The Medicago CDKC;1-CYCLINT;1 kinase complexphosphorylates the carboxy-terminal domainof RNA polymerase II and promotes transcription

Katalin Fulop1,2, Aladar Pettko-Szandtner1, Zoltan Magyar1,3, Pal Miskolczi1, Eva Kondorosi1,2, Denes Dudits1 and

Laszlo Bako1,4,*

1Institute of Plant Biology, Biological Research Center of the Hungarian Academy of Sciences, H-6701 Szeged, Hungary,2Institut des Sciences du Vegetal, CNRS-UPR2355, 91198 Gif sur Yvette, France,3Royal Holloway, School of Biological Sciences, University of London, Egham, TW20 OEX, UK, and4Department of Plant Physiology, Umea Plant Science Center, Umea University, S-901 87 Umea, Sweden

Received 27 October 2004; revised 31 January 2005; accepted 9 March 2005.*For correspondence (fax þ46 90 7866676; e-mail laszlo.bako@plantphys.umu.se).

Summary

The Ms;CDKC;1 kinase is structurally similar to those cyclin-dependent kinases (CDKs) that are not involved

directly in cell cycle regulation. The presence of a PITAIRE motif in Ms;CDKC;1 suggests that it interacts with

cyclins different from known PSTAIRE/PPTALRE kinase regulatory subunits. Here we demonstrate that a

Medicago CYCLINT (CYCT) protein is a specific interactor of Ms;CDKC;1 and the interaction between these two

proteins gives rise to an active kinase complex that localizes to the nucleus and phosphorylates the carboxy-

terminal YSPTSPS heptapeptide repeat domain (CTD) of the largest subunit of RNA polymerase II in vitro.

Mutation of Ser to Ala at position 5 within the heptapeptide repeat abolishes substrate phosphorylation by the

Ms;CDKC;1 kinase complex. Furthermore, our data show that addition of the Medicago CDKC;1-CYCT;1

heterodimer completely restored the transcriptional activity of a HeLa nuclear extract depleted of endogeneous

CDK9 kinase complexes. Together, these results indicate that the Medicago CDKC;1-CYCT;1 complex is a

positive regulator of transcription in plants and has a role similar to the CDK9/cyclin T complex of human

positive transcription elongation factor P-TEFb.

Keywords: cell cycle, CDK-cyclin complex, CTD kinase, P-TEFb, transcription, Medicago.

Introduction

Cyclin-dependent protein kinases (CDKs) play a central role

in the control of eukaryotic cell division (Nigg, 1995). During

different phases of the cell cycle CDKs are found in associ-

ation with defined cyclins and other regulatory proteins

(Draetta, 1994; O’Connell and Nurse, 1994). In yeasts, a

particular CDK enzyme, Cdc2/CDC28, has been shown to

bind members of the Cln and Clb cyclin families (Nasmyth,

1993). In animals, a number of differentially regulated

cyclins are known to form complexes with different CDKs

(Lew and Kornbluth, 1996; Morgan, 1997). Plants also have

multiple CDKs classified into six types from A to F (Joubes

et al., 2000; Vandepoele et al., 2002; reviewed by Dewitte

and Murray, 2003). A-type CDKs are orthologs of the yeast

Cdc2/CDC28 proteins, they contain the classical PSTAIRE

motif in their cyclin-binding site and their complexes display

kinase activity both in G1/S and G2/M transitions (Magyar

et al., 1997). B-type CDKs are plant-specific kinases with

PPTALRE or PPTTLRE motif, they are expressed in a cell

cycle-dependent manner and regulate G2/M transition

(Porceddu et al., 2001). C-type CDKs are PITAIRE kinases that

are constitutively expressed during cell cycle. It has been

proposed recently that members of this family are involved

in the regulation of transcription (Barroco et al., 2003). The

D- and F-type CDKs are CDK-activating kinases (CAKs). In

addition to CAK activity, D-type rice R2 and Arabidopsis

CAK2At and CAK4At kinases also phosphorylate the CTD of

the largest subunit of RNA polymerase II (Shimotohno et al.,

2003; Yamaguchi et al., 1998).

In yeast and metazoans certain CDKs are implicated in

the control of transcriptional processes by altering the

810 ª 2005 Blackwell Publishing Ltd

The Plant Journal (2005) 42, 810–820 doi: 10.1111/j.1365-313X.2005.02421.x

phosphorylation pattern of the CTD of the largest subunit of

RNA polymerase II (Prelich, 2002). RNA polymerase II CTD is

hypophosphorylated in the pre-initiation complex but gets

heavily phosphorylated during initiation and transcript

elongation (Dahmus, 1996). CDK/cyclin complexes known

to be involved in the phosphorylation of the CTD are the

Kin28/Ccl1, Srb10/Srb11, Ctk1/Ctk2 and Bur1/Bur2 com-

plexes in yeast and the CDK7/cyclin H, CDK8/cyclin C and

CDK9/cyclin T and K complexes in metazoans (Kobor and

Greenblatt, 2002). These kinase-cyclin pairs have distinct

biochemical properties and their kinase activity is required at

different phases of the transcription cycle (Ramanathan

et al., 2001; Rickert et al., 1999).

The CDK7/cyclin H/Mat1 trimeric complex was first des-

cribed as a CAK kinase activity in higher eukaryotes (Devault

et al., 1995; Poon et al., 1993; Solomon et al., 1993). Later it

was shown that the heterotrimer forms the catalytical core of

the general transcription factor TFIIH and is responsible for

its associated CTD kinase activity (Roy et al., 1994). TFIIH is

part of the pre-initiation complex and a positive regulator of

transcription as it phosphorylates the CTD shortly after

initiation of transcription (Hengartner et al., 1998). In con-

trast, the CDK8/cyclin C complex and its yeast homolog

Srb10/Srb11 are negative regulators, which inhibit the

formation of transcription pre-initiation complexes by phos-

phorylating the CTD (Hengartner et al., 1998). CDK8 has also

been demonstrated to phosphorylate cyclin H within the

TFIIH complex resulting in the inhibition of its CTD kinase

activity (Akoulitchev et al., 2000). CDK9 was originally iso-

lated as a PITALRE kinase capable of phosphorylating the

retinoblastoma protein (Grana et al., 1994). It was later

identified as the catalytic subunit responsible for the CTD

kinase activity of the previously described positive tran-

scription elongation factor b (P-TEFb) (Marshall and Price,

1992; Zhu et al., 1997). CDK9 forms active complexes with

multiple cyclins including cyclin T1, T2 and cyclin K (Fu et al.,

1999; Peng et al., 1998a). These complexes are involved in

the control of elongation by phosphorylating the CTD of

RNA polymerase II.

With the exception of CDK8, plants appear to contain

homologs of the yeast and metazoan CTD kinases. The

D-type rice R2 kinase is related to human CDK7, the protein is

localized to the nucleus and activated by binding to

CYCLINH, and phosphorylates CDKs and the CTD of RNA

polymerase II (Yamaguchi et al., 2000). These properties

imply that plant CDKD-CYCLINH complexes are likely coun-

terparts of the metazoan CDK7/cyclin H complex, although

their function in transcription is not yet known. The C-type

CDKs carrying the PITAIRE cyclin-binding motif are the

closest homologs of the metazoan CDK9 proteins. Members

of this class were identified from Arabidopsis, alfalfa and

tomato (Barroco et al., 2003; Joubes et al., 2001; Magyar

et al., 1997). A yeast two-hybrid screen with the Arabidopsis

At;CDKC;2 bait identified a protein homologous to animal

cyclin T and K as well as ribonucleoproteins and transcrip-

tion factors suggesting a role for At;CDKC in transcriptional

control (Barroco et al., 2003).

Our work aimed at demonstrating the function of

Ms;CDKC;1 in transcriptional regulation. We have identified

the interacting cyclin partner of Ms;CDKC;1 and investigated

the biochemical properties of the Medicago CDKC;1-CYCT;1

complex. We show here that the complex is localized to the

nucleus and displays cell cycle-independent CTD kinase

activity. Due to its kinase activity the complex can replace the

human CDK9/cyclin T function in depleted HeLa nuclear

extract and promotes transcription in vitro. Thus our results

confirm a novel CDK function in plants by demonstrating the

positive role of the CDKC;1-CYCT;1 complex in the regula-

tion of RNA polymerase II-mediated transcription.

Results

Identification of a CYCLINT protein as specific interactor of

the Ms;CDKC;1 kinase

As cyclins modulate the temporal activity and substrate

specificity of the CDKs, their identification is crucial in the

functional characterization of a given CDK. To identify cyclin

partners of the Ms;CDKC;1 kinase, a yeast two-hybrid inter-

action screen was performed with Ms;CDKC;1 fused to the

GAL4 DNA-binding domain as bait. An M. truncatula cDNA

library constructed in the pAD-GAL4 phagemid vector in

fusion with the GAL4 activation domain (Gyorgyey et al.,

2000) was applied as prey. From the 9 · 105 independent

yeast transformants screened, 78 positive interactors were

detected of which 77 represented various regions of the

same gene. The longest clone was 1390 bp in length and

contained an open reading frame of 1119 bases coding for a

polypeptide of 372 amino acids with a relative molecular

mass of 42.6 kDa. Search of the M. truncatula database with

the cDNA sequence yielded the AW691348 and CA921228

EST clones that perfectly matched to the 5¢ and 3¢ ends of thecDNA, respectively, indicating that the gene is expressed.

The predicted protein was most similar to Arabidopsis

CYCLINT and CYCLINT-like proteins as well as Drosophila

cyclin T and human cyclin T and K proteins (Figure 1).

Between amino acids 137 and 281 the M. truncatula protein

harbors a cyclin box, a helical protein recognition domain

characteristic of cyclins (Noble et al., 1997). To rule out the

possibility that this cyclin is a member of the cyclin K family,

we testedwhether it would rescue the conditional lethality of

the yeast strain Y145 carrying cln1, cln2 and cln3 deletions. It

was shown that expression of human cyclin K in the Y145

background rescues conditional lethality (Edwards et al.,

1998). The M. truncatula cyclin did not restore growth

independently if it was expressed from centromeric or 2 lplasmids in Y145 yeast cells (data not shown). Due to

this observation and to comparative analysis with the

Analysis of the Medicago CDKC;1-CYCLINT;1 complex 811

ª Blackwell Publishing Ltd, The Plant Journal, (2005), 42, 810–820

reannotated set of Arabidopsis cyclins that positioned this

cyclin to the phylogenetic cluster containing human K/L/T-

and Arabidopsis T-type of cyclins (Wang et al., 2004), we

designated this M. truncatula protein as Medtr;CYCLINT;1.

The specificity of interaction between Ms;CDKC;1 and

Mt;CYCT;1was analyzed in pairwise yeast two-hybrid assays

by testing their associations with several known Medicago

CDKs and cyclins. In this experimental system a strong and

reproducible interaction was detected only between the

Ms;CDKC;1 and the Mt;CYCT;1 proteins, independently of

the GAL4-AD or GAL4-BD protein domains they were fused

to (Figure 2a). To test the interaction between Ms;CDKC;1

and Mt;CYCT;1 in vivo, their full-length cDNAs were cloned

in translational fusion with three tandem copies of the

c-myc- or HA-epitopes. Proteins were expressed under the

control of the cauliflower mosaic virus 35S RNA promoter in

Arabidopsis protoplasts and the expression of tagged

proteins was monitored byWestern blotting with antibodies

recognizing the respective epitopes. Complex formationwas

then analyzed in lysates of cotransformed protoplasts by

immunoprecipitation with anti-HA and subsequent Western

blotting with anti-myc antibodies. The results of these

experiments clearly demonstrated the interaction between

Ms;CDKC;1 and Mt;CYCT;1 proteins in vivo (Figure 2b).

Ms;CDKC;1 forms an active kinase complex that

phosphorylates the carboxy-terminal repeat domain

of the largest subunit of RNA polymerase II

For the biochemical characterization of Ms;CDKC;1 kinase

complexes it was first necessary to generate Ms;CDKC;

1-specific antibodies. We expressed the carboxy-terminal

half of Ms;CDKC;1 as a hexahistidine-tagged fusion protein

in bacteria and immunized rabbits with the purified protein.

The affinity-purified antibody recognized a single protein of

the expected size of 56 kDa on Western blots. It was then

used in immunoprecipitation protein kinase assays to test

whether Ms;CDKC;1 forms active kinase complexes. Activity

of the immunocomplexes purified from M. sativa cell

extracts was analyzed with proteins known to be good

substrates of CDK kinases in vitro. In the absence of any

substrate, no radioactive signal was detected suggesting

the lack of Ms;CDKC;1 self-phosphorylation reaction. How-

ever, the immunoprecipitated Ms;CDKC;1 kinase readily

At;CYCT-like1 27 ..................................EEVSRWYFGRKEIEENAt;CYCT-like2 27 ..................................DEVARWYFGRKEIEENMt;CYCT;1 101 ..................................DDDKPIFMSRDDIDRNAt;CYCT 28 ..................................CETSKWYFSREEIERFHsCycT1 6 ..................................NNNKRWYFTREQLE.N

At;CYCT-like1 44 SPSRLDGIDLKKETYLRKSYCTFLQDLGMRLKVFPISPQVTIATAIIFCHAt;CYCT-like2 44 SPSRLDSIDLKKETYLRKSYCTFLQDLGMRLKV....PQVTIATAIIFCHMt;CYCT;1 151 SPSRKDGIDVLHETHLRYSYCAFLQNLGTRLEM....PQTTIGTSMVLCHAt;CYCT 45 SPSRKDGIDLVKESFLRSSYCTFLQRLGMKLHV....SQVTISCAMVMCHHsCycT1 22 SPSRRFGVDPDKELSYRQQAANLLQDMGQRLNV....SQLTINTAIVYMH

At;CYCT-like1 94 RFFFRQSHAKNDRRTIATVCMFLAGKVEETPRPLKDVIFVSYEIINKKDPAt;CYCT-like2 90 RFFIRQSHARNDRRTIATVCMFLAGKVEETPRPLKDVIVVSYEIIHKKDPMt;CYCT;1 197 RFFVRRSHACHDRFLIATAALFLAGKSEESPCPLNSVLRTSSELLHKQDFAt;CYCT 91 RFYMRQSHAKNDWQTIATSSLFLACKAEDEPCQLSSVVVASYEIIYEWDPHsCycT1 68 RFYMIQSFTQFPGNSVAPAALFLAAKVEEQPKKLEHVIKVAHTCLHPQE.

At;CYCT-like1 144 GASQKIKQKEVYEQQKELILNGEKIVLSTLGFDLNVYHPYKPLVEAIKKFAt;CYCT-like2 140 TTAQKIKQKEVYEQQKELILNGEKIVLSTLGFDFNVYHPYKPLVEAIKKFMt;CYCT;1 247 AFLSYWFPVDWFEQYRERVLEAEQLILTTLNFELGVQHPYAPLTSVLNKLAt;CYCT 141 SASIRIHQTECYHEFKEIILSGESLLLSTSAFHLDIELPYKPLAAALNRLHsCycT1 116 ..SLPDTRSEAYLQQVQDLVILESIILQTLGFELTIDHPHTHVVKCTQLV

At;CYCT-like1 194 KVAQNALAQVAWNFVNDGLRTSLCLQFKPHHIAAGAIFLAAKFLKVKLPSAt;CYCT-like2 190 KVAQNALAQVAWNFVNDGLRTSLCLQFKPHHIAAGAIFLAAKFLKVKLPSMt;CYCT;1 297 GLSKTVLVNMALNLVSEGLRSSLWLQFKPHQIAAGAAYLAAKFLNMDLAAAt;CYCT 191 NAWP.DLATAAWNFVHDWIRTTLCLQYKPHVIATATVHLAATFQNAKVGSHsCycT1 165 RASKDLAQTSYFMATNSLHLTTFSLQYTPPVVACVCIHLACKWSNWEIPV

At;CYCT-like1 244 DGEKVWWQEFD...VTPRQLEDVSNQMLELYEQN................At;CYCT-like2 240 DGEKVWWQEFD...VTPRQLEDVSNQMLELYEQN................Mt;CYCT;1 347 YKN..IWQEFQ...ATPSVLQDVSQQLMELF...................At;CYCT 240 RRD..WWLEFG...VTTKLLKEVIQEMCTLIEVD................HsCycT1 215 STDGKHWWEYVDATVTLELLDELTHEFLQILEKT................

Figure 1. Comparison of the Medicago truncatula CYCLINT;1 amino acid sequence (Mt;CYCT;1) with human cyclin T1 (HsCycT1) and Arabidopsis CYCLINT-related

proteins. The alignment shows the cyclin box region only. Amino acids that are identical in all proteins are boxed, the majority is indicated by reverse shading.

Accession numbers for the aligned proteins are: AAD46000 (At;CYCT), BAB11392 (At;CYCT-like1), CAB40377 (At;CYCT-like2), AAR01224 (Mt;CYCT;1) and O60563

(HsCycT1).

812 Katalin Fulop et al.

ª Blackwell Publishing Ltd, The Plant Journal, (2005), 42, 810–820

phosphorylated the myelin basic protein (MBP) and a syn-

thetic polypeptide comprising six copies of the C-terminal

YSPTSPS repeat domain of the largest subunit of RNA

polymerase II. The Ms;CDKC;1 kinase also phosphorylated

the human retinoblastoma protein, albeit to a lesser extent.

In contrast, the typical CDK substrate histone H1 and

beta-casein were not phosphorylated at all indicating that

they are not substrates of Ms;CDKC;1 (Figure 3a).

To obtain further biochemical evidence supporting that

Ms;CDKC;1 has associated CTD kinase activity and to

examine the subcellular localization of the kinase, cytoplas-

mic and nuclear protein fractions were prepared from

cultured M. sativa cells. The amount and activity of

Ms;CDKC;1 was tested in these fractions byWestern blotting

and immunoprecipitation protein kinase assays. The

Ms;CDKC;1 protein as well as the CTD kinase activity were

detectable in the whole cell extract and at a highly enriched

level in the nuclear fraction suggesting a nuclear localization

of Ms;CDKC;1 in cultured M. sativa cells (Figure 3b). To

determine the subcellular localization of the Ms;CDKC;

1-Mt;CYCT;1 kinase complex, subunits tagged with myc- or

HA-epitopes were co-expressed in Arabidopsis protoplasts.

Ms;CDKC;1 and Mt;CYCT;1 proteins were detected with

secondary antibodies conjugated to red and green fluoro-

chromes. Both proteins accumulated exclusively in the

nucleus supporting the results of the biochemical fraction-

ation experiments (Figure 3c).

A;1 A;2 C;1 B1;1 E;1 B2;1

B2;1 B2;2 A2;1 D3;1 D4 D5 T;1

CD

KC

;1C

YC

LIN

T;1

Bai

t:

Medsa;CDK

Medsa;CYC Medtr;CYC

(a)

(b)

++++––––++––––––––––++––––––++++

IPSIPSIPSIPS

75

50

37

HA-CDKC;1

myc-CDKC;1

HA-CYCT;1

myc-CYCT;1

* * * *

Figure 2. Mt;CYCT;1 is a specific interactor of the Ms;CDKC;1 kinase.

(a) To assess specificity, interaction of Ms;CDKC;1 with different cyclins as

well as interaction of Mt;CYCT;1 with various CDK kinases was tested. Yeasts

co-transformed with the respective constructs were grown on selective

medium lacking tryptophan, leucine, histidine and adenine. Classification of

CDKs and cyclins is according to Joubes et al. (2000) and Wang et al. (2004),

respectively.

(b) Ms;CDKC;1 interaction with Mt;CYCT;1 in vivo. Arabidopsis protoplasts

were transfected with constructs expressing epitope-tagged Ms;CDKC;1 and

Mt;CYCT;1 proteins or co-transfected to investigate complex formation.

Proteins were analyzed both in the supernatants (S) and in the anti-HA

precipitated fraction (IP) by immunoblotting with an anti-myc antibody.

Position of the epitope-labeled Ms;CDKC;1 and Mt;CYCT;1 proteins are

marked with filled and open arrowheads, respectively. Cross-reaction of the

immunoglobulin heavy chain with the secondary antibody is labeled by

asterisk. Molecular mass standards are indicated in kilodaltons to the left.

66453629

14

20

97

Cas

ein

(CTD

) 6H

isto

ne H

1M

BP

GST

-Rb

w/o

Sub

stra

te

WCE NECE

α RNAP II

α CDKC;1

(CTD)6-32P

CDKC;1

IIoIIa

(a) (b)

(c)

3 5 7 9 11 13 15 17

205

116

RNAPII

66

45CDKC;1

(CTD)6-32P

myc-CDKC;1

HA-CYCT;1

– + + – –– – + – + +

+

α myc IP α HA IP

(CTD)6-32P

(d)

(e)

myc-CDKC;1 DAPI

TMHA-CYCT;1

Figure 3. Ms;CDKC;1 is a nuclear CTD kinase.

(a) Ms;CDKC;1 was precipitated from Medicago sativa cell extracts and kinase

activity of the immunocomplexes was tested with different substrates.

Phosphorylated proteins were resolved by SDS-PAGE and detected by

autoradiography. Molecular mass standards are indicated in kilodaltons to

the left.

(b) Whole cell extract (WCE) as well as cytosolic (CE) and nuclear (NE) protein

fractions were immunoblotted with anti-RNA polymerase II and anti-

Ms;CDKC;1 antibodies. IIo and IIa denote the hyper- and hypophosphorylated

forms of the largest subunit of RNA polymerase II. CTD kinase activity was

tested in the same protein fractions.

(c) Arabidopsis cells expressing Myc-Ms;CDKC;1 and HA-Mt;CYCT;1 proteins

were dual labeled with TRITC-conjugated anti-myc and Alexa 488-conjugated

anti-HA antibodies. The nucleus was visualized with DAPI staining. TM,

transmission image.

(d) Nuclear proteins were separated according to their size by glycerol

gradient centrifugation. Fractions were analyzed by Western blotting and

immunoprecipitation followed by CTD kinase assays.

(e) Binding of Mt;CYCT;1 to Ms;CDKC;1 results in enhanced CTD kinase

activity. myc-Ms;CDKC;1 and HA-Mt;CYCT;1 proteins were expressed in

Arabidopsis protoplasts and the effect of complex formation was analyzed

by anti-myc and anti-HA immunoprecipitation followed by CTD kinase assays.

Analysis of the Medicago CDKC;1-CYCLINT;1 complex 813

ª Blackwell Publishing Ltd, The Plant Journal, (2005), 42, 810–820

As it is known that certain CTD kinases, like the metazoan

CDK7/cyclin H and CDK8/cyclin C or the yeast Srb10/Srb11

complexes, are associated with the RNA polymerase II

holoenzyme (Chao et al., 1996; Koleske and Young, 1994)

we tested in the nuclear protein fraction whether Ms;CDKC;1

associates with RNA polymerase II. To examine this possi-

bility, nuclear proteins were size-fractionated by glycerol

gradient centrifugation and the fractions were probed for

RNA polymerase II and Ms;CDKC;1 proteins by immuno-

blotting. This experiment revealed that the Ms;CDKC;1

protein and its associated CTD kinase activity sediment in

a complex of smaller size than that of RNA polymerase II

holoenzyme (Figure 3d).

To investigate the effect of Mt;CYCT;1 binding on

Ms;CDKC;1 kinase activity, myc-Ms;CDKC;1 and

HA-Mt;CYCT;1 proteins were expressed either alone or

together in Arabidopsis protoplasts. Epitope-tagged sub-

units and their complexes were then immunoprecipitated

with the respective antibodies and CTD kinase activitieswere

tested. When the Ms;CDKC;1 or Mt;CYCT;1 subunits were

expressed alone, a modest CTD kinase activity of the immu-

nocomplexes was detected. Co-expression of both subunits

resulted in a severalfold increase of CTD kinase activity

indicating a true functional interaction between Ms;CDKC;1

and Mt;CYCT;1 that facilitates catalytic function (Figure 3e).

The level and kinase activity of Ms;CDKC;1 protein

is not cell cycle dependent

We have demonstrated earlier that, similar to Ms;CDKA;1

and Ms;CDKA;2 genes, expression of the Ms;CDKC;1 gene

is constitutive during the cell cycle (Magyar et al., 1997).

However, the histone H1 kinase activity associated with the

Ms;CDKA proteins does oscillate during cell cycle, dis-

playing increased activity at the G1/S and G2/M transitions

(Meszaros et al., 2000). To test whether the Ms;CDKC;1

protein level or its CTD kinase activity would show any cell

cycle-dependent alteration, we used an M. sativa cell cul-

ture synchronized to the G1/S phase by hydroxyurea

treatment. The Ms;CDKC;1 protein level was examined in

protein lysates of the synchronous culture by Western

blotting whereas the kinase activity was tested by immu-

noprecipitation protein kinase assays. Both the Ms;CDKC;1

protein and the CTD kinase activity levels remained almost

constant throughout the S and G2/M phases of the cell

cycle (Figure 4) arguing against a direct involvement of

Ms;CDKC;1 in cell cycle control. As a control for synchro-

nicity, the Ms;CDKB2;1 (formerly Cdc2MsF) kinase, the

protein level of which is known to show a characteristic G2/M

accumulation (Magyar et al., 1997), was also analyzed by

immunoblotting. As expected, the Ms;CDKB2;1 protein

level was almost undetectable during the S-phase, then

gradually increased through G2 and reached a maximum in

mitosis.

The Ms;CDKC;1 kinase targets 5Ser residue within the CTD

The 1YSPTSPS7 heptapeptide repeat contains several

potential phosphorylation sites that are targeted by distinct

protein kinases (Ramanathan et al., 2001). As the effect of

phosphorylation on RNA polymerase II-mediated transcrip-

tion depends mainly on the residue being phosphorylated

(Prelich, 2002), an important step for the characterization of a

CTD kinase is to determine its site specificity within the

heptapeptide repeat. Phosphoamino acid analysis of the

Ms;CDKC;1-phosphorylated CTD peptide revealed the pres-

ence of phosphoserine only (data not shown), suggesting

that Ms;CDKC;1 phosphorylates serines at position 2, 5 or 7.

To establish precisely at which serine residue phosphoryla-

tion takes place, constructs expressing GST fusion proteins

carrying two copies of either the wild-type CTD repeat or

mutated versions, containing the non-phosphorylated Ala

residue instead of Ser, were created and the purified pro-

teins were tested as substrates in immunoprecipitation kin-

ase assays (Figure 5a). In these assays, the control protein

GST was not phosphorylated whereas the GST-CTD fusion

protein was labeled, indicating a specific phosphorylation of

the CTD repeat by Ms;CDKC;1. Similar to the wild-type

repeat, the S2A mutated version where Ala replaced Ser at

position 2 was phosphorylated. In contrast, the S5A muta-

tion completely abolished substrate phosphorylation, indi-

cating that Ms;CDKC;1 phosphorylates the CTD exclusively

on the 5Ser residue. A similar phosphorylation pattern

was observed when the Ms;CDKC;1-Mt;CYCT;1 complex

was immunoprecipitated from Arabidopsis protoplasts

co-expressing the epitope-tagged subunits (data not shown).

(CTD)6-32P

CDKC;1

CDKB2;1

AS bw aw 2 4 6 8 10 12 14 16 18 20 22 24 26

Time (h)

G1/S S G2/M G1

G1/S

S

G2/M

64 61

21

15

38

1

61

36

3

48

51

1

40

53

7

24

66

10

20

41

39

13

36

51

12

13

75

11

13

76

29

17

54

42

19

39

54

22

24

59

20

21

65

21

14

71

8

21

Figure 4. Ms;CDKC;1 protein expression and kinase activity in Medicago

sativa cells synchronized to the S-phase by hydroxyurea treatment. After

release from the block protein extracts were prepared from cells collected at

the indicated time points. Equal amounts of protein extracts were used for

immunoblotting with anti-Ms;CDKC;1 (upper panel) and anti-Ms;CDKB2;1

(bottom panel) antibodies. CTD phosphorylation assays were performed with

Ms;CDKC;1 immunocomplexes purified from the protein extracts. The

phosphorylated peptides were resolved by SDS-PAGE and detected by

autoradiography (middle panel). Distribution of cells in different phases of the

cell cycle was measured by flow cytometry and is indicated (in percentage) in

the table. AS: sample collected from asynchronous cells; bw and aw: samples

collected before and after hydroxyurea removal, respectively.

814 Katalin Fulop et al.

ª Blackwell Publishing Ltd, The Plant Journal, (2005), 42, 810–820

The Ms;CDKC;1-Mt;CYCT;1 complex is a positive regulator

of RNA polymerase II-mediated transcription

The evidence that Ms;CDKC;1 interacts with Mt;CYCT;1 and

the Ms;CDKC;1-Mt;CYCT;1 complex is enriched in the nuc-

lear protein fraction but is not associated with the RNA

polymerase II holoenzyme complex and that Ms;CDKC;1

immunocomplexes displayed a strong and cell cycle-inde-

pendent CTD kinase activity suggests that the complex

might be the plant counterpart of P-TEFb. P-TEFb is a kinase-

cyclin pair that consists of CDK9 and one of the cyclin

T isoforms in animals and facilitates transcriptional elonga-

tion by phosphorylating the C-terminal domain of RNA

polymerase II (reviewed by Price, 2000). To examinewhether

the Ms;CDKC;1-Mt;CYCT;1 complex has a role similar to that

of P-TEFb, we used an in vitro transcription system based on

nuclear extract prepared from HeLa cells (Figure 5b). As

transcription template a plasmid DNA containing the

adenovirus major late promoter in fusion with a G-less

cassette was applied (Sawadogo and Roeder, 1985).

Endogenous CDK9 were depleted from the HeLa extract by

repeated rounds of immunoprecipitation with a CDK9-spe-

cific antibody to render the transcriptional activity of the

extract P-TEFb-dependent. It has been shown that such an

immunodepletion does not reduce the concentration of

other factors needed for transcription (Peng et al., 1998b;

Zhu et al., 1997). When comparedwith the untreated nuclear

extract, depletion of CDK9 almost completely abolished

transcriptional activity and reduced the amount of the radi-

olabeled transcript to a barely detectable level (lanes 2 and 3).

When the transcription reaction mixture was supplemented

either with HA-Mt;CYCT;1 or myc-Ms;CDKC;1 proteins that

were immunoprecipitated from lysates of transfected

Arabidopsis protoplasts, a slight increase in the rate of

transcription was observed (lanes 4 and 5). This slight

increase is probably due to complex formation, a small

fraction of the expressed Medicago subunits being associ-

ated with endogenous Arabidopsis proteins. Addition of

immunoprecipitated Ms;CDKC;1-Mt;CYCT;1 complexes,

however, did not only restore the depleted extract tran-

scriptional activity, but also resulted in the activation of

transcription (lanes 6 and 7). That the Medicago CDKC;

1-CYCT;1 complex is capable of complementing the missing

P-TEFb function suggests that the complex is a functional

ortholog of the human CDK9-cyclin T pair.

Discussion

Metazoan CDK9 is a multifunctional CDK that appears to be

involved in regulation of diverse cellular processes (De Falco

and Giordano, 1998). When bound to cyclin T and K proteins,

the complex functions as P-TEFb and activates transcript

elongation by phosphorylating the CTD of RNA polymerase

II. AsCDK9 isexpressedatahigh level indifferentiatedmouse

tissues, it hasbeensuggested toparticipateeither in cell cycle

arrest or in the initiation of differentiation programs. Over-

expression of a kinase inactive form of CDK9 in HeLa cells

results in growth inhibition and implies yet another role in

cellular growth. We focused our work on the M. sativa

CDKC;1 protein to determine whether functions similar to

that of metazoan CDK9 kinases can be established.

Figure 5. The Ms;CDKC;1 complex phosphorylates 5Ser and functions in

transcription.

(a) Ms;CDKC;1 kinase was immunoprecipitated from Medicago sativa nuclear

extract and phosphorylation site preference was tested using wild type and

mutated versions of CTD fused to GST. Kinase reaction mixtures were

resolved by SDS-PAGE and radioactive substrates were detected by autora-

diography.

(b) Aliquots of untreated (NE) or CDK9-depleted (dNE) HeLa nuclear extract

were supplemented with HA-Mt;CYCT;1 and myc-Ms;CDKC;1 proteins or

myc-Ms;CDKC;1-HA-Mt;CYCT;1 complexes that were immunoprecipitated

from transfected Arabidopsis protoplasts. Transcription reactions were

performed in the presence of [a-32P]UTP and pML(C2AT) plasmid DNA as

template. Reaction products were analyzed on a 5% denaturing gel and

visualized by autoradiography. In the absence of template DNA there was no

detectable transcript production indicating that reactions were specific for the

template added. Arrowhead marks the position of the radiolabeled transcript.

Analysis of the Medicago CDKC;1-CYCLINT;1 complex 815

ª Blackwell Publishing Ltd, The Plant Journal, (2005), 42, 810–820

First we screened Medicago proteins for Ms;CDKC;1

interaction and identified a cyclin protein similar to human

and plant T-type cyclins. Curiously, plant CYCLINT proteins

do not contain any other recognizable sequencemotifs other

than the characteristic N-terminal cyclin box fold. In com-

parison, human cyclin T1, T2a and T2b proteins contain a

histidine-rich stretch that is supposed to facilitate recogni-

tion of the CTD of RNA polymerase II and a carboxy-terminal

PEST sequence known to target proteins for ubiquitination-

dependent degradation (Garriga and Grana, 2004; Rechste-

iner and Rogers, 1996). Instead, protein secondary structure

prediction algorithms (http://cubic.bioc.columbia.edu/pre-

dictprotein) detect a helical domain at the C-terminal part

of all plant CYCLINT proteins. It was shown for Xenopus A

and B cyclins that a second helix located outside the cyclin

box fold contributes to CDK binding and can serve as an

interaction platform for substrate binding (Goda et al.,

2001). Similar structural elements might govern the

Ms;CDKC;1 and Mt;CYCT;1 interaction, as in the yeast two-

hybrid system the two proteins show an extreme interaction

specificity as they bind exclusively to each other. Medicago

cyclins including members of the A-, B- and D-types were

unable to form complexes with the Ms;CDKC;1 protein. A

comparable specificity has been reported for the PITAIRE-

domain kinase Lyces;CDKC;1 from tomato (Joubes et al.,

2001). Similar to Arabidopsis CYCT (Barroco et al., 2003),

Mt;CYCT does not bind to any other kinase subunits

including A-, B- and E-type CDK proteins. Thus it appears,

that the observed interaction specificity is mediated by

unique structural features of both subunits.

The Ms;CDKC;1 complex immunoprecipitated from

M. sativa cells possesses protein kinase activity that phos-

phorylates proteins such as the MBP, the CTD of RNA

polymerase II and the retinoblastoma protein in vitro. Unlike

complexes of plant A- and B-type CDK kinases, the

Ms;CDKC;1 complex fails to phosphorylate the typical CDK

substrate histone H1 suggesting a distinct consensus phos-

phorylation site requirement. This substrate specificity

resembles that of human CDK9/cyclin T complexes that

phosphorylate the MBP, the retinoblastoma protein and the

CTD of RNA polymerase II, the latter two proteins being

in vivo substrates as well (De Falco and Giordano, 1998;

Garriga et al., 1996). Phosphorylation of the retinoblastoma

protein by the human CDK9/cyclin T2 complexwas shown to

activate MyoD-mediated gene transcription and induce

differentiation of muscle cells (Simone et al., 2002). It

remains to be seen if phosphorylation of the retinoblasto-

ma-related protein (RBR) by plant CDKC complexes would

have a similar role in cellular differentiation. Phosphoryla-

tion of the CTD of RNA polymerase II largest subunit by

CDK9/cyclin T complexes is required for the transcription of

most class II genes and is implicated in the transition from

initiation to the elongation phase of the transcription cycle

(Shim et al., 2002; Taube et al., 2002). Because the structure

of RNA polymerase II holoenzyme and the general tran-

scription factors are highly conserved from yeast to plants it

is probable that plant CDKC-CYCT complexes phosphorylate

the CTD in vivo as well. Several lines of evidence support

this idea. First, we demonstrated by cellular fractionation

and immunolocalization experiments that Ms;CDKC;1 and

Mt;CYCT;1 proteins are localized to the nucleus. This implies

that the complex physiological substrates are nuclear pro-

teins and coincides with the observed nuclear localization of

human CDK9/cyclin T complexes (Herrmann and Mancini,

2001). Secondly, it has been shown that the CTD kinase

activity of the human CDK9 complex is not cell cycle

regulated (Garriga et al., 2003), although others suggested

that CDK9 protein stability and thus the activity of the

complex is regulated by the SCFSKP2 ubiquitin ligase in a cell

cycle-dependent fashion (Kiernan et al., 2001). Our results

indicate a cell cycle-independent activity pattern for the

Ms;CDKC;1 complex as neither the protein level of the kinase

subunit nor the activity of its complexes changed during the

cell division cycle. Moreover, when Ms;CDKC;1 and

Mt;CYCT;1 proteins were expressed in Arabidopsis cells

we could not detect any increase in their stability upon

treatment with MG132, which is a specific inhibitor of the

26S proteasome. Thirdly, the Ms;CDKC;1 complex phos-

phorylates 5Ser within the CTD heptapeptide repeat when

tested with short peptide substrates in vitro. This site

preference is consistent with that of known physiological

CTD kinases like the CDK7/cyclin H, CDK8/cyclin C and CDK9/

cyclin T complexes (Ramanathan et al., 2001). However, on

longer peptide substrates comprising 15 YSPTSPS repeat

domains the CDK9/cyclin T complex phosphorylates prefer-

entially the 2Ser residue (Pinhero et al., 2004). Given that

CTD phosphorylation is a dynamic process shifting from5Ser hyper-phosphorylation during initiation to 2Ser hyper-

phosphorylation in elongation (Svejstrup, 2004), phosphory-

lation on 2Ser is more consistent with a role in transcript

elongation. The in vivo site preference of metazoan CDK9

complexes is not yet known but the structurally similar yeast

Ctk1 kinase phosphorylates the CTD of elongating RNA

polymerase II on 2Ser residues (Cho et al., 2001). Finally,

promoter-specific in vitro transcription assays using CDK9-

depleted HeLa nuclear extracts revealed that the Medicago

CDKC;1-CYCT;1 complex is able to substitute for the lack of

CDK9 function. Neither Ms;CDKC;1 nor Mt;CYCT;1 protein

alone restored transcriptional activity of the depleted extract

suggesting that a functional complex with full kinase activity

is necessary for complementation.

In summary, we have identified and characterized the

Medicago CDKC,1-CYCT;1 complex, the biochemical prop-

erties of which are clearly distinguishable from that of

known plant CDK-cyclin complexes. The CDKC;1-CYCT;1

complex mirrors the most important features of yeast and

metazoan CDK9/cyclin T complexes. Namely, the complex is

localized to the nucleus and has cyclin-dependent CTD

816 Katalin Fulop et al.

ª Blackwell Publishing Ltd, The Plant Journal, (2005), 42, 810–820

kinase activity, displays similar substrate range as CDK9/

cyclin T, its activity is not cell cycle-dependent and positively

regulates RNA polymerase II-mediated transcription reac-

tions in vitro. Based on the above evidence we suggest that

the CDKC;1-CYCT;1 kinase is a plant ortholog of P-TEFb.

Experimental procedures

Yeast two-hybrid screen and interaction tests

The yeast strain PJ69-4A (MATa trp1-901, leu2-3,112 ura3-52, his3-200 gal4delta, gal80delta GAL2-ADE2, LYS2::GAL1-HIS3, met2::GAL7-lacZ) was used for library screening and pairwise interactionassays as described (James et al., 1996). Ms;CDKC;1 cDNA wascloned in frame with the DNA-binding domain of pGBT9 to obtainthe bait construct. Transformation of yeast with the bait and theM. truncatula library was performed according to establishedprotocols (Schiestl and Gietz, 1989). Transformation mixtures wereplated on yeast drop-out selection media lacking either leucineand tryptophan to estimate the transformation efficiency or lack-ing leucine, tryptophan and histidine to test protein–proteininteractions. Positive colonies were recovered after 4–6 days andtheir growth were further tested on selective plates lacking leu-cine, tryptophan, histidine and adenine. For pairwise interactionassays full-length cDNAs encoding Medicago CDK kinases andcyclins were subcloned from pBluescript II SK plasmids (Strata-gene, La Jolla, CA, USA) into yeast two-hybrid vectors in frameeither with the GAL4 AD or BD domains. The protein-coding re-gions of Medsa;CDKA;1, CDKE;1, CYCB2;1, CYCB2;2 and CYCA2;1were cloned into the EcoRI-SalI sites of pGAD424 vector (Clontech,Palo Alto, CA, USA). The protein-encoding regions of Medsa;CD-KA;2, CDKB1;1, CDKB2;1 and CYCD3;1 were inserted into theBamHI-SalI sites of pGAD424. The cDNA of Medsa;CDKC;1 wascloned into Cfr9I-BamHI sites of pGAD424 vector. Medicago trun-catula cyclins D4 and D5 were in the pAD-GAL4-2.1 vector (Strat-agene) and obtained by in vivo excision from lambda HybriZAPclones as described (Gyorgyey et al., 2000). Co-transformed yeaststrains were selected on Leu, His, Ade and Trp plates and testedfor b-galactosidase activity by filter and liquid assays.

Bacterial protein expression and generation of antibodies

An EcoRI-XmnI fragment encoding the C-terminal 21 kDa region ofMs;CDKC;1 was cloned into pTrcHisA vector (Invitrogen, Carlsbad,CA,USA) and expressed inEscherichia coli strain BL21 in fusionwithan N-terminal hexahistidine tag. The fusion protein was purified byNi2þ-nitrilotriacetic acid agarose (Qiagen, Hilden, Germany) chro-matography under denaturing conditions according to the protocolprovided by the manufacturer and used to immunize rabbits. Foraffinity purification of the antiserum, the cDNA encoding the wholeMs;CDKC;1 protein was introduced into EcoRV-NotI digested pET-35bvector (Novagen,Madison,WI,USA) andexpressed inE. coli as afusion protein with the cellulose-binding domain. The recombinantprotein was isolated from purified inclusion bodies by boiling in 1%SDS and was used to blot-affinity purify the anti-Ms;CDKC;1 anti-body.

To generate GST fusion proteins containing two copies of thewild type or phosphorylation site-mutated RNAP II C-terminalheptapeptide repeat, the following oligonucleotides were annealed,digested with BamHI and EcoRI enzymes (sites underlined) andinserted into pGEX-4T-2 expression vector (Pharmacia, Uppsala,Sweden): wt upper: 5¢-cgggatcctactccccg-acctccccgtcctactccccga-

cctccccggaattccg, wt lower: 5¢-cggaattccggggaggtcggggagtaggac-ggggaggtcggggagtaggatcccg, S2A upper: 5¢-cgggatcctacgccccga-cctccccgtcctacgccccgacctccccggaattccg, S2A lower: 5¢-cggaattcc-ggggaggtcggggcgtaggacggggaggtcggggcgtaggatcccg, S5A upper:5¢-cgggatcctactccccgaccgccccgtcctactccccgaccgccccggaattccg, S5Alower: 5¢-cggaattccggggcggtcggggagtaggacggggcggtcggggagta-ggatcccg. Recombinant proteins were expressed in E. coli strainBL21 and purified as recommended by the manufacturer.

Plant material

Medicago sativa ssp. varia genotype A2 and Arabidopsis thalianaecotype Columbia cell suspension cultures were maintained byweekly subculturing in MS media (Murashige and Skoog, 1962)supplemented with 1 mg l)1 2,4-D and 0.2 mg l)1 kinetin or240 lg l)1 2,4-D and 14 lg l)1 kinetin, respectively. Synchronizationof the M. sativa A2 cell culture and flow cytometric analysis wereperformed according to Magyar et al. (1997). Plant protein extractswere prepared in extraction buffer containing 25 mM Tris-HCl pH7.6, 15 mM MgCl2, 15 mM EGTA, 75 mM NaCl, 60 mM b-glycero-phosphate, 1 mM DTT, 0.1% NP40, 0.1 mM Na3VO4, 1 mM NaF,1 mM PMSF and protease inhibitors (Complete; Roche, Mannheim,Germany). Cytoplasmic and nuclear protein fractions were isolatedand size fractionated according to Bako et al. (2003).

Kinase assays

For protein kinase assays, the Ms;CDKC;1 protein was immuno-precipitated from 150 lg of total protein extract prepared fromM. sativa A2 cell suspension with 50 ll affinity-purified polyclonalanti-Ms;CDKC;1 antibody and immobilized on protein A agarose.Immunocomplexes were washed three times in TBS plus 0.5%Tween 20 and once with kinase buffer containing 25 mM Tris-HCl,pH 7.8, 15 mM MgCl2, 1 mM DTT. Phosphorylation reactions wereperformed in kinase buffer supplemented with 0.2 mg ml)1 sub-strate protein and 2.5 lCi of [c -32P]ATP for 30 min at room tem-perature then terminated by the addition of SDS loading buffer.Reaction mixtures were separated by SDS-PAGE and phosphory-lation was revealed by autoradiography.

Transient expression of proteins in protoplasts and

immunoprecipitation

Ms;CDKC;1 and Mt;CYCLINT;1 cDNAs without the initial ATG codonwere amplified by PCR reaction using the following primersharboring BamHI and SalI restriction sites (underlined):5¢-ttggatccgcgggtccagggcaattga and 5¢-ttctgcagctactgctgccagcca-tact for Ms;CDKC;1, 5¢-ttggatcctcttttgttcggaattttcaagc and 5¢-gtc-gacctaaaagagctccattaactg for Mt;CYCLINT;1. Amplified fragmentswere digested with BamHI and SalI enzymes and cloned intopRT104-derived vectors (Topfer et al., 1987) modified to incorporatethree tandem copies of the c-myc and HA epitopes as N-terminalfusions. Protoplasts from Arabidopsis cell suspension were pre-pared and transfected as described (Meskiene et al., 2003). From500 lg of total protein extract HA-tagged proteins were immuno-precipitated using 500 ng of 3F10 anti-HA monoclonal antibody(Roche) and 50 ll of Protein G paramagnetic microbeads (MiltenyBiotec, Bergisch Gladbach, Germany) by overnight incubationat 4�C. Beads were then loaded onto columns and washed threetimes with extraction buffer and once with 20 mM Tris, pH 7.5.Bound proteins were eluted with 50 ll of SDS loading buffer.Co-immunoprecipitation of themyc-tagged protein was revealed by

Analysis of the Medicago CDKC;1-CYCLINT;1 complex 817

ª Blackwell Publishing Ltd, The Plant Journal, (2005), 42, 810–820

SDS-PAGE followed by Western blotting using the 9E10 anti-mycmonoclonal antibody (Roche) and enhanced chemiluminescencedetection (SuperSignal WestPico; Pierce, Rockford, IL, USA).

For immunolocalization, Arabidopsis protoplasts were co-trans-fected with HA-tagged Mt;CYCT;1 and myc-tagged Ms;CDKC;1.After 16 h, protoplasts were fixed in 3% paraformaldehyde inmicrotubule stabilization buffer (MSB) containing PBS pH 6.8,10 mM MgSO4, 10 mM EGTA, 0.17 M glucose and 0.17 M mannitolfor 50 min. After five washes with MSB, protoplasts were spreadon slides and dried to immobilize. Cells were permeabilized withMSB containing 0.5% Triton X-100 for 20 min and blocked with 5%normal donkey serum in MSB for 30 min. Slides were thenincubated overnight at 4�C with monoclonal anti-HA (clone16B12; Covance, Berkeley, CA, USA) and polyclonal anti-myc(Molecular Probes, Eugene, OR, USA) antibodies at a 1:200dilution. Cells were washed with MSB and incubated with thesecondary Alexa 488 and TRITC-conjugated antibodies (JacksonImmunochemicals, West Grove, PA, USA) at 1:1000 and 1:150dilutions, respectively, for 1 h at room temperature. Cells werewashed, counterstained with 1 lg ml)1 DAPI for 10 min andmounted using Citifluor (Citifluor Ltd, London, UK). Confocal laserscanning microscopy was performed using a Leica TCS SP2 DM-RXA2 microscope (Leica Microsystems, Mannheim, Germany).

In vitro transcription reactions

Transcription assays were performed with the HeLaScribe nuclearextract (Promega, Madison, WI, USA) according to the protocolprovided by the manufacturer with the following modifications. Astemplate, supercoiled pML(C2AT) plasmid DNA carrying the a-denovirus major late promoter in front of a synthetic 400 bp-long G-less cassette (Sawadogo and Roeder, 1985) was used. Reactionmixtures in a final volume of 25 ll contained 1x transcription buffer,4 mM MgCl2, 100 ng of template DNA and 8 U (50 lg) of HeLanuclear extract were assembled on ice then incubated for 15 min atroom temperature. Reactions were initiated by adding ATP and CTPto 400 lM and UTP containing 5 lCi of [a-32P] UTP (3000 Ci mmol)1;Amersham Biosciences, Uppsala, Sweden) to 15 lM final concen-trations, incubated for 45 min at 30�C then terminated with 175 ll ofstop solution. After phenol/chloroform and chloroform extractionsRNAs were ethanol precipitated, dried, redissolved in formamideloading buffer and resolved on denaturing 5% polyacrylamide 0.5xTBE gels. Radioactive transcription products were detected byautoradiography. When biochemical complementation was tested,CDK9 complexes were depleted from the HeLa nuclear extract bythree consecutive rounds of immunoprecipitation, each for 1 h with2.5 lg of polyclonal anti-CDK9 antibody (sc-484; Santa Cruz Bio-technology, Santa Cruz, CA, USA) immobilized on Dynabeads Pro-tein G paramagnetic particles (Dynal, Oslo, Norway). At the sametime Ms;CDKC;1 and Mt;CYCT;1 proteins as well as CDKC;1-CYCT;1complexes were immunoprecipitated either with anti-myc or withanti-HA antibodies from lysates of Arabidopsis protoplasts expres-sing the epitope-tagged proteins. Aliquots of the depleted nuclearextract were then supplemented with the different immunocom-plexes captured on Protein G Dynabeads and transcriptional activitywas analyzed as described above.

Acknowledgements

We thank S.J. Elledge for providing the Y145 yeast strain; andR.P. Bhalerao and M. Grebe for critical reading of the manuscript.This work was supported by a joint grant of the Swedish andHungarian Academy of Sciences and by grants from the European

Union (ECCO QLG2-CT 1999-00454) and the Hungarian ResearchFund OTKA T038365.

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