non-specific binding sites. Thea-c-myc 9E 10 antibody (Covance Research Products, http://www.crpinc.com) was used at a 1:1000 dilution. The horseradishperoxidase-conjugated goat a-mouse secondary antibody (Sigma)was used at a 1:3000 dilution. Proteins were detected with theenhanced chemiluminescence (ECL) kit (Amersham PharmaciaBiotech, http://www.gelifesciences.com).

For immunoprecipitation, 5 ll a-c-myc 9E 10 antibody was addedto 3 mg protein extracts and was incubated for 1–3 h at 4�C. To collectimmune complexes, 30 ll of protein A agarose beads (Roche, http://www.roche.com) were added and were incubated for from 3 h toovernight. Immune complexes were washed three times in 1 ml ofprotein extraction buffer. Finally, agarose beads were resuspendedin SDS-PAGE sample buffer. Immunoblot analysis was carried out asdescribed above. The a-CDKA;1 antibody was used at a 1:5000dilution. The a-CDKB1;1 and a-CYCD2;1 antibodies (Healy et al.,2001) were used at a 1:3000 dilution. The horseradish peroxidase-conjugated goat a-rabbit secondary antibody (Chemicon Interna-tional, http://www.millipore.com) was used at a 1:2500 dilution.

Acknowledgements

We thank Dirk Inze for the a-CDKA;1 antibody and CYCB1;1:GUSline, William M. Gray for the axr6-3 mutant, the SALK InstituteGenomic Analysis Laboratory, ABRC and GABI-KAT for T-DNAinsertion mutants. This work was supported by grants from theNational Science Foundation (NSF 2010 MCB-0115870) to ME andthe Spanish MEC (BIO2004-01749) to JCdP.

Supplementary material

The following supplementary material is available for this articleonline:

714 Hong Ren et al.


Figure S1. RT-PCR analysis of SKP2a and SKP2b expression in theSKP2a and SKP2b T-DNA insertion mutants.Table S1. Developmental expression of KRP1.This material is available as part of the online article from http://www.blackwell-synergy.comPlease note: Blackwell Publishing are not responsible for thecontent or functionality of any supplementary materials suppliedby the authors. Any queries (other than missing material) should bedirected to the corresponding author for the article.

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Degradation of the cyclin-dependent kinase inhibitor …labs.biology.ucsd.edu/estelle/publications/Ren.et.al...Degradation of the cyclin-dependent kinase inhibitor KRP1 is regulated